Design and total synthesis of a fluorescent phorboxazole a analog for cellular studies

Article abstract of DOI:10.1016/j.bmcl.2005.10.109

To enable studies to elucidate the intracellular processing and targeting of the potent cytostatic/apoptotic anticancer natural products phorboxazoles A and B, a fluorescent derivative has been developed. This involved the total syntheses of the terminal

Full text of DOI:10.1016/j.bmcl.2005.10.109

Bioorganic & Medicinal Chemistry Letters 16 (2006) 901–904
Design and total synthesis of a fluorescent phorboxazole
a analog for cellular studies
Jiehao Chen,a, Lu Ying,a T. Matthew Hansen,a,à Mary M. Engler,a,§ Chi Sing Lee,a,–
James J. La Clairb and Craig J. Forsytha,*
aDepartment of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
bXenobe Research Institute, PO Box 4073, San Diego, CA 92164-4073, USA
Received 11 October 2005; revised 29 October 2005; accepted 31 October 2005
Available online 21 November 2005
Abstract—To enable studies to elucidate the intracellular processing and targeting of the potent cytostatic/apoptotic anticancer
natural products phorboxazoles A and B, a fluorescent derivative has been developed. This involved the total syntheses of the
terminal alkyne 33-O-Me-45,46-dehydrobromophorboxazole A (MDHBPA) and a terminal vinyl iodide derivative of the blue
fluorescent dye N,N,-dimethyl-7-aminocoumarin (DMC). Sonogashira coupling of these partners provided enyne DMC–MDHBPA
in high yield.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Natural products phorboxazole A (1, Fig. 1) and its C13
epimer display potent cytostatic activity against many
human cancer cell lines.1 Synthetic 1 was also found to
induce apoptosis.2 The phorboxazoles arrest human
cancer cell growth during S phase, but their undefined
mechanism of action does not correlate with other
known cytostatic/cytotoxic agents. Hence, their antican-
cer activities may illuminate cellular targets that have
not previously been explicitly exploited. As such, the
phorboxazoles remain active candidates for further
exploration and therapeutic development.
synthesis remains the most reliable option to provide
sufficient quantities of the phorboxazoles5 and more
importantly, designed structural variants6,7 to enable
continued exploration of their scientific and biomedical
potential. Accordingly, we have described a total synthe-
sis,5a structure–activity relationship study,6a and the
preparation of a biotinylated phorboxazole derivative
for target identification.6b Reported here are the design
and synthesis of a fluorescent derivative for in vivo bio-
photonic studies to elucidate intracellular processing
and targeting of the natural product.
The isolation of phorboxazoles from sponges of two dif-
ferent genera collected in the Indian Ocean off western
Australia1a,3 suggests that these natural products may
be biosynthesized by a common symbiotic microorgan-
ism that has yet to be identified.4 To date, laboratory
Our initial structure–activity-relationship study identi-
fied the C33 mixed methyl ketal (2) and the terminal al-
kyne 45,46-dehydrobromophorboxazole A (DHBPA, 3)
as synthetic analogs that maintained the anticancer
activities of 1 (Fig. 1). Accordingly, we had previously
prepared a biotinylated derivative of 3 from synthetic
DHBPA and a biotin-bearing polyethylene glycol linker
for affinity localization and isolation studies.6b This in-
volved Sonogashira coupling8 between the terminal al-
kyne of 3 and a vinyl iodide terminated linker bearing
a biotin moiety. It was recognized that this reliable, late
stage Sonogashira coupling approach for the generation
of phorboxazole analogs could be extended to incorpo-
rate fluorescently labeled probes, such as 4 (Fig. 1).
Keywords: Phorboxazole; Coumarin; Probe; Anticancer; Total
synthesis.
*
Corresponding author. Tel.: +1 612 624 0218; fax: +1 612 626
7541; e-mail: forsyth@chem.umn.edu
Present address: Memorial Sloan-Kettering Cancer Center, New
York, NY 10021, USA.
à
Present address: Abbott Laboratories, Abbott Park, IL 60064-6217,
USA.
§
Present address: SAFC, Madison, WI 53711, USA.
Present address: Shenzhen Graduate School of Peking University,
Shenzhen 518055, China.
–
Derivatives of the blue fluorescent dye N,N-dimethyl-7-
aminocoumarin-4-acetic acid have been demonstrated
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.109

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J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 901–904
to display an ideal profile of abiotic non-interference
upon cellular uptake and protein binding.9 Hence, the
N,N-dimethyl-7-aminocoumarin (DMC) moiety was
selected as the fluorophore.
(DMC–MDHBPA, Fig. 1) obtained from phorboxazole
derivative 5 and DMC analog 6 (Scheme 1).
The analog 33-O-methyl-45,46-dehydrobromo-phorb-
oxazole A (5) is obtainable via our established tri-com-
ponent coupling entry to the phorboxazole
architecture.5a,10 A C31–C46 fragment bearing a car-
boxylate at C31 and an alkyne at C45,4611 was joined
with a C1–C30 amino-alcohol via sequential amide
and oxazole formation. Fluoride-induced cleavage of
C13 and C38 silyl ethers gave terminal alkyne 5.
To merge coumarin fluorophore and bioactive phorbox-
azole derivatives via the mild Sonogashira coupling
reaction requiring no protective groups on either part-
ner, one could consider coupling 1 via its vinyl bromide
terminus with a terminal alkyne derivative of DMC.
However, the terminal alkyne of 3 (Fig. 1) is obtained
more efficiently than the (E)-vinyl bromide of 1 via total
synthesis. Furthermore, a terminal alkyne is a more ro-
bust and versatile functional group for derivatization.
Thus, the obvious roles of vinyl halide and terminal al-
kyne in the Sonogashira coupling process were reversed.
The alkyne would be assigned to the phorboxazole
chemophore and the vinyl halide to the DMC fluoro-
phore. The vinyl halide moiety would reside at the termi-
nus of a polar diether linker to enhance aqueous
solubility. This linker would be attached to the readily
functionalizable 4-position of DMC. The immediate
synthetic precursor to 3 is the corresponding C33 mixed
methyl ketal 5 (Scheme 1). The culmination of these de-
sign considerations resulted in the targeting of N,N-di-
The complementary vinyl iodide 6 (Scheme 1) was pre-
pared from triethylene glycol (7) and N,N-dimethyl-7-
amino-4-methyl-coumarin (9, Scheme 2). Differential
terminal functionalization of 7 involved mono-silylation
followed by reciprocal iodination to yield silyl ether-io-
dide 8. Commercially available 9 was converted into the
extended enolate with LDA/HMPA. Alkylation with io-
dide 8 followed by desilylation yielded primary alcohol
10. Oxidation12 to the corresponding aldehyde followed
by Takai iodo-olefination13 yielded a 3:1 E/Z mixture of
vinyl iodides, from which the (E)-isomer 6 was chro-
matographically isolated.
methyl-7-aminocoumarin–MDHBPA
conjugate
4
Sonogashira coupling of 5 and 6 to generate 4 was
accomplished in 10 min and essentially quantitative
yield on small scale using bis(triphenylphosphine)palla-
dium(II) chloride and CuI in the presence of Et3N and
in THF (Scheme 2).8,14 This remarkable reaction de-
serves comment. First, there are no protective groups
in either coupling partner that required subsequent
removal. Hence, this coupling culminated in the prepa-
ration of the targeted fluorescent phorboxazole probe.
Second, the efficiency of coupling of 5 and 6 may be con-
trasted with the similar coupling of a biotinylated vinyl
iodide linker with DHBPA (3). In the latter case, a con-
siderably lower yield of enyne was obtained.6b This may
be attributable to the presence of Pd-sequestering sulfur
in the biotin moiety, whereas the vinyl iodide DMC
derivative 6 participates in Pd/Cu-mediated enyne for-
mation without complication.
This work provides a novel phorboxazole probe for
in vivo intracellular localization and target elucidation
Figure 1. Structures of phorboxazole A and analogs. Natural 1 bears a
C33 hemiketal and an (E)-vinyl bromide. Synthetic C33 mixed methyl
ketal 2 and terminal alkyne 3 maintain the cytostatic activity of 1.
Coumarin conjugate 4 joins N,N-dimethyl-7-amino-coumarin (DMC)
and 33-O-Me-3 (MDHBPA).
Scheme 1. Sonogashira disconnection of phorboxalog fluoroprobe 4.

J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 901–904
903
OTBDPS
HO
1) n-BuLi; TBDPSCl
O
O
O
OH
O
I
2) Ph3P, I2, Imidazole
(75%, 2-steps)
7
8
O
O
HO
1) LDA / THF / HMPA
O
O
O
2) 8
3) TBAF/ THF
(64%, 2-steps)
Me
O
Me
Me
N
N
Me
Me
10
9
1) TPAP, NMO
2) CHI3, CrCl2
(55%, 2-steps)
I
O
O
5 (MDHBPA)
4
(Ph3P)2PdCl2
CuI /THF / Et3N
(ca. 90%)
O
6 (DMC)
DMC-MDHBPA
O
Me
N
Me
Scheme 2. Synthesis of coumarin label 6 and fluorescent conjugate 4.
6. (a) Uckun, F. M.; Forsyth, C. J. Bioorg. Med. Chem.
Lett. 2001, 11, 1181; (b) Hansen, T. M.; Engler, M.
M.; Forsyth, C. J. Bioorg. Med. Chem. Lett. 2003, 13,
2127.
studies. Results derived from application of this probe
for such studies will be reported in due course.
7. Smith, A. B., III; Razler, T. M.; Pettit, G. R.; Chapuis, J.-
C. Org. Lett. 2005, 7, 4403.
Acknowledgments
8. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 16, 4467.
Financial support from the NIH (R01 CA099950) and
experimental contributions from B, Wang, Y. Lu, C.
Leitheiser, M. S. Ostroski, M. Christmann, F. Ahmed,
and R. D. Cink are gratefully acknowledged.
9. (a) Sandler, J. S.; Fenical, W.; Gulledge, B. M.; Cham-
berlin, A. R.; La Clair, J. J. J. Am. Chem. Soc. 2005, 127,
9320(b) La Clair, J. J.; Alexander, M. D.; Burkart, M. D;
Leonard, M. S.; Portonovo, P.; Liang, B; Ding, X.;
Joullie, M. M.; Gulledge, B. M.; Aggen, J. B.; Chamberlin,
A. R.; Sandler, J.; Fenical, W.; Cui, J.; Gharpure, S. J.;
Polosukhin, A.; Zhang, H.-R.; Evans, P. A.; Richardson,
A. D.; Harper, M. K.; Ireland, C. M.; Vong, B. H.; Brady,
T. P.; Theodorakis, E. A. Submitted for publication.
10. Synthesis and characterization of 5: Lee, C. S., Ph.D.
Thesis, University of Minnesota, Minneapolis, MN, USA,
1999.
11. Ahmed, F.; Forsyth, C. J. Tetrahedron Lett. 1998, 39, 183.
12. Ley, S. V.; Marsden, S. P.; Norman, J.; Griffith, W. P.
Synthesis 1994, 639.
13. Takai, K.; Nitta, K.; Utimoto, K. J. Am. Chem. Soc. 1986,
108, 7408.
14. Preparation of 4: To a stirred, rt solution of vinyl iodide 6
(1.5 mg, 3.3 lmol) and C33-methoxy-45,46-dehydrob-
romophorboxazole A10 (5, 0.5 mg, 0.5 lmol) in freshly
distilled THF (0.5 mL) were added sequentially freshly
distilled triethylamine (0.1 mL), bis(triphenylphos-
phine)palladium dichloride (0.5 mg, 0.7 lmol), and CuI
(0.5 mg, 3 lmol). The resulting mixture was stirred under
argon at rt. After 10 min, TLC showed no remaining 5.
Saturated aqueous NH4Cl (0.5 mL) and ethyl acetate
(1 mL) were added. The aqueous layer was separated and
extracted with ethyl acetate (6· 1 mL). The combined
organic phases were dried over Na2SO4, filtered, and
concentrated under vacuum. The residue was purified by
silica gel column chromatography (ethyl acetate/methanol,
20:1, v/v) followed by preparative thin layer chromatog-
raphy (ethyl acetate/methanol, 10:1, v/v) to provide 4
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J. Chen et al. / Bioorg. Med. Chem. Lett. 16 (2006) 901–904
(0.6 mg, 0.5 lmol, ca. 90%) as a yellow amorphous solid:
4.00 (m, 1H), 3.77 (m, 1H), 3.62 (m, 1H), 3.59 (s, 4H),
3.50–3.56 (m, 3H), 3.48 (m, 2H), 3.41 (m, 1H), 3.34 (s,
3H), 3.31 (s, 3H), 3.27 (s, 3H), 3.09 (m, 1H), 3.06 (s, 6H),
2.97 (s, 1H), 2.80 (t, J = 8.0 Hz, 2H), 2.72 (d, J = 13 Hz,
1H), 2.64 (m, 1H), 2.55 (m, 2H), 2.43 (m, 2H), 2.35 (m,
2H), 2.25 m, 1H), 2.02 (m, 2H), 1.99 (m, 2H), 1.98 (s, 3H),
1.97–1.90 (m, 5H), 1.86 (s, 3H), 1.73 (d, J = 14.5 Hz, 1H),
1.58 (m, 1H), 1.45 (m, 1H), 1.37 (m, 1H), 1.11 (m, 1H),
0.97 (d, J = 7.0 Hz, 3H), 0.77 (d, J = 6.5 Hz, 3H); HRMS
(ESI): calcd for [C74H97N3O17+2Na]2+ 1331.6446, found
1322.6512.
Rf 0.45 (ethyl acetate/methanol, 10:1, v/v); 1H NMR
(500 MHz, CDCl3) d 7.58 (s, 1H), 7.49 (d, J = 8.5 Hz, 1H),
7.43 (s, 1H), 6.69 (m, 1H), 6.63 (d, J = 9 Hz, 1H), 6.54 (s,
1H), 6.30 (d, J = 11 Hz, 1H), 6.28 (s, 1H), 6.16 (d,
J = 12.5 Hz, 1H), 6.12 (dt, J = 5.5, 10.5 Hz, 1H), 5.99 (s,
1H), 5.92 (s, 1H), 5.73 (d, J = 16 Hz, 1H), 5.34 (dd, J = 9,
11.5 Hz, 1H), 5.30 (dd, J = 8, 11 Hz, 1H), 5.47 (d, J = 8.5,
1H), 5.00 (s, 1H), 4.75 (dd, J = 3.5, 10 Hz, 1H), 4.63 (s,
1H), 4.52 (dd, J = 4, 10.5 Hz, 1H), 4.41 (s, 1H), 4.33 (m,
1H), 4.18 (m, 1H), 4.10 (m, 1H), 4.06 (d, J = 5 Hz, 2H),