Total synthesis of the antitumor natural product polycarcin v and evaluation of its DNA binding profile

Article abstract of DOI:10.1021/ol501095w

The convergent total synthesis of polycarcin V, a gilvocarcin-type natural product that shows significant cytotoxicity with selectivity for nonsmall-cell lung cancer, breast cancer, and melanoma cells, has been achieved in 13 steps from 7, 8, and 22; the

Full text of DOI:10.1021/ol501095w

Letter
pubs.acs.org/OrgLett
Total Synthesis of the Antitumor Natural Product Polycarcin V and
Evaluation of Its DNA Binding Profile
Xiao Cai,† Kevin Ng,† Harmanpreet Panesar,† Seong-Jin Moon,† Maria Paredes,† Keishi Ishida,‡
Christian Hertweck,‡ and Thomas G. Minehan*,†
†Department of Chemistry and Biochemistry, California State University, Northridge, 18111 Nordhoff Street, Northridge, California
91330, United States
‡Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology, HKI
Beutenbergstrasse 11a, 07745 Jena, Germany
S
* Supporting Information
ABSTRACT: The convergent total synthesis of polycarcin V, a
gilvocarcin-type natural product that shows significant cytotoxicity
with selectivity for nonsmall-cell lung cancer, breast cancer, and
melanoma cells, has been achieved in 13 steps from 7, 8, and 22; the
sequence features a stereoselective α-C-glycosylation reaction for the
union of protected carbohydrate 7 and naphthol 8. The association
constant for the binding of polycarcin V to duplex DNA is similar to that previously reported for gilvocarcin V.
he gilvocarcin family of C-aryl glycoside natural products1
Thave been shown to exhibit high antitumor activity with low
overall toxicity.2 A likely mode of action of gilvocarcin V, the
most studied member of the gilvocarcins, involves intercalation
of the aromatic chromophore into DNA, followed by UV-
induced covalent linkage of the natural product to DNA by a [2 +
2]-cycloaddition between the vinyl moiety and a thymine
residue.3 Photoactivated gilvocarcin V is also able to selectively
cross-link DNA and the phosphorylated form of histone H3 and
GRP78, a heat shock protein.4 Importantly, gilvocarcins M and E,
both of which bear aliphatic residues instead of a vinyl group at
C.8 of the chromophore, are not cytotoxic.5
Figure 1. Structures of polycarcin V (1) and gilvocarcins V (2), M (3),
and E (4).
Both Waring6 and McGee7 have previously demonstrated that
the presence of a carbohydrate moiety on an intercalating
chromophore contributes positively and significantly to the
binding association with DNA. Studies on the interaction of C-
aryl glycoside natural products with DNA have shown that the
carbohydrate moieties typically occupy the minor groove in the
bound complex, where noncovalent interactions between
functional groups present on the sugar and residues in the
minor groove are established.8 It has been proposed that these
carbohydrate−DNA noncovalent contacts may be largely
responsible for the binding-site sequence selectivity of the C-
aryl glycosides.9 Furthermore, the identity of the sugar
substituent of C-aryl glycosides such as the gilvocarcins may
also be relevant to cell-type specificity, potency, transport, and
pharmacokinetics.10
with selectivity for non-small-cell lung cancer (LXF 1211 L and
LXFL 529L, IC70 < 0.3 ng/mL and 0.3 ng/mL), breast cancer
(MCF7, MDAMB231, MDAMB 468, IC70 from <0.3 ng/mL to 4
ng/mL), and melanoma cells (MEXF 462NL, MEXF 514 L,
MEXF 520L, IC70 from <0.3 ng/mL to 0.4 ng/mL), and its
antiproliferative fingerprint is virtually identical to that of
actinomycin D. With the aim of evaluating the DNA binding
affinity of polycarcin V for comparison with the known
gilvocarcins, we sought to undertake the total synthesis of this
natural product.
Since the Suzuki O → C glycoside rearrangement reaction, a
typical strategy employed for the creation of aryl-glycosidic
carbon−carbon bonds,12 favors the formation of β-glycosidic
linkages, we envisioned that the carbon−carbon bond between
the aromatic and carbohydrate moieties of polycarcin V could be
fashioned instead by a Lewis acid mediated Friedel−Crafts-type
glycosylation13 utilizing a rhamnosyl donor containing a suitable
protecting group at C.2′ to direct the stereochemistry of bond
formation at C.1′ (7, Figure 2). Standard oxidation and selective
Polycarcin V (Figure 1) is a recently isolated gilvocarcin-type
natural product obtained from a culture extract of Streptomyces
polyformus sp. nov. (YIM 33176).11 This substance co-occurs
with gilvocarcin V but possesses an α-linked L-rhamnopyranose
moiety instead of a β-linked D-fucofuranose sugar; indeed, the α-
C-glycosidic linkage is rare among the known C-aryl glycoside
natural products.1d,e Polycarcin V shows significant cytotoxicity
Received: April 15, 2014
Published: May 13, 2014
© 2014 American Chemical Society
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Scheme 2. C-Glycosylation of Naphthalene 8
obtained cleanly in 70% yield with >95:5 α:β stereoselectivity. In
model studies, it was found that similar yields and stereo-
selectivities of glycosylated products could be obtained when
rhamnosyl acetates bearing a C.2′ isobutyryl ester were
employed. Anchimeric participation by the C.2′ ester group
may explain the ease of this C-glycosylation reaction, both
assisting the expulsion of the carbohydrate C.1′ acetate and
shielding the β-face of the resulting oxocarbenium ion from
attack by the naphthyl nucleophile.18
Figure 2. Retrosynthetic analysis of polycarcin V.
hydroxyl group protection would then provide a naphthol
glycoside capable of carbodiimide-mediated coupling with
protected iodoarene carboxylic acid 22, prepared from 3,5-
dihydroxybenzoic acid. Following the precedent of Suzuki,14
palladium-catalyzed intramolecular arylation, deprotection, and
hydroxyl group elimination would then provide the natural
product.
Elaboration of C-glycoside 9 toward the natural product
commenced with formylation of the aromatic ring under
Vilsmeier conditions (Scheme 3).19 After much experimentation,
Scheme 3. Elaboration of C-Glycoside 9 to Naphthol 14
The synthesis commenced with assembly of the carbohydrate
and aromatic fragments for coupling. For the preparation of
diacetate 7,15 L-rhamnose was first treated with allyl alcohol
under acidic conditions (cat. H2SO4, 85 °C, 3 h; Scheme 1) to
Scheme 1. Synthesis of Diacetate 7
provide the corresponding allyl glycoside, which then underwent
regioselective acetonide protection of the syn C.2′ and C.3′
hydroxyl groups (DMP, cat. pTsOH, DMF) to furnish 5 in 90%
overall yield. Benzylation of the C.4′ hydroxyl group and
acetonide hydrolysis according to the protocol of Venkateswar-
lu16 then gave rise to diol 6 in 80% yield, which was
regioselectively benzylated utilizing Hanessian’s dibutyltin
oxide method.17 Acylation of the C.2′ hydroxyl and acetolysis
of the allyl glycoside under acidic conditions then provided 7 in
70% overall yield from 6.
Benzylation of commercially available 1,5-dihydroxynaphtha-
lene under basic conditions provided aromatic coupling partner 8
(Scheme 2). The union of compounds 7 and 8 was accomplished
by treatment of the mixture in CH2Cl2 (0.5 M) with 1.5 equiv of
TMSOTf at room temperature for 30 min.13 C-Glycoside 9 was
it was found that stirring 9 with excess POCl3 and DMF in
toluene at reflux for 6 h gave aldehyde 10 in 70% yield. Baeyer−
Villiger oxidation of 10 under acidic conditions (H2O2, cat.
H2SO4, THF, MeOH)20 then furnishes phenol 11 cleanly in 92%
yield. Sequential exposure of 11 to ceric ammonium nitrate and
sodium dithionite in an oxidation/reduction sequence then
provides diol 12 in 80% yield. At this point, attempts to
selectively methylate the less hindered C.2 hydroxyl group gave
rise to inseparable mixtures of mono- and dimethylated products.
Due to the instability of 12 under basic conditions,
deprotonation of the phenolic hydroxyl groups had to be
performed under low-temperature conditions with NaHMDS in
the presence of the electrophilic methylating agent (NaHMDS,
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MeOTf, THF, −78 to 0 °C). Intriguingly, careful analysis of 1H
NMR data revealed that the major monomethylated product
possessed the methyl group on the C.5 hydroxyl. This result,
together with model studies on naphthyl systems lacking the
carbohydrate moiety demonstrated that the more sterically
hindered hydroxyl at C.5 is in fact more reactive toward
methylation under basic conditions than the less hindered C.2
hydroxyl, likely due to resonance contributions from the C.1
benzyloxy ether.21 It was ultimately found that temporary
protection of the C.5 hydroxyl with chloromethyl ethyl ether
(NaHMDS, THF, −78 °C) provided the corresponding C.5
acetal 13 in 65% yield. Subsequent methylation (Me2SO4, THF,
NaHMDS, −78 °C) of the C.2 hydroxyl and hydrolysis of the
C.5 acetal (catalytic HCl in methanol) gives rise to the desired
naphthol coupling partner 14b in 86% yield from 13.
Scheme 5. Completion of the Synthesis of 1
Next, we envisioned that the iodoarene carboxylic acid
coupling partner could be fashioned from 3,5-dihydroxybenzoic
acid 15 (Scheme 4). Careful methylation of 15 with dimethyl
Scheme 4. Preparation of 22
(PPh3)2PdCl2 and KOAc in DMA gave rise to lactone 24a in 64%
overall ysield. Cleavage of the benzyl ether protecting groups was
next accomplished by exposure of 24a to catalytic quantities of
Pearlman’s catalyst in THF/EtOH under an atmosphere of
hydrogen gas; acylation (Ac2O, pyr) then provided the
tetraacetate 24b in 64% yield. Acetal cleavage under Lewis acidic
conditions (TMSBr, CH2Cl2, −78 °C → −10 °C)14 proceeded
uneventfully to furnish the corresponding primary alcohol 25.
Dehydration under Grieco’s conditions (2-NO2C6H4SeCN,
PBu3, THF; H2O2)24 afforded alkene 26 in 57% yield, which
was identical in its NMR spectroscopic properties to an authentic
sample of polycarcin V tetraacetate prepared by acylation (Ac2O,
Pyr) of the natural product (see the Supporting Information for
comparison of spectra). Deacylation of 26 (NaCN, MeOH) gave
1, which proved to be sensitive to light and concentration. The
1H NMR, 13C NMR, optical rotation, and HRMS of synthetic 1
were in full accord with the data reported for natural polycarcin
V.11
The binding of 1 to duplex DNA was explored by fluorescence
and UV spectroscopies. Excitation of 1 (0.5 μM in 10 mM Tris−
EDTA buffer) at 380 nm in the presence of increasing
concentrations of calf thymus (CT) DNA in the dark resulted
in an enhancement of the fluorescence emission intensity at 470
nm; the addition of CT DNA to 1 also produced a blue shift in
the fluorescence spectrum of 1, an observation consistent with
what has been previously reported for both gilvocarcin V and
M.5,25 Analysis of the fluorescence data by nonlinear regression
based on the orthodox treatment of McGhee and von Hippel26
(see the Supporting Information) gave the association constant
Ka = 1.7 ( 0.1) × 106 M−1, a value which agrees with those
reported for gilvocarcin V by Arce et al. (1.1 × 106 M−1)5 and
Gasparro et al. (6.6 × 105 M−1).25 A similar analysis of 1 in the
presence of increasing concentrations of either poly(dAdT)·
poly(dAdT) or poly(dGdC)·poly(dGdC) revealed that poly-
carcin V binds AT-rich DNA with approximately 1 order of
magnitude greater binding affinity than GC-rich DNA, a finding
also consistent with the known covalent association of
gilvocarcin V with thymine residues.3 Furthermore, thermal
denaturation studies showed a significant (+3 °C) shift in the TM
sulfate (2.2 equiv) and excess K2CO3 in DMF gave rise to a 75%
yield of methyl ester 16, bearing a single phenolic hydroxyl
group.22 Triflation (Tf2O, Pyr), followed by palladium-catalyzed
cross coupling with allyltributyltin, furnished alkene 17 in 80%
overall yield. Next, oxidative cleavage of the olefin was
accomplished in two steps (OsO4, acetone, t-BuOH, NMO;
KIO4, acetone, pH 6.5 buffer) to provide an intermediate
aldehyde, which was immediately reduced with NaBH4 in
methanol to provide primary alcohol 18 in 67% yield. Protection
of the alcohol with chloromethyl ethyl ether then afforded acetal
19 in 77% yield.
Introduction of the iodine atom by directed ortho metalation23
required reduction of the methyl ester to the primary alcohol,
which was accomplished by exposing 19 to LiAlH4 in ether at
room temperature, giving rise to 20 in 98% yield. Treatment of
20 with excess n-BuLi in ether for 3 h at room temperature,
followed by quenching with I2 in THF, furnished a 65% yield of
iodide 21. Finally, sequential oxidation of 21 with PCC (PCC,
DCM, KOAc) and NaClO2 (NaClO2, NaH2PO4, H2O, t-BuOH)
afforded the target carboxylic acid 22 in 80% yield.
Carbodiimide-mediated coupling of naphthol 14b with
carboxylic acid 22 smoothly afforded ester 23 in 98% yield
(Scheme 5). Subsequent intramolecular Heck arylation with
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Expr. 1995, 1261, 195. (d) Prakash, A. S.; Moore, A. G.; Murray, V.;
Matias, C.; McFadyen, W. D.; Wickham, G. Chem.−Biol. Interact. 1995,
95, 17. (e) Kamei, N.; Sekiguchi, T.; Tsuboi, M.; Ishii, S.; Ohtsubo, E.;
Maeda, Y. Nucleic Acids Res. 1994, 31, 87. (f) Sun, D.; Hansen, M.;
Hurley, L. J. Am. Chem. Soc. 1995, 117, 2430.
(10) (a) Greenstein, M.; Monji, T.; Yeung, R.; Maiese, W. M.; White,
R. J. Antimicrob. Agents Chemother. 1986, 29, 861. (b) Singh, K. J.
Antibiot. 1984, 37, 71. (c) Narita, T.; Matsumoto, M.; Mogi, K.; Kukita,
K.; Kawahara, R.; Nakashima, T. J. Antibiot. 1989, 42, 347. (d) Misra, R.;
Tritch, H. R., III; Pandey, R. C. J. Antibiot. 1985, 38, 1280.
(11) Li, Y. Q.; Huang, X. S.; Ishida, K.; Maier, A.; Kelter, G.; Jiang, Y.;
Peschel, G.; Menzel, K. D.; Li, M. G.; Wen, M. L.; Xu, L. H.; Grabley, S.;
Fiebig, H. H.; Jiang, C. L.; Hertweck, C.; Sattler, I. Org. Biomol. Chem.
2008, 6, 3601.
(12) (a) Matsumoto, T.; Katsuki, M.; Suzuki, K. Tetrahedron Lett.
1988, 29, 6935. (b) Matsumoto, T.; Katsuki, M.; Jona, H.; Suzuki, K.
Tetrahedron Lett. 1989, 30, 6185. (c) Matsumoto, T.; Hosoya, T.;
Suzuki, K. Tetrahedron Lett. 1990, 31, 4629. (d) Matsumoto, T.; Katsuki,
M.; Jona, H.; Suzuki, K. J. Am. Chem. Soc. 1991, 113, 6982. (e) Yamauchi,
T.; Watanabe, Y.; Suzuki, K.; Matsumoto, T. Synthesis 2006, 2818.
(13) For previous examples of Lewis acid promoted Friedel−Crafts-
type glycosylations using anomeric acetates, see: (a) Yepremyan, A.;
Salehani, B.; Minehan, T. G. Org. Lett. 2010, 12, 1580. (b) Kalvoda, L.
Collect. Czech. Chem. Commun. 1973, 38, 1679. (c) Vorbruggen, H.;
Kroliliewicz, K.; Bennua, B. Chem. Ber. 1981, 114, 1234.
(14) Hosoya, T.; Takashiro, E.; Matsumoto, T.; Suzuki, K. J. Am. Chem.
Soc. 1994, 116, 1004.
(64 °C) of salmon testes DNA in the presence of 1, even at low
1:DNA ratios (0.05).27 These data suggest that polycarcin V has
a strong noncovalent association with duplex DNA in the
absence of light and that the mode of association of 1 with DNA
is likely similar to that of gilvocarcin V.
In conclusion, we have achieved the total synthesis of the
antitumor α-C-aryl glycoside natural product polycarcin V in
3.2% overall yield from protected carbohydrate 7, naphthol 8,
and arene carboxylic acid 22. This route is easily amenable to the
synthesis of derivatives that incorporate alternate carbohydrate
moieties and/or C.8 aryl substituents. The association constant
for the binding of polycarcin V to CT DNA has been determined
by fluorescence spectroscopy and was found to coincide with Ka
values determined previously for gilvocarcin V. Due to the
photosensitivity of 1, current efforts are directed toward the
synthesis of the C.8 phenyl derivative of polycarcin, which may
retain its strong DNA binding ability without the possibility of
forming covalent adducts with DNA.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed experimental procedures including spectroscopic and
analytical data. This material is available free of charge via the
Internet at http://pubs.acs.org.
(15) For an alternate preparation of 7, see: (a) Pozsgay, V.; Brison, J.-
R.; Jennings, H. J. Can. J. Chem. 1987, 65, 2764. See also: (b) Liptak, A.;
Nanasi, P. Carbohydr. Res. 1981, 93, 43. (c) Chen, Q.; Kong, F.; Cao, L.
Carbohydr. Res. 1993, 240, 107.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: thomas.minehan@csun.edu.
Author Contributions
(16) Swamy, N. R.; Venkateswarlu, Y. Tetrahedron Lett. 2002, 43, 7549.
(17) David, S.; Hanessian, S. Tetrahedron 1985, 41, 643.
(18) The C.2 ester moiety not only directs the anomeric stereo-
chemistry during the carbon−carbon bond-forming step but also
preserves that stereochemistry through other synthetic steps where
anomerization is possible. See ref 13a and also: Schmidt, R. R.; Castro-
Palomino, J. C.; Retz, O. Pure Appl. Chem. 1999, 71, 729.
(19) Meth-Cohn, O.; Stanforth, S. P. Comp. Org. Synth. 1991, 2, 777.
(20) Roy, A.; Reddy, K. B.; Mohanta, P. K.; Ila, J.; Junjappa, H. Synth.
Commun. 1999, 29, 3781.
(21) A similar observation was made by Suzuki: see ref 14.
(22) Nawrat, C. C.; Palmer, L. I.; Blake, A. J.; Moody, C. J. J. Org. Chem.
2013, 78, 5587.
(23) Anctil, E. J.-G.; Snieckus, V. The Directed Ortho-Metallation
Cross-Coupling Nexus. Synthetic Methodology for the Formation of
Aryl−Aryl and Aryl−Heteroatom−Aryl Bonds. In Metal-Catalyzed
Cross Coupling Reactions; De Meijere, A., Diederich, F., Eds.; Wiley-
VCH: Weinheim, 2004; Vol. 2, pp 761−813.
The manuscript was written through contributions of all authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the National Institutes of Health (SC3 GM 096899-
01) for their generous support of our research program.
■
REFERENCES
■
(1) (a) Hua, D. H.; Saha, S. Recl. Trav. Chim. Pay-B 1995, 114, 341.
(b) Nakano, H.; Matsuda, Y.; Ito, K.; Ohkubo, S.; Morimoto, M.;
Tomita, F. J. Antibiot. 1981, 34, 701. (c) Nakano, H.; Matsuda, Y.; Ito,
K.; Ohkubo, S.; Morimoto, M.; Tomita, F. J. Antibiot. 1981, 34, 266.
(d) Hansen, M. R.; Hurley, L. H. Acc. Chem. Res. 1996, 29, 249.
(e) Hacksell, U.; Daves, G. D. Prog. Med. Chem. 1985, 22, 1.
(2) Wei, T. T.; Bryne, K. M.; Warnick-Pickle, D.; Greenstein, M. J.
Antibiot. 1982, 35, 545.
(3) McGee, L. R.; Misra, R. J. Am. Chem. Soc. 1990, 112, 2386.
(4) Matsumoto, A.; Hanawalt, P. C. Cancer Res. 2000, 60, 3921.
(5) Oyola, R.; Arce, R.; Alegria, A. E.; Garcia, C. Photochem. Photobiol.
1997, 65, 802.
(24) Grieco, P. A.; Gilman, S.; Nishizawa, M. J. Org. Chem. 1976, 41,
1485.
(25) Gasparro, F.; Knobler, R.; Edelson, R. Chem.−Biol. Interact. 1988,
67, 255.
(26) McGhee, J.; von Hippel, P. J. Mol. Biol. 1974, 86, 469. Tse, W. C.;
Boger, D. L. Acc. Chem. Res. 2004, 37, 61.
(27) Shi, X.; Chaires, J. B. Nucleic Acids Res. 2006, 34, e14.
(6) Bailly, C.; Colson, P.; Houssier, C.; Rodrigues-Periera, E.;
Prudhomme, M.; Waring, M. J. Mol. Pharmacol. 1998, 53, 77.
(7) Tse-Dinh, Y. C.; McGee, L. R. Biochem. Biophys. Res. Commun.
1987, 143, 808.
(8) (a) Owen, E. A.; Burley, G. A.; Carver, J. A.; Wickham, G.; Keniry,
M. A. Biochem. Bioph. Res. Commun. 2002, 290, 1602. (b) Pavlopoulos,
S.; Bicknell, W.; Wickham, G.; Craik, D. J. J. Mol. Recognit. 1999, 12, 346.
(c) Pavlopoulos, S.; Bicknell, W.; Craik, D. J.; Wickham, G. Biochemistry
1996, 35, 9314. (d) Hansen, M.; Yun, S.; Hurley, L. Chem. Biol. 1995, 2,
229.
(9) (a) Cairns, M. J.; Murray, V. Biochem. Mol. Biol. Int. 1998, 46, 267.
(b) Hardman, L. C.; Murray, V. Biochem. Mol. Biol. Int. 1997, 42, 349.
(c) Murray, V.; Moore, A. G.; Matias, C.; Wickham, G. BBA-Gene Struct.
2965
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