Total synthesis of indole-3-acetonitrile-4-methoxy-2-C-β-d- glucopyranoside. Proposal for structural revision of the natural product

Article abstract of DOI:10.1039/c2ob25821h

Indole-3-acetonitrile-4-methoxy-2-C-β-d-glucopyranoside (1), a novel C-glycoside from Isatis indigotica with important cytotoxic activity, has been prepared in ten steps from ethynyl-β-C-glycoside 3 and 2-iodo-3-nitrophenyl acetate 6. Key steps in the synthesis include a Sonogashira coupling and a CuI-mediated indole formation. NMR spectroscopic data for synthetic 1 differs from that reported for the natural product. A revised structure for the natural product, containing an alternate carbohydrate substituent, is proposed. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25821h

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Total synthesis of indole-3-acetonitrile-4-methoxy-2-C-β-D-glucopyranoside.
Proposal for structural revision of the natural product†
Akop Yepremyan and Thomas G. Minehan*
Received 30th April 2012, Accepted 30th May 2012
DOI: 10.1039/c2ob25821h
Indole-3-acetonitrile-4-methoxy-2-C-β-D-glucopyranoside (1),
a novel C-glycoside from Isatis indigotica with important
cytotoxic activity, has been prepared in ten steps from
ethynyl-β-C-glycoside 3 and 2-iodo-3-nitrophenyl acetate 6.
Key steps in the synthesis include a Sonogashira coupling
and a CuI-mediated indole formation. NMR spectroscopic
Fig. 1 Indole C-glycosides from Isatis indigotica.
data for synthetic 1 differs from that reported for the natural
product. A revised structure for the natural product, contain-
ing an alternate carbohydrate substituent, is proposed.
C-aryl glycosides are a class of natural products that exhibit a
range of important biological properties.1 Numerous members of
this family display potent antitumor, antiviral, and antibiotic
activities,2 and there is ample experimental evidence that C-aryl
glycosides bind duplex DNA.3
Two novel alkaloids recently isolated from the roots of
the plant Isatis indigotica possess an indole-C-glycoside
core.4 Indole-3-acetonitrile-4-methoxy-2-C-β-D-glucopyranoside
(1, Fig. 1) displays cytotoxic activity against human myeloid
leukemia HL60 cells (IC50 = 1.3 mM) and human liver cancer
HepG2 cells (IC50 = 2.1 mM). The structural isomer of 1,
N-methoxy-indole-3-acetonitrile-2-C-β-D-glucopyranoside (2), shows
cytotoxic activity against both HL60 cells (IC50 = 5.1 mM) and
human myeloid leukemia Mata cells (IC50 = 12.1 mM). In view
of its promising biological profile, and with the ultimate aim of
exploring the DNA-binding properties of indole-C-glycosides,
we decided to undertake a total synthesis of 1.
We envisioned that the crucial linkage between the indole and
glycoside moieties could be fashioned from protected alkynyl-C-
glycoside 3 and 2-iodo-3-nitrophenol 6 through a Sonogashira
coupling, nitro group reduction, and intramolecular amine–
alkyne cyclization sequence (Fig. 2). Subsequent installation of
the acetonitrile moiety under standard conditions and deprotec-
tion was envisaged to provide the natural product.
Fig. 2 Retrosynthetic analysis of 1.
The assembly of the alkynyl-C-glycoside coupling partner 3,
possessing the requisite β-stereochemistry at the anomeric
carbon, required efficient access to 2,3,4,6-tetra-O-benzyl glucono-
lactone 5 (Scheme 1).
Allylation of dextrose under acidic conditions,5 followed by
exhaustive benzylation provided allyl glycoside 4 as a mixture of
C-1 anomers in 75% yield. Removal of the allyl ether by stan-
dard base-mediated olefin isomerization and enol ether hydro-
lysis6 furnished 2,3,4,6-tetra-O-benzyl glucose, which upon
Swern oxidation7 provided lactone 5. Following literature pre-
cendent,8,9 reaction of 5 with lithium (trimethylsilyl)acetylide in
the presence of CeCl3 led to an intermediate lactol which was
immediately reduced with BF3·OEt2–Et3SiH to provide the silyl-
protected alkynyl glycoside. Subsequent treatment with aqueous
NaOH gave rise to alkyne 3.9
Department of Chemistry and Biochemistry, California State University,
Northridge, 18111 Nordhoff Street, Northridge, CA 91330, USA.
E-mail: thomas.minehan@csun.edu; Fax: +1 818 677 4062;
Tel: +1 818 677 3315
†Electronic supplementary information (ESI) available: Full experimen-
tal details and characterization data for all compounds reported. See
DOI: 10.1039/c2ob25821h
The synthesis of the aryl iodide coupling partner commenced
from commercially available 2-amino-3-nitrophenol; diazotiza-
tion10 in the presence of NaI furnished iodide 6a, which could
be methylated (K2CO3, CH3I, DMF) or acylated (AcCl, Et3N,
DCM, 0 °C) to give 6b or 6c, respectively.
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Scheme 2 Sonogashira fragment coupling.
Scheme 1 Preparation of coupling fragments 3 and 6.
Initial attempts at coupling methyl ether 6b with alkyne 3
under standard Sonogashira conditions11 met with limited
success (Scheme 2). The major product isolated in these reac-
tions was invariably symmetrical diyne 3d. We hypothesized
that, due to the electron-donating character of the methoxy sub-
stituent, oxidative addition of the palladium catalyst to 6b was
slow relative to alkyne dimerization. Gratifyingly, employing
acetoxyaryl iodide 6c in the cross-coupling reaction cleanly gave
rise to alkyne 7 in 66% yield with minimal formation of dimer
3d. Aminolysis of the acetate ester, followed by hydroxyl
methylation and reduction of the nitro group provided aniline 8b
in 61% yield over three steps.
Next, a base-mediated indolization was attempted using
Knochel’s t-BuOK–NMP system (Scheme 3).12 Treatment of 8b
with potassium tert-butoxide in NMP for 15 minutes furnished
variable yields of indole C-glycoside 9 in the range of 33–55%.
Prolonged exposure to the reaction conditions resulted in signifi-
cant substrate decomposition. A superior protocol for producing
9 reproducibly involved exposure of 8b to excess CuI (2–5
equivalents) at 145 °C in DMF for 2 hours.13 These conditions
allowed indole 9 to be secured in 65% yield.
With 9 in hand, we next tested installation of the acetonitrile
moiety via a three-step protocol.14 Treatment of 9 with formalin,
diethylamine, and acetic acid overnight, isolation and subjection
of the crude amine to methyl iodide in CH2Cl2, followed by
refluxing the resulting quaternary ammonium salt with sodium
cyanide in ethanol (80 °C), gave rise to nitrile 10 in 85% overall
yield. However, attempted deprotection of the benzyl ether pro-
tecting groups by hydrogenolysis over Pearlman’s catalyst15 led
to the formation of significant quantities of amine 11 in addition
to desired nitrile 1. Hydrogenolysis of 9 instead, followed by
alcohol acylation provided peracetate 12, the three-dimensional
structure of which was confirmed by NOESY spectroscopy.
An alternative, higher-yielding route to 1 was secured via
persilyl derivative 13 (Scheme 4), prepared in 67% yield from
9 (H2, Pd(OH)2; TBS-Cl, imid, DMF, 60 °C). Acetonitrile
Scheme 3 C-glycosyl indole synthesis and attempted deprotection;
NOESY correlations for 12.
installation under the aforementioned conditions gave rise to
nitrile 14 in 60% overall yield. Removal of the silyl ether pro-
tecting groups was accomplished in 72% yield by exposure of
14 to TBAF (5 equiv) in THF for 9 hours, providing synthetic 1
as an amorphous white solid. The 1H and 13C NMR data for syn-
thetic 1 (recorded in acetone-d6) differed from those reported by
Hu et al.4 for natural 1 (Fig. 3). Proton and carbon assignments
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1 occur for protons on the carbohydrate substituent and for
protons and carbon atoms near the site of attachment of the
sugar moiety to the indole ring. The 9.1 Hz coupling constant16a
reported for H1′ of the natural product suggests that the carbo-
hydrate moiety is indeed a hexopyranose, and likely a diastereo-
mer of synthetic 1 such as allose (the C-3 epimer) or galactose
(the C-4 epimer).16b
In summary, we have developed a concise route to indole-3-
acetonitrile-4-methoxy-2-C-β-D-glucopyranoside, the proposed
structure of a natural indole C-glycoside from Isatis indigotica.
Comparison of spectroscopic data for synthetic and natural 1
indicate that the natural product likely contains a diastereomeric
hexopyranose moiety. Preparation of the galactopyranose- and
allopyranose-containing indole C-glycosides is underway and
comparisons of their spectroscopic data with that of the natural
material will be reported in due course.
Acknowledgements
Scheme 4 An alternative route to 1 and structure assignment of 15.
We thank the National Institutes of Health (SC3 GM 096899-01)
and the CSUN Office of Research and Sponsored Projects for
their generous support of our research program.
Notes and references
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Fig. 3 Comparison of NMR chemical shift data for natural (ref. 4) and
synthetic 1 recorded in acetone-d6.
9 T. Lowary, M. Meldal, A. Helmboldt, A. Vasella and K. Block, J. Org.
Chem., 1998, 63, 9657.
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for synthetic 1 were obtained from COSY, HSQC, and HMBC
experiments, which also provided support for the hydrogen–
carbon and carbon–carbon connectivity of the molecule. To
further confirm the structure of our synthetic material, compound
1 was converted to peracetate 15 (Ac2O, Pyr, 95%) and COSY
and NOESY experiments were performed. Analysis of the cross-
peaks in the NOESY spectrum of 15 revealed a connectivity
pattern consistent with the findings for compound 12: a gluco-
pyranosyl carbohydrate moiety is attached at the C.2 carbon
atom of the indole ring.
15 W. M. Pearlman, Tetrahedron Lett., 1967, 1663.
16 (a) Hu et al. report two separate coupling constants for H1′ in ref. 2: 9.1
Hz and 9.5 Hz (b) The >9 Hz coupling constant for H1′ in the natural
product is indicative of an equatorial hydroxyl group at C.2 of the carbo-
hydrate; thus a mannopyranose unit is not a likely candidate for the
carbohydrate substituent of the natural product.
Careful study of the spectroscopic data in Fig. 3 indicates that
major differences in chemical shift between synthetic and natural
5196 | Org. Biomol. Chem., 2012, 10, 5194–5196
This journal is © The Royal Society of Chemistry 2012