Synthesis of 3,3′-Di- O -methyl ardimerin and exploration of its DNA binding properties

Article abstract of DOI:10.1021/ol500725e

The 3,3′-di-O-methyl derivative (15) of the bis-C-aryl glycoside natural product ardimerin (1) has been synthesized in 11 steps from 2,3,4,6-tetrabenzylglucose (2) and 1,2,3-trimethoxybenzene (3). Key steps in the synthesis involve a Lewis acid mediated Friedel-Crafts type glycosylation and a Yamaguchi lactonization under Yonemitsu conditions. 3,3′-Di-O-methyl ardimerin aggregates in aqueous solutions at concentrations greater than 1 M, and both UV and fluorescence binding studies indicate that 15 has a low affinity for duplex DNA.

Full text of DOI:10.1021/ol500725e

Letter
pubs.acs.org/OrgLett
Synthesis of 3,3′-Di‑O‑methyl Ardimerin and Exploration of Its DNA
Binding Properties
Miran Mavlan, Kevin Ng, Harmanpreet Panesar, Akop Yepremyan, and Thomas G. Minehan*
Department of Chemistry and Biochemistry, California State University, Northridge 18111 Nordhoff Street, Northridge, California
91330, United States
S
* Supporting Information
ABSTRACT: The 3,3′-di-O-methyl derivative (15) of the bis-C-
aryl glycoside natural product ardimerin (1) has been
synthesized in 11 steps from 2,3,4,6-tetrabenzylglucose (2) and
1,2,3-trimethoxybenzene (3). Key steps in the synthesis involve a
Lewis acid mediated Friedel−Crafts type glycosylation and a
Yamaguchi lactonization under Yonemitsu conditions. 3,3′-Di-O-
methyl ardimerin aggregates in aqueous solutions at concen-
trations greater than 1 μM, and both UV and fluorescence binding studies indicate that 15 has a low affinity for duplex DNA.
lants used in traditional Chinese medicine have yielded a
wealth of chemical constituents with important biological
possibility, we decided to undertake its synthesis and investigate
its DNA binding properties.
We envisioned (Figure 2) that the C−C linkage between
carbohydrate and aromatic moieties could be fashioned by a
P
activities.1 Ardimerin (1a, Figure 1), a dimeric lactone with
Figure 1. Structures of ardimerin and ardimerin digallate.
radical scavenging activity, was isolated from Ardisia japonica by
Ryu et al. in 2002.2 Subsequently, ardimerin digallate (1b) was
isolated from the same species, along with the flavonoid
quercitrin and the terpenoids friedelin, epifriedelinol, baurenol,
and baurenyl acetate.3 The digallate derivative of ardimerin was
shown to inhibit HIV-1 and HIV-2 RNase H in vitro with IC50
values of 1.5 and 1.1 μM, respectively.
C-Aryl glycosides are an important class of naturally
occurring compounds endowed with remarkable stability
toward acid and enzymatic hydrolysis;4 this affords them a
sufficient intracellular lifetime to allow trafficking to the
nucleus, where they bind DNA to form stable complexes.5
The bis-C-aryl glycoside altromycin B has been shown by NMR
studies to associate with DNA via a helix-threading mode of
binding, with carbohydrate moieties positioned in opposite
grooves of the duplex.5e Given that ardimerin is a symmetrical
bis-C-aryl glycoside, we envisioned that, despite the non-
planarity of its aglycone,6 it might also be capable of the
recognition of nucleic acids by a threading mode of
intercalation, with the glucosyl substituents positioned in
both the major and minor grooves of DNA. To assess this
Figure 2. Retrosynthetic analysis of ardimerin.
Lewis acid mediated Friedel−Crafts type C-glycosylation
reaction between protected glucose 2 and 1,2,3-trimethox-
ybenzene (3).9 Aromatic ring carbonylation and selective ortho
methoxy group deprotection would then provide 7, a crucial
substrate for esterification with the derived carboxylic acid
monomer 8. Oxidation, macrocyclization, and protecting group
removal would then provide the natural product.
Received: March 9, 2014
Published: April 9, 2014
© 2014 American Chemical Society
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The carbohydrate coupling partner (2) required for the C-
glycosylation reaction may be prepared in 72% overall yield
from dextrose as previously described.7 Treatment of 2a with a
1:1 solution of trifluoroacetic anhydride and CH2Cl2 for 30
min, followed by evaporation and combination with commer-
cially available 1,2,3-trimethoxybenzene (1.5 equiv) and BF3·
OEt2 (1.1 equiv) in CH2Cl2 at room temperature for 30 min,
afforded coupled product 4 (>20:1 β:α at C.1) in 62% yield
(Scheme 1).8 Interestingly, 2-O-benzyl-1,3-dimethoxybenzene
treatment of 6 with 1.1 equiv of AlCl3 and 1.5 equiv of NaI in
CH3CN (0.25 M) at 80 °C for 1 h gave hydroxy aldehyde 7 in
90% yield. Subsequent benzyl ether formation and oxidation
with NaClO2 gave a 50% overall yield of carboxylic acid 8.
Interestingly, all attempts to cleave the methoxy group meta to
the aldehyde of 7 (corresponding to the C.3/C.3′ position of
the natural product) by extended exposure to AlCl3/NaI (80
°C) resulted in substrate decomposition.
With both 7 and 8 in hand, we set out to identify conditions
for the construction of the eight-membered diolide (Scheme 2).
Scheme 1. Synthesis of C-Glycoside Monomers 7 and 8
Scheme 2. Model Study: Synthesis of Diolide 12
Attempts to directly dimerize the model compound 2,3-
dimethoxysalicylic acid (SOCl2, dilute toluene, reflux; DCC or
EDC, DCM, rt; TFAA, DCM, 0 °C) failed, producing only
uncharacterized oligomers in low yields. In line with literature
precendent,13 coupling of the known acid 9a14 and aldehyde
10a15 was accomplished via direct addition of sodium alkoxide
10b to acid chloride 9b in THF at room temperature;
subsequent oxidation (NaClO2, NH2SO3H, acetone/water)
afforded carboxylic acid 11. Harris has demonstrated13 that
subjection of ortho-benzyl protected carboxylic acid substrates
similar to 11 to refluxing thionyl chloride leads directly to eight-
membered diolides of the type 12, arising from acid chloride
formation, in situ benzyl ether cleavage, and macrolactonization
of the hydroxy acid chloride; however, we observed that
refluxing 11 in SOCl2 for 3 h led only to the intermediate
hydroxy acid chloride, which was sufficiently stable to survive
aqueous reaction workup. Instead, the acid chloride inter-
mediate was diluted in benzene or toluene (0.01 M) and
treated with 3 equiv of DMAP and stirred at room temperature
overnight. In this way, diolide 12 could be secured in 50−60%
yield.
With a method to prepare the diolide core of ardimerin in
hand, we proceeded to explore the similar union of aldehyde 7
and carboxylic acid 8 (Scheme 3). Treatment of compound 8
with oxalyl chloride in the presence of catalytic quantities of
DMF gave rise to the corresponding acid chloride, which was
added to the potassium salt of 7 in THF at 0 °C; the resultant
crude aldehyde 13a was immediately oxidized under Pinnick−
Lindgren−Kraus conditions16 to afford the stable carboxylic
acid 13b. Refluxing 13b in SOCl2 for 3 h gave rise to the
corresponding benzyloxy acid chloride and not the desired
hydroxyl acid chloride; further heating in SOCl2 overnight led
only to extensive substrate decomposition. To effect removal of
the benzyl ether before conversion to the acid chloride,
compound 13b was treated with a 1:1 mixture of TFA and
toluene at room temperature for 5 min.17 The intermediate
(3b) was not a suitable partner for the C-glycosylation reaction,
undergoing rapid decomposition in the presence of either BF3·
OEt2 or TMSOTf. However, the 2-O-tert-butyldiphenylsilyl
derivative 3c coupled efficiently with 2b in the presence of
TMSOTf as a Lewis acid promoter to provide the C-aryl
glycoside product in 72% yield.
Several methods were explored to introduce the aldehyde
moiety on the aromatic ring. Attempted Vilsmeier formylation
(DMF, POCl3, toluene, 100 °C) of 4 resulted in minimal
conversion, even after an extended reaction time (24 h).9
Directed ortho-lithiation10 with n-BuLi/TMEDA and trapping
with DMF gave only low (<10%) yields of carbonyl-containing
products, likely due to intramolecular protonation of the
aryllithium species by the benzyl ether protecting groups on the
carbohydrate.11 Bromination (NBS, CHCl3, reflux) of 4
resulted in the formation of aryl bromide 5 in 90% yield.
Lithium−halogen exchange with n-BuLi, followed by rapid
quenching with DMF, again led to a hydrodebrominated
product arising from the aforementioned intramolecular proton
transfer process. However, magnesiate formation according to
the protocol of Oshima (i-PrMgCl, 2 equiv of n-BuLi, THF, 0
°C to −78 °C, then 5) followed by quenching with DMF led to
a 71% yield of the desired aldehyde 6.12
Selective deprotection of the methoxy group ortho to the
aldehyde initially proved to be problematic. Treatment of 6
with 1−3 equiv of BCl3 in CH2Cl2 at −60 °C (1 h) or room
temperature (overnight) led to significant substrate decom-
position. The combination of 6 with AlCl3 in benzene at 80 °C
or in CH2Cl2 at room temperature gave only poor yields of the
desired hydroxyl aldehyde. Finally it was discovered that
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Scheme 3. Seco-acid Macrolactonization
Figure 3. UV absorption spectra of 15 (10 mM Tris-EDTA) at varying
concentrations; [15] = (0.79, 1.18, 1.95, 2.70, 3.80, 5.56, 8.81, and
11.76) × 10−6 mol L−1 for curves 1−8, respectively.
Furthermore, compound 15 displayed relatively limited ability
to displace bound ethidium bromide from calf thymus DNA as
compared to control compound daunorubicin over the same
concentration range (1 × 10−9 M to 4 × 10−7 M; see
Supporting Information).26 These data suggest that 15 has a
low affinity for duplex DNA, perhaps indicative of the difficulty
in accommodating the bulky chromophore-linked C-glycosyl
moieties in the narrow minor groove, the initial site of small-
molecule binding to DNA.27
In summary, we have developed an 11 step synthesis of the
3,3′-di-O-methyl derivative of the natural product ardimerin
and have shown that this substance readily aggregates in
aqueous solution and has a low apparent affinity for duplex
DNA. Current efforts toward the completion of the synthesis of
ardimerin are centered around deprotection of the C.3 methyl
ether of aldehyde 7.
hydroxy acid was then treated with oxalyl chloride (cat. DMF,
CH2Cl2), and a dilute solution (0.1 M) of the resulting acid
chloride was then added dropwise to a refluxing solution of
DMAP (3 equiv) in benzene. However, this condition gave rise
to the hydroxyacid monomer resulting from DMAP-induced
cleavage of the ester linkage. Standard Yamaguchi lactonization
conditions,18 involving slow addition of the mixed anhydride
(seco acid, 2,4,6-trichlorobenzoyl chloride, Et3N, THF, rt) to a
refluxing solution of DMAP in toluene, gave the same result.
Gratifyingly, attempted macrolactonization under Yonemitsu
conditions (2,4,6-trichlorobenzoyl chloride, Et3N, DMAP, and
the seco acid in benzene, 1 × 10−3 M)19 at room temperature
gave rise to the desired diolide 14 in 50% yield. Hydrogenation
of 14 over Pearlman’s catalyst gave 3,3′-di-O-methyl ardimerin
15 in 90% yield. Interestingly, attempted acylation of 15 (Ac2O,
Pyr, rt, 16 h or Ac2O, i-Pr2NEt, DMAP)20 led to none of the
desired peracetate and the production of numereous side
products. Ultimately, it was found that stirring 15 in neat acetyl
chloride21 overnight led to formation of peracetate 16 in 85%
yield. The β-stereochemistry of the glucosyl moieties was
indicated by the 8.5 Hz coupling constant of the C.1 proton,
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.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: thomas.minehan@csun.edu.
Notes
1
and the connectivity of the molecule was verified by H−1H
COSY and NOESY experiments (see Supporting Information).
In our attempts to access ardimerin by selective cleavage of
the C.3 and C.3′ methyl ethers, treatment of 14 with BCl3/
CH2Cl2 (rt, overnight), AlCl3/NaI (80 °C, CH3CN, 3 h), or
MgI2 (50−80 °C, toluene)22 initially led to no starting material
conversion, but after a prolonged reaction and an increase in
the number of equivalents of Lewis acid, extensive decom-
position products, arising from diolide cleavage, were formed.
Similarly, use of sodium ethanethiolate in DMF (100 °C, 2 h)23
also led to dissolution of the bislactone moeity. These data
indicate that removal of the requisite methyl ethers is likely to
be successful only on substrates prior to formation of the
diolide core of the natural product.
The binding of 15 to duplex DNA was explored by UV and
fluorescence spectroscopies. A concentration-dependent red
shift in the absorption at λmax = 214 nm in the ultraviolet
spectrum of 15 suggested that self-association/aggregation was
occurring in an aqueous buffer solution (10 mM Tris-EDTA) at
concentrations >1 μM (Figure 3).24 Thermal denaturation
studies showed no significant shift in the TM (68 °C) of salmon
testes DNA in the presence of 15 at low ligand/DNA ratios.25
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.
■
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