Doxorubicin and Aclarubicin: Shuffling Anthracycline Glycans for Improved Anticancer Agents

Article abstract of DOI:10.1021/acs.jmedchem.0c01191

Anthracycline anticancer drugs doxorubicin and aclarubicin have been used in the clinic for several decades to treat various cancers. Although closely related structures, their molecular mode of action diverges, which is reflected in their biological activity profile. For a better understanding of the structure-function relationship of these drugs, we synthesized ten doxorubicin/aclarubicin hybrids varying in three distinct features: Aglycon, glycan, and amine substitution pattern. We continued to evaluate their capacity to induce DNA breaks, histone eviction, and relocated topoisomerase IIα in living cells. Furthermore, we assessed their cytotoxicity in various human tumor cell lines. Our findings underscore that histone eviction alone, rather than DNA breaks, contributes strongly to the overall cytotoxicity of anthracyclines, and structures containing N,N-dimethylamine at the reducing sugar prove that are more cytotoxic than their nonmethylated counterparts. This structural information will support further development of novel anthracycline variants with improved anticancer activity.

Full text of DOI:10.1021/acs.jmedchem.0c01191

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Article
Doxorubicin and Aclarubicin: Shuffling Anthracycline Glycans for
Improved Anticancer Agents
Dennis P. A. Wander, Sabina Y. van der Zanden, Gijsbert A. van der Marel, Herman S. Overkleeft,
́
Jacques Neefjes,* and Jeroen D. C. Codee*
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ABSTRACT: Anthracycline anticancer drugs doxorubicin and aclarubicin have been used in the clinic for several decades to treat
various cancers. Although closely related structures, their molecular mode of action diverges, which is reflected in their biological
activity profile. For a better understanding of the structure−function relationship of these drugs, we synthesized ten doxorubicin/
aclarubicin hybrids varying in three distinct features: aglycon, glycan, and amine substitution pattern. We continued to evaluate their
capacity to induce DNA breaks, histone eviction, and relocated topoisomerase IIα in living cells. Furthermore, we assessed their
cytotoxicity in various human tumor cell lines. Our findings underscore that histone eviction alone, rather than DNA breaks,
contributes strongly to the overall cytotoxicity of anthracyclines, and structures containing N,N-dimethylamine at the reducing sugar
prove that are more cytotoxic than their nonmethylated counterparts. This structural information will support further development
of novel anthracycline variants with improved anticancer activity.
by poisoning topoisomerase IIα (TopoIIα).16,17 Aclarubicin is
INTRODUCTION
■
capable of evicting histones as well, but targets TopoIIα without
Anthracyclines comprise one of the most successful classes of
inducing DNA double-strand breaks.17−19 In addition, it has
natural product chemotherapeutic agents. Two archetypal
been shown that aclarubicin affects cell viability by reducing the
anthracyclines are doxorubicin (1) and aclarubicin (12, Figure
mitochondrial respiratory activity.20 Histone eviction induced
1), both effective anticancer agents isolated from nature.1,2
by anthracycline drugs results in epigenetic and transcriptional
Doxorubicin has been in use in the clinic for more than five
changes, which are thought to then induce apoptosis.17 We
decades and is prescribed worldwide to about a million patients
recently showed that anthracyclines that induce both DNA
annually for the treatment of a variety of cancers.3−5 Aclarubicin
double-strand break formation and histone eviction are
in contrast is prescribed exclusively in Japan and China, mainly
cardiotoxic. Aclarubicin and N,N-dimethyldoxorubicin (3)
for the treatment of acute myeloid leukemia (AML). Although
both lack DNA damage activity but are able to induce histone
doxorubicin is very effective, its use coincides with cardiotox-
eviction, and can thus be used as effective anticancer drugs
icity, formation of secondary tumors, and infertility.6−9
without cardiotoxicity.15 The structural basis causing this
Therefore, clinical use with doxorubicin is generally limited to
difference in biological activities, however, is still lacking.
a cumulative dose of 450−550 mg/m2.7,10,11 The formation of
Therefore, better insight into the structure−function relation-
reactive oxygen species (ROS) by these drugs has been
ship of these molecules is needed.
considered as a major mechanism mediating anthracycline-
induced cardiotoxicity.12,13 However, aclarubicin, which has a
higher redox potential than doxorubicin,14 displays fewer
Received: July 9, 2020
cardiotoxic side effects, and recent findings in our labs suggested
that this difference in cardiotoxicity relates to significant
differences in the mode of action of these two compounds.15
Doxorubicin causes chromatin damage by inducing histone
eviction, as well as the formation of DNA double-strand breaks
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Common mechanisms of resistance toward anthracycline
drugs are reduced expression or activity of TopoIIα and
overexpression of membrane transporters such as P-glycopro-
tein (P-gp) and multidrug resistance-associated protein (MRP),
both of which decrease the cellular accumulation of the drugs via
increased drug export.23−25
Although the structures of doxorubicin (1) and aclarubicin
(12) are quite similar (they both contain an anthraquinone and a
sugar containing a basic amine), three differences can be
identified: (i) variation in the substitution and oxidation pattern
of the anthraquinone aglycon, (ii) variation in the size of the
carbohydrate part, and (iii) the methylation pattern of the amine
of the first sugar attached to the anthraquinone. Doxorubicin
features an α-L-daunosamine as the single monosaccharidic
carbohydrate appendage, while aclarubicin features an α-L-
rhodosamine (N,N-dimethyldaunosamine) that is further
glycosylated at the 4-hydroxyl with a disaccharide composed
of α-L-oliose and α-L-cinerulose A. Thousands of analogues of
doxorubicin and aclarubicin have been isolated from bacterial
sources or prepared through organic synthesis.26 In spite of this,
the chemical space between doxorubicin and aclarubicin has not
been fully explored. Although some doxorubicin/aclarubicin
hybrids have been prepared (including compounds 2,27 3,15,28
4,29 8,30 10,31 and 1132), the reported methods of synthesis are
fragmented and the complete set, as shown in Figure 1, has not
been evaluated in the context of the different modes of action
described above. We therefore set out to generate a
comprehensive set of doxorubicin/aclarubicin hybrid structures,
systematically varying the structural elements in which the two
anthracyclines differ. Based on these structural differences
between doxorubicin and aclarubicin, we envisaged the set of
doxorubicin/aclarubicin hybrids 2−11 (Figure 1) that com-
prises anthracyclines composed of either of the two aglycons,
additionally featuring either a monosaccharide, a disaccharide,
or a trisaccharide glycan composed of the sugar configurations
also found in the parent structures, and bearing either no or two
N-methyl substituents. Altogether, they fill the chemical space
between doxorubicin (1) and aclarubicin (12). Furthermore, we
probed this coherent set of anthracycline hybrid structures for
their DNA damaging, TopoIIα relocalization, histone evicting,
and cytotoxic activities to get a better understanding of the
structural basis underlying the observed difference for the
anticancer activity of these compounds. These new insights
could ultimately lead to the development of new anthracycline
variants with improved anticancer activity.
RESULTS
■
Synthesis of Doxorubicin/Aclarubicin Hybrid Mono-
saccharides 2 and 4. For the assembly of the set of
anthracyclines, we used Biao Yu’s gold(I)-mediated condensa-
tion33 of the glycans and aglycons, as these mild glycosylation
conditions are compatible with the lability and reactivity of the
deoxy sugars that are to be appended to the anthraquinones. The
anthraquinone aglycons were readily obtained by acidic
hydrolysis of the drugs doxorubicin (1) and aclarubicin (12).
This yielded aklavinone (14)34 and, following protection of the
primary alcohol in doxorubicinone as the tert-butyldimethylsilyl
(TBS) ether, 14-O-TBS-doxorubicinone 1635 (Scheme 1).
Condensation of daunosaminyl cyclopropylethynylbenzoate
(ABz) 13 (see Schemes S1 and S2 (Supporting Information)
for a complete description of the syntheses of the building
blocks) and aklavinone (14) under Yu’s conditions provided
anthracycline 15 in a stereoselective manner (Scheme 1). The
Figure 1. Chemical structures of doxorubicin (1), aclarubicin (12), and
hybrid structures (2−11), subject of the here-presented studies.
In addition to the treatment-limiting side effects, development
of resistance constitutes to be a frequent clinical limitation for
the treatment of patients with anthracycline drugs.21,22
B
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a
Scheme 1. Synthesis of Hybrid Monosaccharide Anthracyclines 2−4
a
Reagents and conditions: (a) 0.2 M aqueous (aq) HCl, 90 °C, quant.; (b) PPh3AuNTf2 (10 mol %), dichloromethane (DCM), −20 °C, 73%
(>20:1 α/β); (c) (i) Pd(PPh3)4, NDMBA, DCM, (ii) HF·pyridine, pyr., 40% over two steps; (d) (i) Pd(PPh3)4, NDMBA, DCM, (ii) aq CH2O,
NaBH(OAc)3, EtOH, (iii) HF·pyridine, pyr., 43% over three steps; (e) (i) aq HCl, 90 °C, (ii) TBS-Cl, imidazole, dimethylformamide (DMF),
97% over two steps.
stereoselectivity of this glycosylation can be accounted for by
long-range participation36,37 of the allyl carbamate, as well as the
conformation of the intermediate oxocarbenium ion that can be
substituted in a stereoselective manner on the α-face.38 The
yield of this glycosylation reaction (73%) compares favorably to
the yields (50−60%) reported by Pearlman et al., who used
glycal donors in combination with Brønsted acid catalysis.39 The
N-Alloc group in 15 was then removed using a catalytic amount
of Pd(PPh3)4 and N,N-dimethylbarbituric acid (NDMBA) as
the allyl scavenger.40 This was followed by desilylation using an
HF·pyr complex to give the first hybrid structure 2.41 The
corresponding dimethylamine 4 could be prepared by perform-
ing reductive alkylation with formaldehyde and NaBH(OAc)3
after the removal of the Alloc functionality, and finally a
desilylation. The third monosaccharide anthracycline 3 was
obtained as we previously described.15
Synthesis of Hybrid Disaccharides 5−8. We then turned
our attention to the four disaccharidic antracyclines 5−8. This
required the synthesis of disaccharide donor 21, which is
depicted in Scheme 2A. Compound 21 was constructed through
an iodonium di-collidinium perchlorate (IDCP)42-mediated
glycosylation of L-olioside thioglycoside donor 18, protected as
the tetraisopropyldisiloxane ether, which effectively shields the
β-face to facilitate the stereoselective introduction of the desired
α-linkage. The reaction between donor 18 and acceptor 17
delivered the desired disaccharide 19 in excellent yield and
stereoselectivity. Triphenylphosphine was added to the reaction
mixture to reduce the in situ formed sulfenamide that was
formed from the Alloc carbamate and the generated phenyl-
sulfenyl iodide.43,44 The chemoselective removal of the
anomeric p-methoxyphenolate (PMP) protective group in 19
was achieved using silver(II) hydrogen dipicolinate (Ag-
(DPAH)2),45,46 and the anomeric alcohol thus liberated was
then condensed with carboxylic acid 20 under Steglich
conditions,47 to deliver the disaccharide alkynylbenzoate
donor 21. The coupling to the two aglycone acceptors 14 and
16 is outlined in Scheme 2B. Treatment of a mixture of donor 21
and doxorubicinone acceptor 16 with PPh3AuNTf2 proceeded
stereoselectively to give 22 in 64% yield. Ensuing Alloc removal
proceeded quantitatively to give 23, after which HF·pyridine-
mediated desilylation yielded the first disaccharide anthracycline
5. To introduce the dimethylamino functionality, amine 23 was
treated with formaldehyde and a substoichiometric amount of
NaBH(OAc)3 to prevent reduction of the hydroxyketone
function on the aglycone.28 A final desilylation resulted in
dimethylated 7. Subjecting donor 21 and aklavinone 14 to
gold(I)-mediated glycosylation also proceeded stereoselectively
to give the protected disaccharide anthracycline, of which the
Alloc group was removed to give 24 in 87% yield over the two
steps. Removal of the disiloxane moiety with HF·pyridine then
gave disaccharide anthracycline 6. A double-reductive N-
methylation was performed on fully deprotected 6 to give 8.
Synthesis of Hybrid Trisaccharides 9−11. To complete
the set of target compounds, trisaccharide anthracyclines 9−11
were prepared. These required trisaccharide alkynylbenzoate
donor 30, the synthesis of which is shown in Scheme 3A. First,
protected daunosaminyl acceptor 17 and oliosyl donor 25 were
condensed using the conditions described for the synthesis of
disaccharide 18 to provide disaccharide 26. This glycosylation
proceeded with excellent stereoselectivity, which can be
attributed to the structure of the intermediate oxocarbenium
ion.38 Removal of the benzoyl protective group in 26 gave
acceptor 27.
Elongation of this disaccharide was achieved using an IDCP-
mediated glycosylation using L-rhodinoside donor 28 to
stereoselectively provide the protected trisaccharide. Removal
of the benzoyl ester gave the alcohol, which was oxidized using a
Dess−Martin oxidation to install the required ketone function-
ality in 29. The trisaccharide was converted to the correspond-
ing Yu donor with the oxidation−Steglich esterification
sequence, as described earlier, to give 30. Of note, the silver(II)
reagent used to remove the anomeric para-methoxyphenol
moiety left the para-methoxybenzyl-protecting group un-
scathed. Treatment of aglycon 16 and donor 30 with
PPh3AuNTf2 led to the stereoselective formation of the first
protected trisaccharide anthracycline, of which the para-
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a
Scheme 2. (A) Synthesis of Disaccharide Alkynylbenzoate Donor 21; (B) Synthesis of Hybrid Disaccharide Anthracyclines 5−
b
8
a
Reagents and conditions: (a) IDCP, Et2O, 1,2-dichloroethane (DCE) (4:1 v/v), then PPh3, 89%; (b) (i) Ag(II)(hydrogen dipicolinate)2, NaOAc,
MeCN, H2O, 0 °C, (ii) 20, EDCI·HCl, N,N-diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP), DCM, 84% over two steps (1:8
b
α/β). Reagents and conditions: (c) 16, PPh3AuNTf2 (10 mol %), DCM, 64% (>20:1 α/β); (d) Pd(PPh3)4, NDMBA, DCM, quant.; (e) HF·
pyridine, pyr., 76% for 5, 81% for 7; (f) aq CH2O, NaBH(OAc)3, EtOH, 71%; (g) (i) 14, PPh3AuNTf2 (10 mol %), −20 °C, DCM, (ii) Pd(PPh3)4,
NDMBA, DCM, 87% over two steps (>20:1 α/β); (h) HF·pyridine, pyr., 41%; (i) aq CH2O, NaBH(OAc)3, EtOH, 34%.
methylbenzyl (PMB) group was removed to give partially
protected anthracycline 31 in 57% yield, over two steps (Scheme
3B). This represents a significant improvement over a previous
synthesis, reported by Tanaka et al.,32 who combined a
trisaccharide bromide and the aglycone acceptor in a TBABr/
collidine-mediated glycosylation to give the trisaccharide
anthracycline in 22% yield. Removal of the Alloc group and
desilylation of 31 then afforded 9. A double-reductive amination
on 31 followed by desilylation provided hybrid anthracycline 11.
For the synthesis of 10, a mixture of 30 and 14 was treated with
PPh3AuNTf2 at −20 °C to afford 32 as a single diastereoisomer
in 71% yield. Removal of the Alloc and PMB groups finally gave
10. The analytical data for the compounds described previously
in the literature (2,27 3,28 4,29 8,30 10,31 1132) were in good
agreement with the reported data.
DNA Double-Strand Breakage and Histone Eviction.
Since the main difference in biological activity between
doxorubicin and aclarubicin is their capacity to induce DNA
double-strand breaks, we tested the ability of hybrid structures
2−11 in comparison to their parental drugs 1 and 12 to induce
DNA damage. Anthracyclines are often used in the treatment of
acute myeloid leukemia; therefore, human chronic myelogenous
leukemia cells (K562 cells) were incubated for 2 h with 10 μM
1−12, and etoposide as a positive control for DNA double-
strand break formation.48,49 These concentrations are corre-
sponding to physiological serum peak levels of cancer patients at
standard treatment.17,50 DNA break formation was analyzed by
measuring phosphorylation of H2AX (γH2AX), a well-known
marker for DNA double-strand breaks, by Western blot (Figure
2A,B) as well as by constant-field gel electroporation (Figure
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a
Scheme 3. (A) Synthesis of Trisaccharide Alkynylbenzoate Donor 30; (B) Synthesis of Hybrid Trisaccharide Anthracyclines 9−
b
11
a
Reagents and conditions: (a) IDCP, Et2O/DCE (4:1 v/v), then PPh3; (b) NaOMe, MeOH, 78% over two steps (>20:1 α/β); (c) IDCP, Et2O/
DCE (4:1 v/v), then PPh3, 100% (>20:1 α/β); (d) (i) NaOMe, MeOH, 85%, (ii) Dess−Martin periodinane, NaHCO3, CH2Cl2, 97%; (e) (i)
Ag(II)(hydrogen dipicolinate)2, NaOAc, MeCN/H2O (1:1, v/v), 0 °C, (ii) 20, EDCI·HCl, DIPEA, DMAP, CH2Cl2, 75% over the two steps (1:7
b
α/β). Reagents and conditions: (f) (i) 16, PPh3AuNTf2 (10 mol %), DCM, (ii) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), DCM, pH 7
phosphate buffer (18:1, v/v), 57% over two steps (>20:1 α/β); (g) Pd(PPh3)4, NDMBA, DCM, 81% from 31, 61% for 10; (h) HF·pyridine, pyr.,
73% for 9, 73% for 11; (i) aq CH2O, NaBH(OAc)3, EtOH, 52%; (j) 14, PPh3AuNTf2 (10 mol %), DCM, −20 °C, 71% (>20:1 α/β); (k) DDQ,
DCM/pH 7 phosphate buffer (18:1, v/v), 90%.
2C).51 Only doxorubicin (1) and hybrid structure 9 induced
DNA double-strand breaks, as is evident from both assays
(Figure S1A−C, Supporting Information). None of the other
compounds induced phosphorylated H2AX and thus resemble
the activity of aclarubicin (12). Subsequently, compounds 1−12
were tested for their ability to induce histone eviction. To
visualize histone eviction, the release of photoactivated green
fluorescent protein-labeled histone H2A (PAGFP-H2A) was
followed in the adherent human melanoma MelJuSo cell line
using time-lapse confocal microscopy, as previously de-
scribed.15,17 Compounds 3, 8, and 11 are equally potent at
evicting histones to their parent structures doxorubicin (1) and
aclarubicin (12). Compounds 4, 6, and 7 are able to evict
histones, but do so less efficiently than 1 and 12, while
E
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Figure 2. Evaluation of DNA break capacity and histone evicting activity of hybrid structures 2−11 and parent compounds doxorubicin (1) and
aclarubicin (12). (A) K562 cells were treated for 2 h with 10 μM of the indicated drugs, etoposide was used as a positive control for DNA double-strand
breaks. γH2AX levels were examined by Western blot. Actin was used as a loading control, and molecular weight markers are as indicated. (B)
Quantification of the γH2AX signal normalized to actin. Results are presented as mean standard deviation (SD) of three independent experiments.
Ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test; ns, not significant; ****P < 0.0001. (C) Quantification of
broken DNA relative to intact DNA as analyzed by constant-field gel electrophoresis (CFGE). Etoposide was used as a positive control for DNA
double-strand breaks. Results are presented as mean SD of three independent experiments. Ordinary one-way ANOVA with Dunnett’s multiple
comparison test; *P < 0.05, ****P < 0.0001 is indicated, all others are not significant. (D) Quantification of the release of fluorescent PAGFP-H2A
from the photoactivated nuclear regions after administration of 10 μM of the indicated drugs. Results are shown as mean SD of 10−20 cells from at
least three independent experiments. Ordinary two-way ANOVA with Dunnett’s multiple comparison test; ns, not significant; ****P < 0.0001. See
also Figures S1 and S2.
compounds 2, 5, 9, and 10 fail to evict histones (Figures 2D and
S2).
compounds 5, 7, and 9 were more poorly taken up by K562
cells compared to doxorubicin (1). A similar observation is made
for compounds 4, 6, 8, and 10, which were taken up more
efficiently than aclarubicin (12), whereas uptake of compound 2
is significantly less compared to aclarubicin (12). Nevertheless,
when drug uptake is plotted against the IC50 in K562 cells or
drug uptake in MelJuSo cells against histone eviction speed, no
correlation between uptake of the hybrid structures with
cytotoxicity or histone eviction was observed (Figure S3F,G,
Supporting Information). Of note here is that, while the uptake
of compound 5 is similar to that of doxorubicin (1), this
compound is not able to induce DNA double-strand breaks or
evict histones. Consequently, this compound is one of the least
cytotoxic hybrids from this set of compounds (Figure 3H). As
anthracycline drugs target TopoII, we decided to validate if the
lack of cytotoxicity of compound 5 can be caused by the loss of
ability to interfere with the catalytic cycle of TopoII. Therefore,
we transiently overexpressed GFP-tagged TopoIIα in MelJuSo
cells and followed the protein localization over time upon
treatment with 10 μM of the different doxorubicin/aclarubicin
hybrid compounds. At steady state, TopoIIα is localized in the
nucleus where it accumulates in nucleoli, but upon treatment
with the hybrid anthracyclines, the protein rapidly relocalizes
(Figure S4A,B). While most of the hybrid compounds are able to
relocate TopoIIα, compound 5 does not. Furthermore,
relocalization of TopoIIα by compounds 2, 6, and 10 was less
efficient than by the other compounds, which might explain why
these four are the least cytotoxic hybrid variants from this set of
compounds.
Cytotoxicity and Cellular Uptake. To test the cell
cytotoxicity of the panel of hybrid anthracyclines, K562 cells
were treated for 2 h with compounds 1−12 at physiological
relevant concentrations, and cell survival was measured 72 h
post-treatment using a CellTiter-Blue assay (Figure 3A,B).17,50
Compounds 3, 8, and 11 were effectively killing K562 cells.
While compounds 3 and 8 showed cytotoxicity in the same
range as their parental drugs doxorubicin (1) and aclarubicin
(12), respectively, compound 11 was ∼13 times more cytotoxic
than doxorubicin and 2.5 times more than aclarubicin.
Compounds 4, 7, 9, and 10 were only effective at higher
concentrations, while compounds 2, 5, and 6 did not show any
cytotoxicity (Figures 3A,B and S3A). The observed cytotoxicity
is not specific for this acute myeloid leukemia cell line (K562)
because similar toxicity profiles were observed for these
compounds when tested in the melanoma cell line MelJuSo,
the colorectal carcinoma cell line HCT116, the two prostate
cancer cell lines PC3 and DU145, and the glioblastoma cell line
U87 (Figure 3C−G). To validate that the differences in DNA
damage, chromatin damage induction, and effective cytotoxicity
are not caused by differences in cellular uptake of the different
hybrid structures, we performed drug uptake experiments for
compounds 1−12 utilizing the inherent fluorescent property of
the anthraquinone moieties found in the anthracycline drugs.52
K562 and MelJuSo cells were treated with 1 μM of the indicated
compounds for 2 h, and fluorescence was then measured by flow
cytometry (Figure S3B−E, Supporting Information). The
fractional increase/decrease in fluorescence was compared to
the parental drugs with that of the corresponding anthraquinone
aglyconthe fluorophore within the anthracyclines. Significant
differences in uptake of the different hybrid structures were
observed. Compounds 3 and 11 are taken up ∼6 and 4 times
more efficiently than doxorubicin (1), respectively, while
Correlation between N,N-Dimethylation and Cytotox-
icity. Although no clear correlation is observed between the
structural features of the compounds and their IC50 values
(Figure S5A−C, Supporting Information), there is a strong
relationship between the rate of histone eviction and cell toxicity
(Figure 4A,B). In general, N,N-dimethylation of the sugar
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Figure 3. Cytotoxicity of compounds 1−12. (A, B) K562 cells were treated for 2 h at the indicated doses (higher doses in (A), lower doses in (B)) of
the various hybrid compounds followed by drug removal. Cell survival in MelJuSo (C), human colorectal carcinoma cell line HCT116 (D), human
prostate tumor cell line PC3 (E) and DU145 (F), and human glioblastoma cell line U87 (G). Cells were treated for 2 h at indicated dose followed by
drug removal. Cell viability was measured by a Cell-Titerblue assay 72 h post-treatment. Data are shown as mean SD from three different
experiments. (H) Table showing the IC50 values for the different doxorubicin/aclarubicin hybrid compounds for the indicated cell lines. See also Figure
S3A, Supporting Information.
Figure 4. Cytotoxicity correlates with N,N-dimethylation and efficiency of histone eviction. (A) Histone eviction speed (time at which 25% of the
initial signal is reduced) versus IC50 of the various hybrid compounds is plotted. Two-tailed Spearman r correlation, *P < 0.05. (B) Zoom-in of data
plotted in (A). (C) N,N-Dimethylation of the first sugar over no methylation gives improved IC50 in K562 cells (1 versus 3/2 versus 4/5 versus 7/6
versus 8/9 versus 11/10 versus 12). IC50 is plotted for the corresponding hybrid structures without (no; N) and with (yes; Y) N,N-dimethylation. The
fold change of IC50 improvement as a result of N,N-dimethylation is indicated above the bars. IC50 could not be determined for compounds 2, 5, and 6
(gray bars) and was therefore depicted as the highest concentration tested (10 μM).
attached to the anthraquinone strongly improves histone
eviction and enhances cytotoxicity of these compounds (Figure
4C). This observation could be very useful in the development
of more effective anthracycline drugs, since (with the exception
of aclarubicin) all anthracycline drugs currently used in the clinic
(doxorubicin, daunorubicin, epirubicin, and idarubicin) contain
a primary amine on their sugar moiety.
DISCUSSION AND CONCLUSIONS
■
Although anthracycline anticancer drugs are known to induce
severe side effects, these effective chemotherapeutic drugs have
been one of the cornerstones in oncology for over five decades.
Following the discovery of doxorubicin, many anthracycline
variants have been isolated, prepared, and evaluated with the aim
of reducing their toxicity, but this has not led to any effective and
less cardiotoxic variants to enter clinical practice other than
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aclarubicin (12). Remarkably, this drug is only used in Japan and
China.3 It has long been thought that the cytotoxic activity of
anthracyclines was due to their DNA double-strand breaking
capacity;53 however, we have previously shown that histone
eviction activity is likely the main mechanism of cytotox-
icity.15,17−19 Here, we have developed synthetic chemistry to
assemble a complete set of doxorubicin/aclarubicin hybrid
structures varying at the anthraquinone aglycon, the nature of
the carbohydrate portion, and the alkylation pattern of the
amine on the first sugar moiety. The set of doxorubicin/
aclarubicin hybrids was assembled using Yu’s gold-catalyzed
glycosylation of the anthracycline aglycons, which in all cases
proceeded with excellent stereoselectivity. The required di- and
trisaccharides were generated using fully stereoselective IDCP-
mediated glycosylations. Overall, the developed synthetic
strategy proved to be broadly applicable and delivered the set
of anthracyclines in a highly efficient manner. Furthermore, we
have subjected these hybrid structures to detailed biological
evaluation, including cellular uptake, TopoIIα relocalization
capacity, DNA damage, and histone eviction assays. Although
no clear correlation was found between the anthraquinone
aglycon moiety and the number of carbohydrate fragments with
the observed cytotoxicity of the compounds, a clear relationship
between histone eviction efficiency and cytotoxicity was
revealed. The coherent set of hybrid structures yielded three
compounds that were more cytotoxic than doxorubicin (3, 8,
and 11). Across the board, N,N-dimethylation of the
carbohydrate appended to the anthraquinone aglycon consid-
erably improved cytotoxicity (3 and 4 outperform 1 and 2; 7 and
8 outperform 5 and 6, and 11 and 12 outperform 9 and 10).
How exactly N,N-dimethylation of the amino sugar improves
cytotoxicity is not yet fully understood, but the addition of the
methyl groups makes those compounds slightly more hydro-
phobic, which might influence their uptake. Furthermore, it has
been shown that N-methylation of anthracyclines modulates
their transport by the membrane transporter P-glycoprotein (P-
gp).54 It has been suggested that the steric hindrance created by
the methyl groups can impair the interaction between the
positively charged amino group with the active site of the P-gp
exporter, which leads to better intracellular drug accumulation.
This would also indicate that the various N,N-dimethylated
hybrid variants might be effective drugs for the treatment of
multidrug-resistant tumors, in which elevated expression of the
P-gp exporter is often observed.23,55 A third option for the
enhanced effectivity of the N,N-dimethylation amino sugar
variants might be a change in the interaction dynamics of the
anthracycline drugs with the DNA. It is known that
doxorubicin−DNA aminal adducts can form between the 3′-
NH2 of the doxorubicin sugar, the N2 of the guanine base, and
formaldehyde.56−59 The addition of two methyl groups to the
critical amino sugar might convert these drugs from a covalent
DNA intercalator into a reversible DNA intercalator, affecting
the dynamics by which these drugs perturb the DNA−histone
organization.
doxorubicin and twice as fast as for aclarubicin. The subsequent
difference in cytotoxicity between compound 11 and doxor-
ubicin or aclarubicin can therefore only partially be explained by
the enhanced histone eviction efficacy. However, besides the
difference in histone eviction efficacy, it has been shown that
various anthracycline drug can have selectivity for distinct (epi-
)genomic regions (and can therefore be considered different
drugs because of different genomic targets).18 The different
targeted (epi-)genomic regions by these drugs can subsequently
have divergent downstream effects, which may explain the
improved cytotoxicity for compound 11 over doxorubicin (1)
and aclarubicin (12).
In summary, in this study, we have developed highly effective
and broadly applicable synthetic chemistry, which was used to
prepare a set of ten doxorubicin/aclarubicin hybrid structures
and studied their specific biological activities in cells. This has
given us better insights into the structure−activity relationship
for this extensively used group of chemotherapeutics, which can
help to direct the development of new effective anticancer drugs.
Interestingly, the most potent compounds identified from the
systematic library of compounds (3, 8, and 11) do not exert their
activity through the induction of DNA double-strand break
formation following inhibition of TopoIIα, but rather through
the induction of histone eviction, indicating that histone eviction
by anthracyclines could be the dominant factor for the
cytotoxicity of this class of anticancer drugs.
EXPERIMENTAL SECTION
■
Chemistry. Doxorubicin was obtained from Accord Healthcare
Limited, U.K., aclarubicin from Santa Cruz Biotech, and etoposide from
Pharmachemie, Haarlem, The Netherlands. For the synthesis of the
doxorubicin/aclarubicin hybrid compounds, all reagents were of
commercial grade and used as received. Traces of water from reagents
were removed by coevaporation with toluene in reactions that required
anhydrous conditions. All moisture/oxygen-sensitive reactions were
performed under an argon atmosphere. DCM used in the glycosylation
reactions was dried with flamed 4 Å molecular sieves before being used.
Reactions were monitored by thin-layer chromatography (TLC)
analysis with detection by UV (254 nm) and, where applicable, by
spraying with 20% sulfuric acid in EtOH or with a solution of
(NH4)6Mo7O24·4H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10 g/L)
in 10% sulfuric acid (aq) followed by charring at ∼150 °C. Flash
column chromatography was performed on silica gel (40−63 μm). 1H
and 13C spectra were recorded on Bruker AV 400 and Bruker AV 500
spectrometers in CDCl3, CD3OD, pyridine-d5, or D2O. Chemical shifts
(δ) are given in parts per million (ppm) relative to tetramethylsilane
(TMS) as internal standard (1H NMR in CDCl3) or the residual signal
of the deuterated solvent. Coupling constants (J) are given in hertz. All
13C spectra are proton-decoupled. Column chromatography was
carried out using silica gel (0.040−0.063 mm). Size-exclusion
chromatography was carried out using a Sephadex LH-20, using
DCM/MeOH (1:1, v/v) as the eluent. Neutral silica was prepared by
stirring regular silica gel in aqueous ammonia, followed by filtration,
washing with water, and heating at 150 °C overnight. High-resolution
mass spectrometry (HRMS) analysis was performed with an LTQ
Orbitrap mass spectrometer (Thermo Finnigan), equipped with an
electrospray ion source in positive mode (source voltage, 3.5 kV; sheath
gas flow, 10 mL/min; capillary temperature, 250 °C) with resolution R
= 60 000 at m/z 400 (mass range m/z = 150−2000) and dioctyl
phthalate (m/z = 391.28428) as a “lock mass”, or with a Synapt G2-Si
(Waters), equipped with an electrospray ion source in positive mode
(electrospray ionization time-of-flight (ESI-TOF)), injection via
NanoEquity system (Waters), with LeuEnk (m/z = 556.2771) as
“lock mass”. Eluents used: MeCN/H2O (1:1 v/v) supplemented with
0.1% formic acid. The high-resolution mass spectrometers were
calibrated prior to measurements with a calibration mixture (Thermo
In addition to N,N-dimethylation of the sugar moiety, the
doxorubicin anthraquinone aglycon appears to be slightly better
than the aclarubicin anthraquinone aglycon and the aclarubicin
trisaccharide improves cytotoxicity over the doxorubicin
monosaccharide. A combination of these structural features is
found in compound 11, the most cytotoxic compound in the
focused library, being 13 times more cytotoxic than doxorubicin
and 2.5 times more than aclarubicin in K562 cells. Histone
eviction by compound 11 is approximately three times as fast as
H
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1
Finnigan). Purity of all compounds is >95%, as determined by H
NMR.
Syntheses of the monosaccharide donors/acceptors are described in
the Supporting Information.
(tetrafluoroethylene) (PTFE) tube, after which HF·pyr complex (70
wt % HF, 125 μL) was added at 0 °C. Over the course of 4 h, additional
HF·pyr complex (70 wt % HF, 125 μL each time) was added five times.
Solid NaHCO3 was added to quench, and the mixture was stirred until
cessation of effervescence. It was then filtered off, and the filtrate was
partitioned between DCM and H2O. The organic layer was dried over
Na2SO4 and concentrated in vacuo. Column chromatography on
neutral silica (DCM; 20:80 MeOH/DCM) gave the title compound as
a yellow solid (7.9 mg, 13.9 μmol, 43% over three steps). 1H NMR (500
MHz, chloroform-d) δ 12.70 (s, 1H), 12.01 (s, 1H), 7.83 (dd, J = 7.5,
1.1 Hz, 1H), 7.77−7.66 (m, 2H), 7.31 (dd, J = 8.4, 1.2 Hz, 1H), 5.55 (d,
J = 3.9 Hz, 1H), 5.29−5.20 (m, 1H), 4.27 (s, 1H), 4.16−4.03 (m, 2H),
3.87 (s, 1H), 3.70 (s, 3H), 2.54 (dd, J = 15.2, 4.5 Hz, 1H), 2.45 (s, 6H),
2.33 (d, J = 15.2 Hz, 1H), 2.05 (td, J = 13.1, 12.6, 4.2 Hz, 1H), 1.89 (dd,
J = 12.9, 4.6 Hz, 1H), 1.76 (dq, J = 14.6, 7.3 Hz, 1H), 1.52 (dq, J = 14.5,
7.3 Hz, 1H), 1.38 (dd, J = 6.5, 2.1 Hz, 3H), 1.09 (t, J = 7.3 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 192.9, 181.4, 171.3, 162.8, 162.3, 142.8,
137.6, 133.6, 133.1, 131.2, 125.0, 121.1, 120.4, 115.9, 114.9, 101.1, 71.9,
71.4, 67.0, 65.8, 61.1, 57.2, 52.7, 42.0, 34.0, 32.2, 27.8, 17.0, 6.8. HRMS:
[M + H]+ calcd for C30H36NO10 570.2339; found 570.2921.
General Procedure A: p-Methoxyphenolate Oxidative Depro-
tection. To a solution of p-methoxyphenyl glycoside in 1:1 MeCN/
H2O (0.02 M, v/v) were added NaOAc (10 equiv) and then
Ag(DPAH)2·H2O60 (2.1 equiv for trisaccharides, 4 equiv for
monosaccharides) portionwise over 30 min at 0 °C. The mixture was
stirred until disappearance of the starting material, after which it was
poured into sat. aq NaHCO3. This was then extracted with DCM thrice,
dried over MgSO4, and concentrated in vacuo to give the crude lactols.
General Procedure B: Alkynylbenzoate Esterification. A solution of
ortho-cyclopropylethynylbenzoic acid methyl ester47 in tetrahydrofuran
(THF) (5 mL/mmol) and 1 M NaOH (5 mL/mmol) was stirred at 50
°C for at least 5 h. It was then poured into 1 M HCl (6 mL/mmol) and
extracted with DCM thrice. The combined organic layers were then
dried over MgSO4 and concentrated in vacuo. The resultant acid was
then used without further purification.
To a solution of the above crude lactol in DCM (0.1 M) were added
DIPEA (9 equiv), DMAP (1 equiv), EDCI·HCl (3.2 equiv), and the
above carboxylic acid (3 equiv). After stirring overnight, the mixture
was diluted with DCM and washed with sat. aq NaHCO3 and brine.
Drying over MgSO4, concentration in vacuo, and column chromatog-
raphy of the residue (EtOAc/pentane) gave the alkynylbenzoates.
General Procedure C: Au(I)-Catalyzed Glycosylation. To a solution
of the glycosyl donor and the required anthracycline acceptor (1−2
equiv) in DCM (0.05 M), activated molecular sieves (4 Å) were added.
The mixture was stirred for 30 min. Subsequently, a freshly prepared 0.1
M DCM solution of PPh3AuNTf2 (prepared by stirring 1:1 PPh3AuCl
and AgNTf2 in DCM for 30 min) (0.1 equiv) in DCM was added
dropwise at the designated temperature. After stirring for 30 min (for
room temperature (RT)) or overnight (−20 °C or lower), the mixture
was filtered and concentrated in vacuo. Column chromatography
(EtOAc/pentane or Et2O/pentane and then acetone/toluene)
followed by (if required) size-exclusion chromatography (Sephadex
LH-20, 1:1 DCM/MeOH v/v) gave the glycosides.
7-[α-L-Daunosamino]-aklavinone (2). To a solution of 15 (60 mg,
0.081 mmol) in DCM (8.1 mL) were added N,N-dimethylbarbituric
acid (38 mg, 0.24 mmol, 3 equiv) and tetrakis(triphenylphosphine)-
palladium(0) (4.6 mg, 4.1 μmol, 0.05 equiv). After stirring for 2.5 h, the
mixture was concentrated in vacuo. Column chromatography (DCM;
2:98 MeOH/DCM) gave the crude amine. This was then redissolved in
pyridine (6 mL) in a PTFE tube, after which HF·pyr complex (70 wt %
HF, 710 μL) was added at 0 °C. After 3.5 and 5.5 h, additional HF·pyr
complex (70 wt % HF, 355 μL each time) was added. After stirring for a
total of 6.5 h, solid NaHCO3 was added to quench, and the mixture was
stirred until cessation of effervescence. It was then filtered off, and the
filter cake was rinsed thoroughly with MeOH/DCM (9:1 v/v). The
combined filtrates were then concentrated in vacuo. Column
chromatography (DCM; 20:80 MeOH/DCM) gave the title
compound as a yellow solid (18 mg, 33 μmol, 41% over two steps).
1H NMR (500 MHz, methanol-d4) δ 7.77−7.61 (m, 2H), 7.53 (s, 1H),
7.31−7.20 (m, 1H), 5.49 (s, 1H), 5.14 (d, J = 4.7 Hz, 1H), 4.27 (q, J =
6.5 Hz, 1H), 4.08 (s, 1H), 3.73 (s, 2H), 3.67 (d, J = 2.8 Hz, 1H), 3.57−
3.47 (m, 1H), 2.52 (dd, J = 15.0, 5.2 Hz, 1H), 2.32 (d, J = 15.0 Hz, 1H),
2.03 (td, J = 12.9, 4.0 Hz, 1H), 1.99−1.90 (m, 1H), 1.76 (dq, J = 14.7,
7.4 Hz, 1H), 1.56 (dq, J = 13.9, 7.1 Hz, 1H), 1.31 (d, J = 6.6 Hz, 3H),
1.11 (t, J = 7.4 Hz, 3H). 13C NMR (126 MHz, MeOD) δ 193.6, 182.3,
172.6, 163.7, 143.8, 138.5, 134.7, 134.0, 125.8, 121.2, 120.8, 117.0,
115.8, 101.7, 72.5, 72.1, 68.4, 68.1, 58.2, 53.0, 49.8, 48.4, 35.8, 33.3,
30.1, 17.0, 7.1. HRMS: [M + H]+ calcd for C28H32NO10 542.2026;
found 542.2031.
Synthesis of Anthracycline Disaccharides 5−8. p-Methoxyphen-
yl-2-deoxy-3,4-tetraisopropyldisiloxyl-α-L-fucopyranosyl-(1 → 4)-3-
N-allyloxycarbonyl-2,3-dideoxy-α-L-fucopyranoside (19). To a sol-
ution of the glycosyl acceptor 17 (901 mg, 2.67 mmol, 1 equiv) and the
glycosyl donor 18 (1.80 g, 3.73 mmol, 1.3 equiv) in Et2O/DCE (70 mL,
4:1 v/v), activated molecular sieves (4 Å) were added. The mixture was
stirred for 30 min, and then, at 10 °C, iodonium dicollidine perchlorate
(5.00 g, 10.7 mmol, 4 equiv) was added. After 30 min,
triphenylphosphine (1.40 g, 5.34 mmol, 2 equiv) was added, and the
mixture was stirred for an additional hour. It was then diluted with
EtOAc and filtered; washed with 10% aq Na2S2O3, 1 M CuSO4 solution
twice, and H2O; and then dried over MgSO4. Concentration in vacuo
and column chromatography (5:95−10:90 EtOAc/pentane) of the
residue gave the title compound as a white foam (1.69 g, 2.38 mmol,
89%). 1H NMR (500 MHz, chloroform-d) δ 7.05−6.93 (m, 2H), 6.93−
6.70 (m, 2H), 6.16 (d, J = 7.9 Hz, 1H), 5.92 (ddt, J = 16.1, 10.9, 5.6 Hz,
1H), 5.52 (d, J = 3.2 Hz, 1H), 5.30 (dq, J = 17.2, 1.6 Hz, 1H), 5.20 (dq, J
= 10.5, 1.4 Hz, 1H), 4.93 (d, J = 3.7 Hz, 1H), 4.58 (qdt, J = 13.3, 5.6, 1.4
Hz, 2H), 4.41 (ddd, J = 12.2, 4.6, 2.5 Hz, 1H), 4.37−4.25 (m, 1H),
4.14−4.04 (m, 2H), 4.01 (s, 1H), 3.77 (s, 3H), 3.54 (s, 1H), 2.19−2.05
(m, 2H), 1.99 (dd, J = 12.6, 4.6 Hz, 1H), 1.89 (td, J = 12.7, 3.5 Hz, 1H),
1.34 (d, J = 6.4 Hz, 3H), 1.18 (d, J = 6.5 Hz, 3H), 1.14−0.83 (m, 28H).
13C NMR (126 MHz, CDCl3) δ 155.6, 154.4, 150.9, 132.8, 117.4,
Synthesis of Anthracycline Monosaccharides 2−4. The synthesis
of 3 is described in ref 15.
7-[3-N-Allyloxycarbonyl-2,3-dideoxy-α-L-fucopyranoside]-aklavi-
none (15). Prepared according to General Procedure C from donor 13
and aklavinone 14 (2 equiv) at RT to give after column
chromatography (4:96 Et2O/pentane and then 1.5:98.5 acetone/
toluene) the title compound as a yellow solid (149 mg, 0.201 mmol,
73%). 1H NMR (400 MHz, chloroform-d) δ 12.66 (s, 1H), 12.04 (s,
1H), 7.83 (dd, J = 7.5, 1.2 Hz, 1H), 7.77−7.64 (m, 2H), 7.31 (dd, J =
8.4, 1.2 Hz, 1H), 5.86 (ddt, J = 16.3, 10.8, 5.6 Hz, 1H), 5.46 (d, J = 3.8
Hz, 1H), 5.28−5.12 (m, 3H), 4.63 (d, J = 8.8 Hz, 1H), 4.58−4.41 (m,
2H), 4.21 (s, 1H), 4.15−4.01 (m, 2H), 3.86 (dq, J = 8.7, 4.1 Hz, 1H),
3.78 (s, 1H), 3.69 (s, 3H), 2.50 (dd, J = 15.0, 4.4 Hz, 1H), 2.34 (d, J =
15.0 Hz, 1H), 1.92 (td, J = 12.8, 4.1 Hz, 1H), 1.81−1.68 (m, 2H), 1.49
(dq, J = 14.3, 7.3 Hz, 1H), 1.36−1.18 (m, 3H), 1.08 (t, J = 7.3 Hz, 3H),
0.99 (t, J = 7.9 Hz, 9H), 0.66 (qd, J = 7.9, 2.1 Hz, 6H). 13C NMR (101
MHz, CDCl3) δ 192.9, 181.5, 171.6, 162.7, 162.3, 155.2, 142.9, 137.5,
133.7, 133.0, 132.9, 131.3, 124.9, 121.1, 120.3, 117.8, 115.9, 114.8,
101.6, 71.5, 71.4, 71.1, 67.6, 65.6, 57.2, 52.6, 47.4, 34.0, 32.2, 30.4, 17.6,
7.2, 6.8, 5.4. HRMS: [M + Na]+ calcd for C38H49NO12SiNa 774.2533;
found 774.2525.
7-[α-L-Rhodosamino]-aklavinone (4). To a solution of 15 (23.7 mg,
0.032 mmol) in DCM (3.2 mL) were added N,N-dimethylbarbituric
acid (15 mg, 0.096 mmol, 3 equiv) and tetrakis(triphenylphosphine)-
palladium(0) (1.8 mg, 1.6 μmol, 0.05 equiv). After stirring for 2.5 h, the
mixture was concentrated in vacuo. Column chromatography (DCM;
2:98 MeOH/DCM) gave the crude amine. This was then redissolved in
EtOH (7.7 mL), and 37% aq CH2O (79 μL, 30 equiv) was added
NaBH(OAc)3 (67 mg, 0.32 mmol, 10 equiv). The mixture was stirred
for 2.5 h before being quenched by addition of sat. aq NaHCO3. It was
then poured into H2O and extracted with DCM, dried over Na2SO4,
and concentrated in vacuo to give the crude dimethylated amine. This
was then redissolved in pyridine (3.2 mL) in a poly-
117.2, 114.4, 101.8, 96.2, 81.2, 73.0, 69.8, 68.0, 67.4, 65.4, 55.5, 46.4,
I
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33.1, 31.5, 17.6, 17.5, 17.4, 17.3, 17.3, 17.2, 17.2, 17.2, 17.1, 17.1, 14.1,
13.9, 13.0, 12.4. HRMS: [M + Na]+ calcd for C35H59NO10Si2Na
732.35752; found 732.3587.
7-[2-Deoxy-α-L-fucopyranosyl-(1 → 4)-3-amino-2,3-dideoxy-α-L-
fucopyranoside]-doxorubicinone (5). To a solution of 23 (30.5 mg,
29.6 μmol) in pyridine (3.0 mL) in a PTFE tube was added HF·pyr
complex (70 wt % HF, 232 μL) at 0 °C. Over the course of 4 h, two
additional such portions of HF·pyr complex were added. Then, solid
NaHCO3 was added to quench, and the mixture was stirred until
cessation of effervescence. It was then filtered off and concentrated in
vacuo. Column chromatography on neutral silica (0:100−20:80
MeOH/DCM) gave the title compound as a red solid (15.1 mg, 22.4
μmol, 76%). 1H NMR (500 MHz, pyridine-d5) δ 7.78 (d, J = 7.7 Hz,
1H), 7.46 (t, J = 8.1 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 5.52 (d, J = 3.0
Hz, 1H), 5.17 (d, J = 3.9 Hz, 1H), 5.12 (d, J = 2.3 Hz, 2H), 5.06 (d, J =
3.8 Hz, 1H), 4.36 (dt, J = 12.1, 3.9 Hz, 1H), 4.33−4.19 (m, 2H), 3.80
(d, J = 2.9 Hz, 1H), 3.68 (s, 3H), 3.54 (s, 1H), 3.41 (t, J = 8.7 Hz, 1H),
3.34−3.12 (m, 2H), 2.51 (d, J = 14.4 Hz, 1H), 2.30 (td, J = 12.2, 3.9 Hz,
1H), 2.22 (dd, J = 14.3, 5.1 Hz, 1H), 2.08 (dd, J = 12.3, 4.9 Hz, 1H),
1.97 (dd, J = 9.2, 2.8 Hz, 2H), 1.27 (d, J = 6.4 Hz, 3H), 1.06 (d, J = 6.4
Hz, 3H). 13C NMR (126 MHz, Pyr) δ 215.4, 187.5, 161.9, 157.5, 156.2,
135.2, 121.6, 120.1, 119.9, 112.3, 112.0, 101.9, 101.9, 81.6, 77.1, 72.4,
70.9, 69.0, 68.8, 66.7, 66.2, 57.1, 48.0, 37.9, 34.6, 34.4, 34.2, 18.1.
HRMS: [M + H]+ calcd for C33H40NO14 674.24488; found 674.2456.
7-[2-Deoxy-α-L-fucopyranosyl-(1 → 4)-3-dimethylamino-2,3-di-
deoxy-α-L-fucopyranoside]-doxorubicinone (7). To a solution of 23
(102 mg, 99 μmol) in EtOH (20 mL) and 37% aq CH2O (245 μL, 30
equiv) was added NaBH(OAc)3 (40 mg, 0.193 mmol, 1.95 equiv). The
mixture was stirred for 1.5 h before being poured into sat. aq NaHCO3.
This was extracted with DCM, dried over Na2SO4, and concentrated in
vacuo. Column chromatography chromatography (3:97 acetone/
toluene) gave the dimethylated amine as a red solid (75 mg, 70.9
μmol, 71%). 1H NMR (500 MHz, chloroform-d) δ 13.92 (s, 1H), 13.24
(s, 1H), 8.01 (dd, J = 7.7, 1.0 Hz, 1H), 7.77 (t, J = 8.1 Hz, 1H), 7.43−
7.37 (m, 1H), 5.54 (d, J = 3.8 Hz, 1H), 5.25 (dd, J = 4.1, 2.1 Hz, 1H),
5.01 (d, J = 3.4 Hz, 1H), 4.98−4.84 (m, 2H), 4.79 (s, 1H), 4.49−4.34
(m, 2H), 4.09 (s, 3H), 3.95 (t, J = 1.8 Hz, 1H), 3.91 (q, J = 6.5 Hz, 1H),
3.75 (s, 1H), 3.38−3.35 (m, 1H), 3.18 (dd, J = 18.9, 1.9 Hz, 1H), 2.98
(d, J = 18.8 Hz, 1H), 2.32 (dt, J = 14.7, 2.3 Hz, 1H), 2.19 (s, 6H), 2.17−
2.06 (m, 3H), 2.06−1.96 (m, 2H), 1.89 (td, J = 12.8, 4.0 Hz, 1H), 1.80
(dd, J = 13.0, 4.1 Hz, 1H), 1.26 (d, J = 6.6 Hz, 3H), 1.19 (d, J = 6.4 Hz,
3H), 1.07 (ddt, J = 9.4, 7.4, 4.6 Hz, 24H), 0.96 (s, 9H), 0.14 (d, J = 2.9
Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 211.4, 187.2, 186.8, 161.1,
156.6, 156.0, 135.8, 135.6, 134.3, 134.2, 121.0, 119.9, 118.5, 111.5,
111.4, 101.5, 99.9, 74.1, 73.8, 70.6, 69.6, 68.8, 67.3, 66.7, 61.8, 56.8,
43.5, 35.7, 34.1, 33.4, 26.0, 18.1, 17.8, 17.8, 17.7, 17.6, 17.6, 17.5, 17.5,
17.4, 17.4, 14.4, 14.3, 13.2, 12.7. HRMS: [M + H]+ calcd for
C53H84NO15Si3 1058.51488; found 1058.51488. To a solution of the
above compound (38 mg, 35.9 μmol) in pyridine (3.6 mL) in a PTFE
tube was added HF·pyr complex (70 wt % HF, 282 μL) at 0 °C. Over
the course of 4.5 h, three additional such portions of HF·pyr complex
were added. Then, solid NaHCO3 was added to quench, and the
mixture was stirred until cessation of effervescence. It was then filtered
off and concentrated in vacuo. Column chromatography on neutral
silica (DCM; 10:90 MeOH/DCM) gave the title compound as a red
solid (20.3 mg, 28.9 μmol, 81%). 1H NMR (500 MHz, chloroform-d +
MeOD) δ 8.02 (d, J = 7.6 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.42 (t, J =
7.3 Hz, 1H), 5.55 (d, J = 4.0 Hz, 1H), 5.28 (s, 1H), 5.05 (d, J = 3.9 Hz,
1H), 4.76 (d, J = 5.6 Hz, 2H), 4.41 (q, J = 6.6 Hz, 1H), 4.14−4.03 (m,
4H), 3.97 (q, J = 6.6 Hz, 1H), 3.83 (d, J = 6.5 Hz, 1H), 3.24 (dd, J =
18.9, 5.9 Hz, 1H), 3.02 (dd, J = 19.2, 6.3 Hz, 1H), 2.39−2.08 (m, 8H),
2.07−1.80 (m, 4H), 1.29 (d, J = 6.7 Hz, 3H), 1.21 (d, J = 6.6 Hz, 3H).
13C NMR (126 MHz, CDCl3 + MeOD) δ 213.6, 187.2, 186.8, 161.1,
o-Cyclopropylethynylbenzoyl-2-deoxy-3,4-tetraisopropyldisilox-
ane-α-L-fucopyranosyl-(1 → 4)-3-N-allyloxycarbonyl-2,3-dideoxy-L-
fucopyranoside (21). Prepared according to General Procedure A and
B from 19 (1.69 g, 2.38 mmol) to give after column chromatography
(10:90−20:80 EtOAc/pentane) the title compound as a white foam
(1.54 g, 1.99 mmol, 84% over two steps, α/β 1:8). 1H NMR (500 MHz,
chloroform-d) δ 8.00−7.85 (m, 1H), 7.47 (dd, J = 7.8, 1.4 Hz, 1H), 7.41
(ddd, J = 9.1, 6.0, 1.4 Hz, 1H), 7.35−7.24 (m, 1H), 6.35 (d, J = 7.6 Hz,
1H), 5.99 (dd, J = 10.0, 2.3 Hz, 1H), 5.96−5.84 (m, 1H), 5.36−5.15
(m, 2H), 4.93 (d, J = 3.9 Hz, 1H), 4.56 (qdt, J = 13.3, 5.6, 1.5 Hz, 2H),
4.45 (ddd, J = 12.1, 4.5, 2.5 Hz, 1H), 4.11−4.06 (m, 1H), 4.01 (d, J =
2.5 Hz, 1H), 3.87 (dddd, J = 12.1, 7.1, 4.1, 2.6 Hz, 1H), 3.85−3.79 (m,
1H), 3.48−3.44 (m, 1H), 2.22 (ddd, J = 11.9, 4.1, 2.2 Hz, 1H), 2.14 (td,
J = 12.4, 4.0 Hz, 1H), 1.99 (dd, J = 12.4, 4.6 Hz, 1H), 1.85 (td, J = 12.3,
10.0 Hz, 1H), 1.51 (tt, J = 7.2, 5.7 Hz, 1H), 1.36−1.30 (m, 6H), 1.13−
0.81 (m, 28H). 13C NMR (126 MHz, CDCl3) δ 164.3, 155.8, 134.2,
133.0, 132.0, 131.1, 130.8, 127.0, 125.1, 117.7, 102.3, 99.8, 93.2, 80.6,
74.5, 73.3, 73.0, 69.9, 68.4, 65.7, 50.1, 33.3, 32.2, 17.8, 17.8, 17.6, 17.5,
17.5, 17.5, 17.4, 17.3, 14.3, 14.2, 13.2, 12.7, 9.0, 8.9, 0.8. HRMS: [M +
Na]+ calcd for C40H61NO10Si2Na 794.37317; found 794.3749.
7-[2-Deoxy-3,4-tetraisopropyldisiloxyl-α-L-fucopyranosyl-(1 →
4)-3-N-allyloxycarbonyl-2,3-dideoxy-α-L-fucopyranoside]-14-O-
tert-butyldimethylsilyl-doxorubicinone (22). Prepared according to
General Procedure C from donor 21 (722 mg, 1.00 mmol) and 14-O-
tert-butyldimethylsilyl-doxorubicinone 16 (793 mg, 1.50 mmol, 1.5
equiv) to give after column chromatography (5:95−20:80 EtOAc/
pentane−4:96 acetone/toluene) the title compound as a red solid (714
mg, 0.640 mmol, 64%). 1H NMR (500 MHz, chloroform-d) δ 13.83 (s,
1H), 13.09 (s, 1H), 7.93 (dd, J = 7.7, 1.0 Hz, 1H), 7.72 (t, J = 8.1 Hz,
1H), 7.43−7.32 (m, 1H), 6.07 (d, J = 7.8 Hz, 1H), 5.91−5.78 (m, 1H),
5.50 (d, J = 3.8 Hz, 1H), 5.27−5.18 (m, 2H), 5.13 (dq, J = 10.5, 1.4 Hz,
1H), 4.98−4.86 (m, 3H), 4.61−4.37 (m, 4H), 4.13 (q, J = 6.5 Hz, 1H),
4.05 (d, J = 24.2 Hz, 6H), 3.90−3.77 (m, 1H), 3.55 (s, 1H), 3.09 (dd, J
= 18.8, 2.0 Hz, 1H), 2.81 (d, J = 18.7 Hz, 1H), 2.29 (d, J = 14.6 Hz, 1H),
2.22−2.05 (m, 2H), 2.05−1.95 (m, 1H), 1.92 (dd, J = 13.1, 4.5 Hz,
1H), 1.78 (td, J = 12.9, 4.0 Hz, 1H), 1.30 (dd, J = 16.4, 6.4 Hz, 6H),
1.16−0.82 (m, 37H), 0.15 (d, J = 2.7 Hz, 6H). 13C NMR (126 MHz,
CDCl3) δ 211.4, 186.8, 186.4, 161.0, 156.3, 155.7, 135.7, 135.3, 134.0,
133.9, 132.9, 120.7, 119.8, 118.5, 117.5, 111.3, 111.2, 101.9, 101.0, 81.0,
73.2, 69.9, 69.7, 68.2, 68.0, 66.7, 65.5, 56.7, 46.6, 35.7, 34.0, 33.3, 31.3,
26.0, 18.7, 17.8, 17.7, 17.6, 17.5, 17.5, 17.5, 17.4, 17.3, 17.2, 14.3, 14.1,
13.1, 12.6, −5.2, −5.3. HRMS: [M + Na]+ calcd for C55H83NO17Si3Na
1136.48665; found 1136.4866.
7-[2-Deoxy-3,4-tetraisopropyldisiloxyl-α-L-fucopyranosyl-(1 →
4)-3-amino-2,3-dideoxy-α-L-fucopyranoside]-14-O-tert-butyldime-
thylsilyl-doxorubicinone (23). A solution of 22 (704 mg, 0.631 mmol)
and N,N-dimethylbarbituric acid (440 mg, 2.84 mmol, 4.5 equiv) in
DCM (63 mL) was degassed for 5 min. Then, Pd(PPh3)4 (36.5 mg,
0.032 mmol, 0.05 equiv) was added and the mixture was allowed to stir
for 20 min. It was then directly subjected to column chromatography
(pentane, then 0:100−50:50 acetone/toluene) to give the title
1
compound as a red solid (650 mg, 0.631 mmol, 100%). H NMR
(500 MHz, chloroform-d) δ 7.93 (dd, J = 7.8, 1.0 Hz, 1H), 7.73 (t, J =
8.1 Hz, 1H), 7.42−7.33 (m, 1H), 5.53−5.41 (m, 1H), 5.21 (dd, J = 4.1,
2.2 Hz, 1H), 4.98 (d, J = 3.7 Hz, 1H), 4.96−4.81 (m, 2H), 4.65 (s, 1H),
4.42 (ddd, J = 12.1, 4.6, 2.5 Hz, 1H), 4.15 (q, J = 6.5 Hz, 1H), 4.10−3.93
(m, 5H), 3.53 (s, 1H), 3.40−3.20 (m, 3H), 3.18−3.00 (m, 2H), 2.82 (d,
J = 18.7 Hz, 1H), 2.29 (dt, J = 14.8, 2.2 Hz, 1H), 2.21−2.09 (m, 2H),
2.05−1.93 (m, 1H), 1.76 (ddd, J = 27.6, 14.0, 4.2 Hz, 1H), 1.29 (d, J =
6.5 Hz, 3H), 1.23 (d, J = 6.5 Hz, 3H), 1.13−0.75 (m, 36H), 0.15 (d, J =
1.4 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 211.2, 186.7, 186.4, 161.0,
156.3, 155.6, 135.7, 135.3, 134.0, 132.1, 132.1, 128.6, 120.7, 119.7,
118.5, 111.3, 101.3, 101.1, 81.5, 73.3, 70.1, 69.6, 68.3, 67.8, 66.6, 56.7,
46.8, 35.6, 33.8, 33.4, 25.9, 18.7, 17.7, 17.7, 17.6, 17.6, 17.5, 17.5, 17.4,
17.3, 17.2, 14.2, 14.1, 13.1, 12.6. HRMS: [M + H]+ calcd for
C51H80NO15Si3 1030.48358; found 1030.4855.
155.9, 155.3, 135.9, 135.4, 133.8, 133.5, 120.8, 119.8, 118.6, 111.6,
111.4, 100.9, 99.2, 73.6, 71.0, 69.2, 68.6, 66.6, 65.4, 65.2, 61.7, 56.6,
43.0, 35.5, 33.8, 32.3, 28.7, 17.9, 16.6. HRMS: [M + H]+ calcd for
C35H44NO14 702.27619; found 702.2769.
7-[2-Deoxy-3,4-tetraisopropyldisiloxyl-α-L-fucopyranosyl-(1 →
4)-3-amino-2,3-dideoxy-α-L-fucopyranoside]-aklavinone (24). Pre-
pared according to General Procedure C from donor 21 (623 mg, 0.806
mmol) and aklavinone 14 (665 mg, 1.61 mmol, 2.00 equiv) at −20 °C
to give after column chromatography (10:90 EtOAc/pentane and then
2:98−10:90 acetone/toluene) of the residue an inseparable mixture of
J
https://dx.doi.org/10.1021/acs.jmedchem.0c01191
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the disaccharide anthracycline and acceptor, which was continued to
the next step. A solution of the above mixture and N,N-
dimethylbarbituric acid (562 mg, 3.60 mmol, 2.2 equiv) in DCM (81
mL) was degassed for 5 min. Then, Pd(PPh3)4 (23 mg, 0.040 mmol,
0.025 equiv) was added and the mixture was allowed to stir for 30 min.
It was then directly subjected to column chromatography (pentane,
then 0:100−25:75 acetone/toluene) to give the title compound as a
yellow solid (636 mg, 0.700 mmol, 86% over two steps). 1H NMR (400
MHz, chloroform-d) δ 7.76 (d, J = 7.5 Hz, 1H), 7.70−7.58 (m, 2H),
7.25 (d, J = 8.4 Hz, 1H), 5.47 (d, J = 2.8 Hz, 1H), 5.25 (dd, J = 4.1, 1.8
Hz, 1H), 4.97 (d, J = 3.6 Hz, 1H), 4.42 (ddd, J = 12.0, 4.7, 2.5 Hz, 1H),
4.19−4.05 (m, 3H), 4.00 (d, J = 2.6 Hz, 1H), 3.70 (s, 3H), 3.51 (d, J =
2.5 Hz, 1H), 3.24 (qt, J = 9.3, 6.6, 5.6 Hz, 1H), 2.52 (dd, J = 15.0, 4.3
Hz, 1H), 2.36−2.28 (m, 1H), 2.17−2.08 (m, 1H), 2.01 (dd, J = 12.3,
4.6 Hz, 1H), 1.86−1.68 (m, 3H), 1.49 (dd, J = 14.1, 7.0 Hz, 1H), 1.30
(d, J = 6.4 Hz, 3H), 1.23 (d, J = 6.5 Hz, 3H), 1.17−0.85 (m, 31H).13C
NMR (101 MHz, CDCl3) δ 192.6, 181.2, 171.4, 162.5, 162.1, 142.7,
137.4, 133.4, 132.9, 131.2, 124.8, 120.9, 120.2, 115.7, 114.6, 101.7,
101.1, 81.7, 73.3, 71.6, 70.9, 70.2, 68.1, 67.8, 57.1, 52.6, 46.8, 33.9, 33.4,
32.2, 17.7, 17.7, 17.6, 17.5, 17.5, 17.4, 17.3, 17.3, 14.3, 14.1, 13.1, 12.6,
6.8. HRMS: [M + H]+ calcd for C46H68NO14Si2 914.4178; found
914.4173.
7-[2-Deoxy-α-L-fucopyranosyl-(1 → 4)-3-amino-2,3-dideoxy-α-L-
fucopyranoside]-aklavinone (6). To a solution of 24 (91 mg, 0.10
mmol) in pyridine (10 mL) in a PTFE tube was added HF·pyr complex
(70 wt % HF, 393 μL) at 0 °C. Over the course of 4.5 h, three additional
such portions of HF·pyr complex were added. Then, solid NaHCO3
was added to quench, and the mixture was stirred until cessation of
effervescence. It was then filtered off and partitioned between DCM
and H2O. The organic layer was washed with brine, dried over Na2SO4,
and concentrated in vacuo. Column chromatography on neutral silica
(DCM; 20:80 MeOH/DCM) followed by size-exclusion chromatog-
raphy (Sephadex LH-20; eluent, DCM/MeOH, 1:1) gave the title
compound as a yellow solid (27.5 mg, 40.9 μmol, 41%). 1H NMR (400
MHz, chloroform-d + MeOD) δ 7.79 (dd, J = 7.5, 1.3 Hz, 1H), 7.74−
7.57 (m, 2H), 7.32−7.23 (m, 1H), 5.47 (t, J = 2.5 Hz, 1H), 5.27−5.20
(m, 1H), 4.97 (d, J = 3.5 Hz, 1H), 4.20−4.01 (m, 4H), 3.70 (s, 3H),
3.64 (d, J = 3.0 Hz, 2H), 3.61−3.52 (m, 2H), 3.11 (dd, J = 10.6, 6.7 Hz,
1H), 2.53 (dd, J = 15.0, 4.4 Hz, 1H), 2.27 (d, J = 15.0 Hz, 1H), 1.97
(ddd, J = 22.5, 12.3, 4.2 Hz, 2H), 1.86−1.64 (m, 3H), 1.50 (dt, J = 14.6,
7.4 Hz, 1H), 1.28 (d, J = 6.4 Hz, 3H), 1.23 (d, J = 6.5 Hz, 3H), 1.07 (q, J
= 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 192.6, 181.4, 171.4,
162.5, 162.0, 142.6, 137.5, 133.5, 132.9, 131.1, 124.9, 121.0, 120.3,
115.8, 114.7, 101.3, 100.8, 81.1, 71.6, 70.9, 70.8, 68.0, 67.4, 65.4, 57.0,
52.6, 46.7, 34.1, 33.2, 32.7, 32.2, 17.3, 16.9, 6.7. HRMS: [M + H]+ calcd
for C34H42NO13 672.2656; found 672.2645.
7-[2-Deoxy-α-L-fucopyranosyl-(1 → 4)-3-dimethylamino-2,3-di-
deoxy-α-L-fucopyranoside]-aklavinone (8). To a solution of 6 (26.2
mg, 37.4 μmol) in EtOH (3.7 mL) and 37% aq CH2O (200 μL, 60
equiv) was added NaBH(OAc)3 (85 mg, 0.374 mmol, 10 equiv). The
mixture was stirred for 2.5 h before being poured into sat. aq NaHCO3.
This was extracted with DCM, dried over Na2SO4, and concentrated in
vacuo. Column chromatography on neutral silica (3:97−10:90 MeOH/
DCM) gave the title compound as a yellow solid (8.8 mg, 12.6 μmol,
34%). 1H NMR (500 MHz, chloroform-d) δ 12.69 (s, 1H), 12.04 (s,
1H), 7.83 (dd, J = 7.5, 1.2 Hz, 1H), 7.78−7.60 (m, 2H), 7.31 (dd, J =
8.4, 1.2 Hz, 1H), 5.51 (d, J = 3.7 Hz, 1H), 5.27 (dd, J = 4.3, 1.9 Hz, 1H),
5.01 (s, 1H), 4.53 (dd, J = 14.2, 7.7 Hz, 1H), 4.17−4.05 (m, 2H), 4.00
(q, J = 6.6 Hz, 1H), 3.74 (d, J = 8.5 Hz, 1H), 3.63 (d, J = 3.1 Hz, 1H),
2.52 (dd, J = 15.0, 4.3 Hz, 1H), 2.29 (dd, J = 16.9, 9.2 Hz, 1H), 2.25−
2.11 (m, 6H), 2.07 (dt, J = 10.9, 5.4 Hz, 1H), 1.87−1.79 (m, 1H), 1.75
(dq, J = 14.6, 7.7, 7.3 Hz, 1H), 1.51 (dq, J = 14.3, 7.2 Hz, 1H), 1.28 (d, J
= 6.5 Hz, 3H), 1.20 (d, J = 6.5 Hz, 3H), 1.09 (t, J = 7.4 Hz, 3H). 13C
NMR (126 MHz, CDCl3) δ 192.9, 181.5, 162.7, 162.3, 142.8, 137.5,
133.6, 133.1, 124.9, 121.1, 120.3, 116.0, 114.8, 101.7, 99.2, 71.8, 71.7,
70.8, 68.5, 66.3, 66.0, 61.7, 57.3, 52.7, 43.4, 33.9, 33.2, 32.3, 18.0, 16.8,
6.8. HRMS: [M + H]+ calcd for C36H46NO13 700.2969; found
700.2966.
bonyl-2,3-dideoxy-α-L-fucopyranoside (27). To a solution of the
glycosyl acceptor 17 (169 mg g, 0.5 mmol, 1 equiv) and the glycosyl
donor 25 (325 mg, 0.7 mmol, 1.4 equiv) in 4:1 Et2O/DCE (15 mL, v/
v), activated molecular sieves (4 Å) were added. The mixture was
stirred for 30 min and then, at 10 °C, iodonium dicollidine perchlorate
(937 mg, 2.00 mmol, 4 equiv) was added. After 30 min,
triphenylphosphine (262 mg, 1.00 mmol, 2 equiv) was added, and
the mixture was stirred for an additional hour. It was then diluted with
EtOAc and filtered; washed with 10% aq Na2S2O3, 1 M CuSO4 solution
twice, and H2O; and then dried over MgSO4. Concentration in vacuo
and column chromatography (15:85−20:80 EtOAc/pentane) of the
residue gave the disaccharide. This was then dissolved in MeOH (8.8
mL) and DCM (8.8 mL), after which NaOMe was added to pH 10.
After stirring for a week, it was neutralized by addition of dry ice and
concentrated in vacuo. Column chromatography (20:80−50:50
EtOAc/pentane) gave the title compound as a clear oil (232 mg,
0.39 mmol, 78% over two steps). 1H NMR (400 MHz, chloroform-d) δ
7.28 (d, J = 6.7 Hz, 2H), 7.05−6.96 (m, 2H), 6.96−6.87 (m, 2H),
6.87−6.77 (m, 2H), 6.21 (d, J = 8.2 Hz, 1H), 5.92 (ddt, J = 16.4, 10.9,
5.5 Hz, 1H), 5.51 (d, J = 3.1 Hz, 1H), 5.37−5.25 (m, 1H), 5.20 (dt, J =
10.4, 1.4 Hz, 1H), 5.00−4.92 (m, 1H), 4.62−4.52 (m, 4H), 4.39−4.25
(m, 1H), 4.11 (q, J = 7.8, 7.1 Hz, 1H), 4.08−4.01 (m, 1H), 3.97 (td, J =
8.4, 3.1 Hz, 1H), 3.86 (s, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.56 (s, 1H),
2.21 (s, 1H), 2.13 (dd, J = 12.6, 4.5 Hz, 1H), 2.08−2.00 (m, 2H), 1.86
(td, J = 12.7, 3.5 Hz, 1H), 1.38 (d, J = 6.6 Hz, 3H), 1.17 (d, J = 6.5 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 159.6, 155.9, 154.7, 151.1, 133.0,
130.0, 129.5, 117.6, 117.5, 114.6, 114.1, 101.4, 96.4, 81.5, 72.7, 70.2,
68.2, 67.5, 67.2, 65.7, 55.8, 55.4, 46.6, 31.8, 30.3, 17.4, 16.8. HRMS: [M
+ Na]+ calcd for C31H41NO10Na 610.2628; found 610.2632.
p-Methoxyphenyl-2,3-dideoxy-4-ulo-α-L-fucopyranosyl-(1 → 4)-
2-deoxy-3-O-p-methoxybenzyl-α-L-fucopyranosyl-(1 → 4)-3-azido-
2,3-dideoxy-α-L-fucopyranoside (29). To a solution of the glycosyl
acceptor 27 (120 g, 2.04 mmol) and the glycosyl donor 28 (1.01 g, 2.86
mmol, 1.4 equiv) in 4:1 Et2O/DCE (62.5 mL, v/v), activated molecular
sieves (4 Å) were added. The mixture was stirred for 30 min and then, at
10 °C, iodonium dicollidine perchlorate (3.82 g, 8.16 mmol, 4 equiv)
was added. After 35 min, triphenylphosphine (1.07 g, 4.08 mmol, 2.00
equiv) was added, and the mixture was stirred for an additional hour. It
was then diluted with EtOAc and filtered; washed with 10% aq
Na2S2O3, 1 M CuSO4 solution twice, and H2O; and then dried over
MgSO4. Concentration in vacuo and column chromatography (10:90−
30:70 EtOAc/pentane) of the residue gave the trisaccharide benzoate
1
as a thick clear oil (1.59 g, 1.97 mmol, 97%). H NMR (400 MHz,
chloroform-d) δ 8.12−8.05 (m, 2H), 7.61−7.54 (m, 1H), 7.51−7.37
(m, 2H), 7.28 (d, J = 2.2 Hz, 2H), 7.04−6.94 (m, 2H), 6.92−6.85 (m,
2H), 6.85−6.76 (m, 2H), 6.16 (d, J = 8.3 Hz, 1H), 5.92 (ddt, J = 16.3,
10.8, 5.6 Hz, 1H), 5.49 (d, J = 2.7 Hz, 1H), 5.34−5.16 (m, 2H), 5.04 (s,
1H), 5.03−4.94 (m, 2H), 4.72−4.50 (m, 5H), 4.40−4.25 (m, 1H),
4.17−4.01 (m, 2H), 3.99−3.88 (m, 2H), 3.79 (s, 3H), 3.77 (s, 3H),
3.56 (s, 1H), 2.29−2.15 (m, 2H), 2.14−1.98 (m, 3H), 1.94 (d, J = 14.0
Hz, 1H), 1.88−1.76 (m, 2H), 1.31 (d, J = 6.5 Hz, 3H), 1.16 (d, J = 6.5
Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
166.3, 159.2, 155.9, 154.7, 151.1, 133.1, 130.6, 130.5, 129.8, 129.0,
128.5, 117.7, 117.6, 114.6, 113.9, 101.5, 98.7, 96.4, 81.1, 77.5, 77.4,
77.2, 76.8, 74.9, 72.7, 70.6, 70.3, 70.3, 68.8, 67.5, 65.7, 65.7, 65.7, 55.8,
55.4, 46.6, 31.8, 31.3, 24.5, 23.1, 17.5, 17.2. HRMS: [M + Na]+ calcd for
C44H55NO13Na 828.3571; found 828.3586.
The above benzoate (1.20 g, 2.04 mmol) was dissolved in MeOH
(40 mL) and DCM (40 mL), after which NaOMe was added to pH 10.
After stirring for a week, it was neutralized by addition of dry ice and
concentrated in vacuo. Column chromatography (50:50−75:25
EtOAc/pentane) gave the alcohol as a white foam (1.21 g, 1.72
1
mmol, 85%). H NMR (400 MHz, chloroform-d) δ 7.32−7.19 (m,
2H), 7.05−6.95 (m, 2H), 6.93−6.85 (m, 2H), 6.85−6.75 (m, 2H), 6.15
(d, J = 8.3 Hz, 1H), 5.97−5.86 (m, 1H), 5.49 (d, J = 3.1 Hz, 1H), 5.30
(dq, J = 17.2, 1.6 Hz, 1H), 5.20 (dq, J = 10.6, 1.5 Hz, 1H), 4.99 (q, J =
1.5 Hz, 1H), 4.92 (d, J = 3.2 Hz, 1H), 4.70−4.46 (m, 4H), 4.43−4.34
(m, 1H), 4.31 (dt, J = 7.8, 4.3 Hz, 1H), 4.09 (q, J = 6.3 Hz, 1H), 4.01 (q,
J = 6.6 Hz, 1H), 3.96−3.86 (m, 2H), 3.80 (s, 3H), 3.77 (s, 3H), 3.54 (s,
1H), 3.52 (s, 1H), 2.17 (td, J = 12.1, 3.7 Hz, 1H), 2.12−1.90 (m, 4H),
Synthesis of Trisaccharides 9−11. p-Methoxyphenyl-2-deoxy-3-
O-p-methoxybenzyl-α-L-fucopyranosyl-(1 → 4)-3-N-allyloxycar-
K
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1.82 (td, J = 12.6, 3.5 Hz, 1H), 1.78−1.66 (m, 3H), 1.29 (d, J = 6.6 Hz,
3H), 1.14 (d, J = 6.5 Hz, 3H), 0.91 (d, J = 6.5 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 159.2, 155.9, 154.6, 151.1, 133.0, 130.6, 129.0, 117.7,
117.5, 114.6, 113.8, 101.4, 98.7, 96.4, 81.0, 74.9, 72.7, 68.9, 67.6, 67.5,
66.6, 55.8, 55.4, 46.6, 31.8, 31.3, 25.8, 23.6, 17.5, 17.1. HRMS: [M +
Na]+ calcd for C37H51NO12Na 724.3309; found 724.3322.
4.58−4.41 (m, 4H), 4.19−4.10 (m, 3H), 4.09 (s, 3H), 3.93−3.82 (m,
1H), 3.78−3.70 (m, 2H), 3.58 (s, 1H), 3.20 (dd, J = 18.7, 1.8 Hz, 1H),
2.97 (d, J = 18.9 Hz, 1H), 2.55−2.39 (m, 3H), 2.29 (d, J = 14.8 Hz, 1H),
2.24−2.02 (m, 4H), 1.92 (ddd, J = 14.0, 10.0, 3.8 Hz, 2H), 1.83−1.72
(m, 1H), 1.37−1.22 (m, 10H), 0.96 (s, 9H), 0.14 (d, J = 2.2 Hz, 6H).
13C NMR (101 MHz, CDCl3) δ 211.5, 209.9, 187.2, 186.8, 161.1,
To a solution of the above alcohol (351 mg, 0.500 mmol) in DCM
(20 mL) were added NaHCO3 (840 mg, 5.00 mmol, 10 equiv) and
Dess−Martin periodinane (530 mg, 1.25 mmol, 2.5 equiv). After
stirring for 1.5 h, 10% aq Na2S2O3 (20 mL) was added, and the mixture
was stirred for a further 30 min. Then, it was washed with sat. aq
NaHCO3, dried over MgSO4, and concentrated in vacuo. Size-
exclusion chromatography (Sephadex LH-20; eluent, 1:1 DCM/
MeOH) gave the title compound as a white solid (341 mg, 0.487
156.5, 156.0, 155.6, 135.8, 135.6, 134.2, 134.0, 133.0, 121.0, 119.9,
118.5, 117.6, 111.6, 111.4, 101.6, 100.9, 100.3, 82.2, 81.1, 72.0, 69.8,
67.9, 66.8, 65.6, 65.0, 56.8, 46.6, 35.8, 34.4, 34.2, 33.5, 31.4, 27.6, 26.0,
18.7, 17.5, 16.9, 14.9. HRMS: [M + Na]+ calcd for C49H65NO18SiNa
1006.3869; found 1006.3876.
7-[2,3-Dideoxy-4-ulo-α-L-fucopyranosyl-2-deoxy-α-L-fucopyra-
nosyl-(1 → 4)-3-amino-2,3-dideoxy-α-L-fucopyranoside]-doxorubi-
cinone (9). A solution of 31 (159 mg, 0.162 mmol) and N,N-
dimethylbarbituric acid (115 mg, 0.729 mmol, 4.5 equiv) in DCM (16.3
mL) was degassed for 5 min. Then, Pd(PPh3)4 (9.0 mg, 81 μmol, 0.05
equiv) was added, and the mixture was allowed to stir for 20 min. It was
then directly subjected to column chromatography on neutral silica
(0:100−3:97 MeOH/DCM) to give the free amine as a red solid (118
mg, 0.131 mmol, 81%). 1H NMR (500 MHz, chloroform-d) δ 13.90 (s,
1H), 7.97 (dd, J = 7.7, 1.1 Hz, 1H), 7.75 (t, J = 8.1 Hz, 1H), 7.38 (dd, J =
8.7, 1.1 Hz, 1H), 5.48 (d, J = 3.7 Hz, 1H), 5.23 (dd, J = 4.1, 2.2 Hz, 1H),
5.10 (t, J = 6.1 Hz, 1H), 5.01 (d, J = 3.6 Hz, 1H), 4.94−4.81 (m, 2H),
4.50 (q, J = 6.7 Hz, 1H), 4.25 (q, J = 6.6 Hz, 1H), 4.13 (ddd, J = 12.2,
4.7, 2.7 Hz, 1H), 4.08 (s, 3H), 4.03 (q, J = 6.4 Hz, 1H), 3.73 (s, 1H),
3.52 (d, J = 2.5 Hz, 1H), 3.13 (dd, J = 18.8, 1.9 Hz, 1H), 3.00 (ddd, J =
12.4, 4.7, 2.4 Hz, 1H), 2.89 (d, J = 18.7 Hz, 1H), 2.56−2.38 (m, 3H),
2.30 (dt, J = 14.8, 2.1 Hz, 1H), 2.23−2.00 (m, 3H), 1.89 (td, J = 12.4,
3.8 Hz, 1H), 1.75 (td, J = 12.9, 3.9 Hz, 1H), 1.68 (dd, J = 13.1, 4.5 Hz,
1H), 1.33 (d, J = 6.8 Hz, 3H), 1.28 (d, J = 6.5 Hz, 3H), 1.22 (d, J = 6.5
Hz, 3H), 0.96 (s, 9H), 0.14 (d, J = 1.2 Hz, 6H). 13C NMR (126 MHz,
CDCl3) δ 211.2, 210.0, 186.9, 186.6, 161.1, 156.4, 155.8, 135.7, 135.5,
134.1, 120.8, 119.8, 118.5, 111.4, 101.4, 100.8, 100.2, 82.3, 81.7, 71.9,
69.6, 68.4, 67.4, 66.6, 65.2, 56.7, 46.8, 35.6, 34.4, 33.9, 33.5, 27.7, 26.0,
18.7, 17.7, 17.2, 14.8. HRMS: [M + H]+ calcd for C45H62NO16Si
900.3838; found 900.3836.
1
mmol, 97%). H NMR (400 MHz, chloroform-d) δ 7.32−7.20 (m,
2H), 7.06−6.99 (m, 2H), 6.92−6.85 (m, 2H), 6.85−6.76 (m, 2H), 6.16
(d, J = 8.2 Hz, 1H), 5.92 (ddd, J = 17.3, 10.6, 5.4 Hz, 1H), 5.50 (d, J =
3.1 Hz, 1H), 5.36−5.15 (m, 2H), 5.10 (t, J = 4.3 Hz, 1H), 5.00 (dd, J =
3.7, 1.7 Hz, 1H), 4.72−4.45 (m, 5H), 4.38−4.25 (m, 1H), 4.08 (dq, J =
13.3, 6.4 Hz, 2H), 4.03−3.88 (m, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 3.56
(s, 1H), 2.60 (ddd, J = 15.0, 8.9, 5.7 Hz, 1H), 2.41 (ddd, J = 15.6, 7.6,
5.5 Hz, 1H), 2.30 (ddt, J = 14.1, 8.9, 5.2 Hz, 1H), 2.25−1.99 (m, 4H),
1.84 (td, J = 12.7, 3.5 Hz, 1H), 1.33 (d, J = 6.5 Hz, 3H), 1.15 (d, J = 6.4
Hz, 3H), 0.97 (d, J = 6.7 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
210.7, 158.9, 155.4, 154.3, 150.7, 132.7, 130.0, 128.7, 117.3, 117.2,
114.2, 113.5, 101.1, 97.6, 96.0, 80.7, 74.7, 72.1, 71.5, 69.9, 68.2, 67.1,
65.3, 55.4, 55.0, 46.2, 33.6, 31.4, 30.7, 29.1, 17.1, 17.0, 14.5. HRMS: [M
+ Na]+ calcd for C37H49NO12Na 722.3153; found 722.3165.
o-Cyclopropylethynylbenzoyl-2,3-dideoxy-4-ulo-α-L-fucopyrano-
syl-(1 → 4)-2-deoxy-3-O-p-methoxybenzyl-α-L-fucopyranosyl-(1 →
4)-3-azido-2,3-dideoxy-L-fucopyranoside (30). Prepared according to
General Procedures A and B from 29 (1.06 g, 1.51 mmol) to give the
title compound as a white foam (872 mg, 1.14 mmol, 75% over two
steps, α/β 1:7). Spectral data for the β-anomer: 1H NMR (400 MHz,
chloroform-d) δ 7.94 (dd, J = 7.9, 1.4 Hz, 1H), 7.48 (dd, J = 7.9, 1.4 Hz,
1H), 7.42 (td, J = 7.5, 1.4 Hz, 1H), 7.37−7.16 (m, 3H), 6.93−6.79 (m,
2H), 6.36 (d, J = 8.0 Hz, 1H), 5.98 (dd, J = 10.0, 2.2 Hz, 1H), 5.90 (ddd,
J = 16.3, 10.7, 5.4 Hz, 1H), 5.37−5.15 (m, 2H), 5.10 (t, J = 4.4 Hz, 1H),
5.03−4.97 (m, 1H), 4.75−4.45 (m, 5H), 4.08 (q, J = 6.6 Hz, 1H),
4.03−3.95 (m, 2H), 3.90 (ddt, J = 12.4, 7.4, 4.1 Hz, 1H), 3.85−3.78 (m,
2H), 3.76 (s, 3H), 3.49 (s, 1H), 2.60 (ddd, J = 15.0, 8.8, 5.7 Hz, 1H),
2.42 (ddd, J = 15.7, 7.7, 5.4 Hz, 1H), 2.31 (ddt, J = 13.9, 8.8, 5.2 Hz,
1H), 2.24−2.15 (m, 2H), 2.10 (tt, J = 10.4, 5.5 Hz, 2H), 1.81 (td, J =
12.3, 9.9 Hz, 1H), 1.50 (tt, J = 7.8, 5.4 Hz, 1H), 1.36−1.27 (m, 6H),
0.97 (d, J = 6.7 Hz, 3H), 0.87 (dd, J = 7.6, 5.3 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 211.1, 164.3, 159.3, 155.8, 134.3, 132.9, 132.0, 130.3,
129.1, 127.0, 125.2, 117.7, 113.9, 101.8, 99.8, 98.0, 93.2, 80.3, 75.1,
74.5, 72.9, 72.4, 71.9, 70.3, 68.7, 65.7, 55.4, 50.0, 34.0, 32.2, 31.1, 29.5,
17.4, 14.8, 9.0, 0.8. HRMS: [M + Na]+ calcd for C42H51NO12Na
784.3309; found 784.3322.
7-[2,3-Dideoxy-4-ulo-α-L-fucopyranosyl-2-deoxy-3-p-methoxy-
benzyl-α-L-fucopyranosyl-(1 → 4)-3-amino-2,3-dideoxy-α-L-fuco-
pyranoside]-14-O-tert-butyldimethylsilyl-doxorubicinone (31). Pre-
pared according to General Procedure C from donor 30 (422 mg, 0.552
mmol) and doxorubicinone acceptor 1635 (1.5 equiv) to give after
column chromatography (20:80−100:0 EtOAc/pentane) the crude
anthracycline trisaccharide. To a solution of the above trisaccharide in
DCM (93 mL) and phosphate buffer (9.3 mL, pH = 7) was added DDQ
(1.25 g, 5.52 mmol, 10 equiv) at 0 °C, after which the mixture was
stirred at that temperature for 45 min. It was then stirred at room
temperature for an additional 2.5 h, after which it was diluted with
DCM and washed with H2O four times. The organic layer was then
dried over Na2SO4 and concentrated in vacuo. Column chromatog-
raphy (5:95−12:88 acetone/toluene) gave the free 3″-hydroxyl
anthracycline trisaccharide as a red solid (310 mg, 0.315 mmol, 57%
over two steps). 1H NMR (400 MHz, chloroform-d) δ 13.93 (s, 1H),
13.24 (s, 1H), 8.03 (dd, J = 7.8, 1.0 Hz, 1H), 7.78 (t, J = 8.1 Hz, 1H),
7.39 (dd, J = 8.6, 1.1 Hz, 1H), 6.02 (d, J = 7.9 Hz, 1H), 5.84 (ddt, J =
16.2, 10.8, 5.5 Hz, 1H), 5.51 (d, J = 3.7 Hz, 1H), 5.26 (td, J = 3.4, 1.7 Hz,
1H), 5.23−5.05 (m, 2H), 4.99−4.93 (m, 1H), 4.90 (d, J = 2.8 Hz, 2H),
To a solution of the above compound (19.7 mg, 21.9 μmol) in
pyridine (0.7 mL) and THF (1.4 mL) in a PTFE tube was added HF·
pyr complex (70 wt % HF, 86 μL) at 0 °C. After 3 h, an additional such
portion of HF·pyr complex was added. After stirring one more hour,
solid NaHCO3 was added to quench, and the mixture was stirred until
cessation of effervescence. It was then filtered off, and the filtrate was
poured into DCM/H2O. The organic layer was dried over Na2SO4 and
concentrated in vacuo. Column chromatography on neutral silica
(DCM; 4:96 MeOH/DCM) gave the title compound as a red solid
1
(12.7 mg, 16.2 μmol, 74%). H NMR (500 MHz, chloroform-d) δ
13.94 (s, 1H), 8.13−7.89 (m, 1H), 7.78 (t, J = 8.1 Hz, 1H), 7.52−7.31
(m, 1H), 5.51 (d, J = 3.8 Hz, 1H), 5.36−5.27 (m, 1H), 5.09 (t, J = 6.1
Hz, 1H), 5.01 (d, J = 3.7 Hz, 1H), 4.81−4.68 (m, 2H), 4.49 (q, J = 6.6
Hz, 1H), 4.23 (q, J = 6.4 Hz, 1H), 4.16−4.05 (m, 4H), 4.01 (q, J = 6.5
Hz, 1H), 3.72 (s, 1H), 3.52 (s, 1H), 3.25 (dd, J = 18.9, 2.0 Hz, 1H),
3.08−2.96 (m, 2H), 2.46 (dtt, J = 17.8, 10.3, 5.8 Hz, 4H), 2.32 (dt, J =
14.5, 2.1 Hz, 1H), 2.25 (t, J = 7.6 Hz, 1H), 2.22−2.05 (m, 4H), 1.89 (td,
J = 12.3, 3.7 Hz, 1H), 1.76 (td, J = 12.9, 3.9 Hz, 1H), 1.70 (d, J = 4.5 Hz,
1H), 1.33 (d, J = 6.5 Hz, 3H), 1.28 (d, J = 6.4 Hz, 3H), 1.22 (d, J = 6.7
Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 213.9, 210.0, 187.2, 186.8,
161.2, 156.4, 155.8, 135.9, 135.6, 134.0, 133.7, 121.0, 120.0, 118.6,
111.7, 111.5, 101.3, 100.9, 100.3, 82.4, 81.7, 72.0, 69.2, 68.5, 67.5, 65.6,
65.3, 56.8, 46.8, 35.6, 34.5, 34.1, 33.6, 27.7, 17.8, 17.2, 14.9. HRMS: [M
+ H]+ calcd for C39H48NO16: 786.2973; found 786.2982.
7-[2,3-Dideoxy-4-ulo-α-L-fucopyranosyl-2-deoxy-α-L-fucopyra-
nosyl-(1 → 4)-3-dimethylamino-2,3-dideoxy-α-L-fucopyranoside]-
doxorubicinone (11). A solution of 31 (159 mg, 0.162 mmol) and N,N-
dimethylbarbituric acid (115 mg, 0.729 mmol, 4.5 equiv) in DCM (16.3
mL) was degassed for 5 min. Then, Pd(PPh3)4 (9.0 mg, 81 μmol, 0.05
equiv) was added, and the mixture was allowed to stir for 20 min. It was
then directly subjected to column chromatography on neutral silica
(0:100−3:97 MeOH/DCM) to give the free amine as a red solid (118
mg, 0.131 mmol, 81%).
L
https://dx.doi.org/10.1021/acs.jmedchem.0c01191
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
To a solution of the above amine (48.0 mg, 53.3 μmol) in EtOH
(10.8 mL) and 37% aq CH2O (132 μL, 30 equiv) was added
NaBH(OAc)3 (21.5 mg, 0.101 mmol, 1.9 equiv). The mixture was
stirred for 1.5 h before being poured into sat. aq NaHCO3. This was
repetitively extracted with DCM, dried over Na2SO4, and concentrated
in vacuo. Column chromatography on neutral silica (10:90−40:60
acetone/toluene) followed by size-exclusion chromatography (Sepha-
dex LH-20, 1:1 DCM/MeOH v/v) gave the dimethylamine as a red
solid (25.8 mg, 27.8 μmol, 52%). 1H NMR (500 MHz, chloroform-d) δ
13.93 (s, 1H), 13.24 (s, 1H), 8.01 (dt, J = 7.7, 1.5 Hz, 1H), 7.83−7.70
(m, 1H), 7.45−7.36 (m, 1H), 5.53 (d, J = 3.8 Hz, 1H), 5.26 (dd, J = 4.1,
2.1 Hz, 1H), 5.10−5.06 (m, 1H), 5.03 (d, J = 3.4 Hz, 1H), 4.97−4.82
(m, 2H), 4.77 (s, 1H), 4.55 (q, J = 6.4 Hz, 1H), 4.50 (q, J = 6.7 Hz, 1H),
4.09 (d, J = 3.3 Hz, 4H), 3.92 (q, J = 6.6 Hz, 1H), 3.75 (s, 1H), 3.72−
3.58 (m, 2H), 3.18 (dd, J = 18.9, 2.0 Hz, 1H), 2.98 (d, J = 18.8 Hz, 1H),
2.53−2.38 (m, 3H), 2.32 (dt, J = 14.6, 2.2 Hz, 1H), 2.26−2.01 (m,
10H), 1.94−1.73 (m, 4H), 1.33 (d, J = 6.8 Hz, 3H), 1.31−1.20 (m,
7H), 1.17 (d, J = 6.4 Hz, 3H), 0.96 (s, 9H), 0.14 (d, J = 2.8 Hz, 6H). 13C
NMR (126 MHz, CDCl3) δ 211.4, 210.3, 187.1, 186.7, 161.1, 156.6,
155.9, 135.8, 135.6, 134.3, 134.1, 124.9, 121.0, 119.9, 118.5, 111.5,
111.4, 101.5, 100.3, 99.6, 83.1, 74.1, 71.9, 69.7, 68.6, 66.7, 65.4, 61.7,
56.8, 43.4, 35.6, 34.4, 34.0, 33.6, 30.4, 29.8, 27.7, 26.0, 18.1, 17.1, 14.9.
HRMS: [M + H]+ calcd for C47H66NO16Si: 928.4151; found 928.4157.
To a solution of the above compound (20.6 mg, 22.2 μmol) in
pyridine (1.4 mL) and THF (1.4 mL) in a PTFE tube was added HF·
pyr complex (70 wt % HF, 87 μL) at 0 °C. Four more additional such
amounts of HF·pyr complex were added over the course of 4.5 h. Then,
solid NaHCO3 was added to quench, and the mixture was stirred until
cessation of effervescence. It was then filtered off, and the filtrate was
poured into DCM/H2O. The organic layer was dried over Na2SO4 and
concentrated in vacuo. Column chromatography on neutral silica
(DCM − 10:90 MeOH/DCM) gave the title compound as a red solid
34.0, 32.2, 31.6, 31.1, 29.5, 17.4, 17.3, 14.8, 6.8. HRMS: [M + Na]+
calcd for C52H61NO18Na 1010.3786; found 1010.3796.
3′,3′-Didesmethyl-aclarubicin (10). To a biphasic mixture of 32
(210 mg, 0.213 mmol) in DCM (36 mL) and phosphate buffer (3.6 mL,
pH = 7) was added DDQ (484 mg, 2.13 mmol, 10 equiv) at 0 °C after
which the mixture was stirred at that temperature for 90 min. It was
diluted with DCM and washed with H2O four times, after which the
organic layer was dried over Na2SO4 and concentrated in vacuo.
Column chromatography (5:95−10:90 acetone/toluene) gave the
intermediate free 3″-hydroxyl as a yellow solid (155 mg, 0.179 mmol,
84%). 1H NMR (400 MHz, chloroform-d) δ 12.65 (s, 1H), 12.00 (s,
1H), 7.81 (dd, J = 7.5, 1.2 Hz, 1H), 7.75−7.60 (m, 2H), 7.32−7.25 (m,
1H), 6.05 (d, J = 7.8 Hz, 1H), 5.83 (ddt, J = 16.3, 10.7, 5.5 Hz, 1H), 5.46
(d, J = 3.8 Hz, 1H), 5.27−5.06 (m, 4H), 4.95 (d, J = 3.5 Hz, 1H), 4.53−
4.38 (m, 3H), 4.28−4.18 (m, 2H), 4.18−4.06 (m, 3H), 3.86 (dd, J =
12.2, 6.5 Hz, 1H), 3.81−3.72 (m, 2H), 3.70 (s, 3H), 3.55 (s, 1H), 2.59−
2.38 (m, 4H), 2.31 (d, J = 15.0 Hz, 1H), 2.24−2.06 (m, 2H), 2.01 (dd, J
= 12.9, 4.6 Hz, 1H), 1.92 (td, J = 12.4, 3.8 Hz, 1H), 1.83−1.68 (m, 2H),
1.49 (dq, J = 14.7, 7.2 Hz, 1H), 1.36−1.24 (m, 9H), 1.08 (t, J = 7.2 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 209.9, 192.8, 181.4, 171.5, 162.6,
162.2, 155.5, 142.7, 137.4, 133.6, 133.0, 133.0, 131.1, 124.8, 121.0,
120.3, 117.5, 115.9, 114.8, 101.6, 101.6, 100.3, 82.1, 81.2, 71.9, 71.5,
71.4, 67.9, 67.7, 65.5, 65.0, 57.1, 52.6, 46.6, 34.4, 34.0, 33.5, 32.2, 31.6,
27.6, 17.3, 16.9, 14.8, 6.8. HRMS: [M + Na]+ calcd for C44H53NO17Na
890.3211; found 890.3220.
A solution of the above compound (155 mg, 0.179 mmol) and N,N-
dimethylbarbituric acid (125 mg, 0.806 mmol, 4.5 equiv) in DCM (18
mL) was degassed for 5 min. Then, Pd(PPh3)4 (10.0 mg, 0.0090 mmol,
0.05 equiv) was added, and the mixture was allowed to stir for 15 min. It
was then directly subjected to column chromatography on neutral silica
(0:100−3:97 MeOH/DCM), followed by size-exclusion chromatog-
raphy (Sephadex LH-20; eluent, 1:1 DCM/MeOH) twice and finally
column chromatography on neutral silica (3:97 MeOH/DCM) to give
1
(13.3 mg, 16.3 μmol, 73%). H NMR (500 MHz, chloroform-d) δ
1
the title compound as a yellow solid (86 mg, 0.11 mmol, 61%). H
13.95 (s, 1H), 13.26 (s, 1H), 8.03 (dd, J = 7.7, 1.0 Hz, 1H), 7.79 (dd, J =
8.5, 7.7 Hz, 1H), 7.40 (dd, J = 8.7, 1.1 Hz, 1H), 5.55 (d, J = 3.8 Hz, 1H),
5.32−5.28 (m, 1H), 5.08 (dd, J = 7.0, 5.6 Hz, 1H), 5.03 (s, 1H), 4.92 (s,
1H), 4.76 (d, J = 1.0 Hz, 2H), 4.54 (d, J = 6.6 Hz, 1H), 4.49 (q, J = 6.7
Hz, 1H), 4.16−4.03 (m, 4H), 3.91 (q, J = 6.5 Hz, 1H), 3.76 (s, 1H),
3.71−3.60 (m, 2H), 3.26 (dd, J = 18.8, 2.0 Hz, 1H), 3.03 (d, J = 18.8 Hz,
1H), 2.54−2.40 (m, 3H), 2.34 (dt, J = 14.6, 2.2 Hz, 1H), 2.24−2.12 (m,
7H), 2.10 (dd, J = 12.1, 4.6 Hz, 1H), 2.03 (d, J = 15.0 Hz, 1H), 1.83 (td,
J = 12.2, 3.8 Hz, 3H), 1.33 (d, J = 6.7 Hz, 3H), 1.27 (d, J = 6.6 Hz, 3H),
1.17 (d, J = 6.4 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 213.9, 210.3,
187.3, 186.9, 161.2, 156.5, 155.9, 135.9, 135.6, 134.2, 133.7, 121.1,
119.9, 118.5, 111.7, 111.5, 101.4, 100.3, 99.6, 83.1, 74.1, 71.9, 69.3,
68.8, 66.9, 65.6, 65.4, 61.8, 56.8, 43.5, 35.6, 34.4, 34.1, 33.7, 27.8, 18.2,
17.1, 14.9. HRMS: [M + H]+ calcd for C41H52NO16: 814.3286; found
814.3301.
NMR (500 MHz, chloroform-d + MeOD) δ 7.81 (dt, J = 7.4, 2.0 Hz,
1H), 7.74−7.62 (m, 2H), 7.30 (d, J = 1.2 Hz, 1H), 5.47 (t, J = 2.5 Hz,
1H), 5.26 (dd, J = 4.4, 1.8 Hz, 1H), 5.10 (t, J = 6.2 Hz, 1H), 4.99 (d, J =
3.6 Hz, 1H), 4.52 (q, J = 6.7 Hz, 1H), 4.19 (q, J = 6.7 Hz, 1H), 4.17−
4.04 (m, 3H), 3.74 (s, 1H), 3.70 (s, 3H), 3.50 (d, J = 2.4 Hz, 1H), 2.99
(ddd, J = 10.9, 6.3, 2.4 Hz, 1H), 2.56−2.37 (m, 4H), 2.30 (dt, J = 14.9,
1.8 Hz, 2H), 2.21−2.12 (m, 1H), 2.09 (dd, J = 12.4, 4.6 Hz, 1H), 1.91
(td, J = 12.4, 3.8 Hz, 1H), 1.75 (ddd, J = 14.1, 9.4, 5.7 Hz, 3H), 1.50 (dp,
J = 13.8, 7.0 Hz, 1H), 1.32 (d, J = 6.8 Hz, 3H), 1.29 (d, J = 6.4 Hz, 3H),
1.23 (d, J = 6.5 Hz, 3H), 1.08 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz,
CDCl3 + MeOD) δ 210.3, 192.7, 181.4, 171.4, 162.4, 162.0, 142.6,
137.4, 133.5, 133.0, 131.2, 124.8, 120.9, 120.2, 115.8, 114.7, 101.6,
100.9, 100.0, 81.9, 81.8, 71.9, 71.6, 70.9, 68.1, 67.5, 65.1, 57.1, 52.6,
46.6, 34.2, 34.2, 33.8, 33.5, 32.1, 27.6, 17.4, 17.0, 14.7, 6.6. HRMS: [M +
H]+ calcd for C40H50NO15 784.3181; found 784.3196.
Cell Culture. K562 cells (B. Pang, Stanford University), HCT116
cells (T. van Hall, LUMC, The Netherlands), and PC3 and DU145 cells
(C. Robson, Newcastle University, U.K.) were maintained in Roswell
Park Memorial Institute (RPMI)-1640 medium supplemented with 8%
fetal bovine serum (FCS). Wild-type MelJuSo cells were maintained in
IMDM (IMDM = Iscove’s Modified Dulbecco’s Medium) supple-
mented with 8% FCS. MelJuSo cells stably expressing PAGFP-H2A
were maintained in IMDM supplemented with 8% FCS and G-418, as
described.17 U87 cells (ATCC HTB-14) were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
8% FCS. Cell lines were maintained in a humidified atmosphere of 5%
CO2 at 37 °C and regularly tested for the absence of mycoplasma.
Western Blot and Constant-Field Gel Electrophoresis (CFGE).
Cells were treated with drugs at indicated doses for 2 h. These
concentrations and treatment times correspond to physiological serum
peak concentrations in cancer patients under standard treatment.17,50
Subsequently, drugs were removed by extensive washing and cells were
collected at indicated time points after drug removal and processed
immediately for the assay. Cells were lysed directly in sodium dodecyl
sulfate (SDS) sample buffer (2% SDS, 10% glycerol, 5% β-
mercaptoethanol, 60 mM Tris−HCl pH 6.8, and 0.01% bromophenol
7-[2,3-Dideoxy-4-ulo-α-L-fucopyranosyl-2-deoxy-3-O-p-methox-
ybenzyl-α-L-fucopyranosyl-(1 → 4)-3-N-allyloxycarbonyl-2,3-di-
deoxy-α-L-fucopyranoside]-aklavinone (32). Prepared according to
General Procedure C from donor 30 (211 mg, 0.276 mmol) and
aklavinone 1434 (2 equiv) at −20 °C to give after column
chromatography (10:90 EtOAc/pentane and then 2:98−20:80
acetone/toluene) the title compound as a yellow solid (210 mg,
1
0.213 mmol, 77%). H NMR (400 MHz, chloroform-d) δ 12.66 (s,
1H), 12.01 (s, 1H), 7.82 (dd, J = 7.5, 1.1 Hz, 1H), 7.72−7.61 (m, 2H),
7.34−7.21 (m, 2H), 6.93−6.82 (m, 2H), 6.07 (d, J = 7.8 Hz, 1H), 5.83
(ddt, J = 16.0, 10.8, 5.6 Hz, 1H), 5.46 (d, J = 3.8 Hz, 1H), 5.30−5.06 (m,
4H), 4.98 (s, 1H), 4.71−4.62 (m, 1H), 4.62−4.49 (m, 2H), 4.46 (ddt, J
= 6.9, 5.5, 1.5 Hz, 2H), 4.22 (s, 2H), 4.12 (s, 1H), 4.09−3.90 (m, 3H),
3.87 (d, J = 7.1 Hz, 1H), 3.82 (s, 3H), 3.70 (s, 3H), 3.55 (s, 1H), 2.66−
2.47 (m, 2H), 2.42 (ddd, J = 15.7, 7.6, 5.5 Hz, 1H), 2.36−2.25 (m, 2H),
2.25−2.04 (m, 3H), 2.00 (dd, J = 12.9, 4.5 Hz, 1H), 1.74 (dq, J = 13.5,
6.0, 4.3 Hz, 2H), 1.50 (dq, J = 14.6, 7.1 Hz, 1H), 1.30 (d, J = 6.6 Hz,
3H), 1.28−1.24 (m, 3H), 1.08 (t, J = 7.3 Hz, 3H), 0.98 (d, J = 6.7 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 211.1, 192.8, 181.4, 171.5, 162.6,
162.2, 159.3, 155.5, 142.7, 137.4, 133.5, 133.0, 133.0, 131.1, 130.3,
129.1, 124.8, 121.0, 120.3, 117.5, 115.8, 114.8, 113.9, 101.6, 101.5, 98.0,
80.9, 75.0, 72.5, 71.8, 71.4, 70.3, 68.5, 67.7, 65.5, 57.1, 55.4, 52.6, 46.5,
M
https://dx.doi.org/10.1021/acs.jmedchem.0c01191
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
blue). Lysates were resolved by SDS-polyacrylamide gel electrophoresis
(PAGE) followed by Western blotting. Primary antibodies used for
blotting were γH2AX (1:1000, 05-036, Millipore) and β-actin
(1:10000, A5441, Sigma). DNA double-strand breaks were visualized
by constant-field gel electrophoresis, as described.51 Images were
quantified with ImageJ.
Herman S. Overkleeft − Leiden Institute of Chemistry, Leiden
University, 2333 CC Leiden, The Netherlands; orcid.org/
0000-0001-6976-7005
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01191
Microscopy. PAGFP-H2A photoactivation and time-lapse confocal
imaging were performed as described17 on a Leica SP8 confocal
microscope system, 63× lens, equipped with a climate chamber. Loss of
fluorescence after different treatments was quantified using ImageJ
software. For TopoIIα live cell imaging, MelJuSo cells were transiently
transfected with a construct encoding TopoIIα-GFP.17 Fractional
distance calculations for the TopoIIα relocalization were done using
LAS X software (Leica).
Cell Viability Assay. Cells were seeded into 96-well plates. Twenty-
four hours after seeding, the cells were treated with indicated drugs for 2
h. Subsequently, drugs were removed and cells were left to grow for an
additional 72 h. Cell viability was measured using the CellTiter-Blue
viability assay (Promega). Relative survival was normalized to the
untreated control and corrected for background signal.
Author Contributions
D.P.A.W. and S.Y.v.d.Z. contributed equally to this work.
D.P.A.W., S.Y.v.d.Z., H.S.O., J.N., and J.D.C.C. conceived the
experiments. D.P.A.W. under supervision of H.S.O., G.A.v.d.M.,
and J.D.C.C. performed the synthesis. S.Y.v.d.Z. under super-
vision of JN performed all biochemical and cellular experiments.
The manuscript was written by D.P.A.W. and S.Y.v.d.Z. with
input of all authors.
Funding
This work was supported by grants from the Dutch Cancer
Society KWF (J.N.) and by the Institute for Chemical
Immunology, an NWO Gravitation project funded by the
Ministry of Education, Culture and Science of the Netherlands
to H.S.O. and J.N.
Flow Cytometry for Measuring Drug Uptake in Cells. Cells
were treated with 1 μM of the indicated drugs for 2 h. The samples were
washed, collected, and fixed with paraformaldehyde. The samples were
analyzed by flow cytometry using a BD FACS Aria II, with a 561 nm
laser and a 610/20 nm detector.
Notes
The authors declare the following competing financial
interest(s): J.N. is a shareholder in NIHM that aims to produce
aclarubicin for clinical use.
ASSOCIATED CONTENT
* Supporting Information
■
sı
ABBREVIATIONS
AML, acute myeloid leukemia; TopoIIα, topoisomerase IIα;
PAGFP-H2A, photoactivated GFP-H2A
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01191.
■
Detailed synthetic procedures of the monosaccharide
building blocks 13, 17, 18, 25, and 28; NMR and other
analytical data of all new compounds including com-
pounds 3−11; evaluation of DNA break capacity of
compounds 1−12; confocal microscopy of PAGFP-H2A-
tagged histones to visualize chromatin damage by
compounds 1−12; confocal microscopy of TopoIIα
relocalization by compounds 1−12; and figures on
correlation of anthracycline structural features versus
cytotoxicity of compounds 1−12 (PDF)
REFERENCES
■
(1) Arcamone, F.; Franceschi, G.; Penco, S.; Selva, A. Adriamycin (14-
Hydroxydaunomycin), a Novel Antitumor Antibiotic. Tetrahedron Lett.
1969, 10, 1007−1010.
̈
(2) Rothig, H. J.; Kraemer, H. P.; Sedlacek, H. H. Aclarubicin:
Experimental and Clinical Experience. Drugs Exp. Clin. Res. 1985, 11,
123−125.
(3) Weiss, R. B. The Anthracyclines: Will We Ever Find a Better
Doxorubicin? Semin. Oncol. 1992, 19, 670−686.
(4) Rizvi, S. F. A.; Tariq, S.; Mehdi, M. Anthracyclines: Mechanism of
Action, Classification, Pharmacokinetics and Future − A Mini Review.
Int. J. Biotechnol. Bioeng. 2018, 4, 81−85.
Molecular formula strings and tabulated biological assays
data (CSV)
(5) Rayson, D.; Richel, D.; Chia, S.; Jackisch, C.; van der Vegt, S.;
Suter, T. Anthracycline−Trastuzumab Regimens for HER2/Neu-
Overexpressing Breast Cancer: Current Experience and Future
Strategies. Ann. Oncol. 2008, 19, 1530−1539.
AUTHOR INFORMATION
Corresponding Authors
Jacques Neefjes − Department of Cell and Chemical Biology,
ONCODE Institute, Leiden University Medical Center, 2333 CZ
Leiden, The Netherlands; orcid.org/0000-0001-6763-2211;
Email: j.j.c.neefjes@lumc.nl
■
(6) Sadurska, E. Current Views on Anthracycline Cardiotoxicity in
Childhood Cancer Survivors. Pediatr. Cardiol. 2015, 36, 1112−1119.
(7) Lotrionte, M.; Biondi-Zoccai, G.; Abbate, A.; Lanzetta, G.;
D’Ascenzo, F.; Malavasi, V.; Peruzzi, M.; Frati, G.; Palazzoni, G. Review
and Meta-Analysis of Incidence and Clinical Predictors of Anthracy-
cline Cardiotoxicity. Am. J. Cardiol. 2013, 112, 1980−1984.
(8) Felix, C. A. Secondary Leukemias Induced by Topoisomerase-
Targeted Drugs. Biochim. Biophys. Acta, Gene Struct. Expression 1998,
1400, 233−255.
́
Jeroen D. C. Codee − Leiden Institute of Chemistry, Leiden
University, 2333 CC Leiden, The Netherlands; orcid.org/
0000-0003-3531-2138; Email: jcodee@chem.leidenuniv.nl
Authors
(9) Mistry, A. R.; Felix, C. A.; Whitmarsh, R. J.; Mason, A.; Reiter, A.;
Dennis P. A. Wander − Leiden Institute of Chemistry, Leiden
University, 2333 CC Leiden, The Netherlands; orcid.org/
0000-0003-3881-5240
Sabina Y. van der Zanden − Department of Cell and Chemical
Biology, ONCODE Institute, Leiden University Medical Center,
2333 CZ Leiden, The Netherlands; orcid.org/0000-0001-
5587-1514
Gijsbert A. van der Marel − Leiden Institute of Chemistry, Leiden
University, 2333 CC Leiden, The Netherlands; orcid.org/
0000-0001-9577-2123
̀
Cassinat, B.; Parry, A.; Walz, C.; Wiemels, J. L.; Segal, M. R.; Ades, L.;
Blair, I. A.; Osheroff, N.; Peniket, A. J.; Lafage-Pochitaloff, M.; Cross, N.
C. P.; Chomienne, C.; Solomon, E.; Fenaux, P.; Grimwade, D. DNA
Topoisomerase II in Therapy-Related Acute Promyelocytic Leukemia.
N. Engl. J. Med. 2005, 352, 1529−1538.
(10) Lefrak, E. A.; Pit’ha, J.; Rosenheim, S.; Gottlieb, J. A. A
Clinicopathologic Analysis of Adriamycin Cardiotoxicity. Cancer 1973,
32, 302−314.
(11) Jones, R. L.; Swanton, C.; Ewer, M. S. Anthracycline
Cardiotoxicity. Expert Opin. Drug Saf. 2006, 5, 791−809.
N
https://dx.doi.org/10.1021/acs.jmedchem.0c01191
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(12) Cappetta, D.; De Angelis, A.; Sapio, L.; Prezioso, L.; Illiano, M.;
Quaini, F.; Rossi, F.; Berrino, L.; Naviglio, S.; Urbanek, K. Oxidative
Stress and Cellular Response to Doxorubicin: A Common Factor in the
Complex Milieu of Anthracycline Cardiotoxicity. Oxid. Med. Cell.
Longevity 2017, 2017, 1−13.
(30) Lu, W.; Leimkuhler, C.; Oberthu
̈
r, M.; Kahne, D.; Walsh, C. T.
AknK Is An L-2-Deoxyfucosyltransferase in the Biosynthesis of the
Anthracycline Aclacinomycin A. Biochemistry 2004, 43, 4548−4558.
(31) Oki, T.; Kitamura, I.; Matsuzawa, Y.; Shibamoto, N.; Ogasawara,
T.; Yoshimoto, A.; Inui, T.; Naganawa, H.; Takeuchi, T.; Umezawa, H.
Antitumor Anthracycline Antibiotics, Aclacinomycin A and Analogues.
II. Structural Determination. J. Antibiot. 1979, 32, 801−819.
(32) Tanaka, H.; Yoshioka, T.; Shimauchi, Y.; Matsushita, Y.;
Matsuzawa, Y.; Oki, T.; Ishikura, T. Chemical Modification of
Anthracycline Antibiotics. IV. Synthesis of New Anthracyclines with
Trisaccharide. J. Antibiot. 1982, 35, 312−320.
(13) Songbo, M.; Lang, H.; Xinyong, C.; Bin, X.; Ping, Z.; Liang, S.
Oxidative Stress Injury in Doxorubicin-Induced Cardiotoxicity. Toxicol.
Lett. 2019, 307, 41−48.
(14) Hashimoto, K.; Ito, K.; Ishimori, Y. Novel DNA Sensor for
Electrochemical Gene Detection. Anal. Chim. Acta 1994, 286, 219−
224.
̀
(15) Qiao, X.; van der Zanden, S. Y.; Wander, D. P. A.; Borras, D. M.;
(33) Yu, B. Gold(I)-Catalyzed Glycosylation with Glycosyl o-
Alkynylbenzoates as Donors. Acc. Chem. Res. 2018, 51, 507−516.
(34) Smith, A.; Toward, A. General Method for the Construction of
Anthracycline Antibiotics. Ph.D. Thesis, Princeton University, 1998.
(35) Horton, D.; Priebe, W.; Valera, O. Synthesis and Antitumour
Activity of 3′-Deamino-3′-Hydroxydoxorubicin. J. Antibiot. 1984, 37,
853−857.
(36) Hansen, T.; Elferink, H.; van Hengst, J. M. A.; Houthuijs, K. J.;
Remmerswaal, W. A.; Kromm, A.; Berden, G.; van der Vorm, S.; Rijs, A.
M.; Overkleeft, H. S.; Filippov, D. V.; Rutjes, F. P. J. T.; van der Marel,
Song, J.-Y.; Li, X.; van Duikeren, S.; van Gils, N.; Rutten, A.; van
Herwaarden, T.; van Tellingen, O.; Giacomelli, E.; Bellin, M.; Orlova,
V.; Tertoolen, L. G. J.; Gerhardt, S.; Akkermans, J. J.; Bakker, J. M.;
Zuur, C. L.; Pang, B.; Smits, A. M.; Mummery, C. L.; Smit, L.; Arens, R.;
Li, J.; Overkleeft, H. S.; Neefjes, J. Uncoupling DNA Damage from
Chromatin Damage to Detoxify Doxorubicin. Proc. Natl. Acad. Sci.
U.S.A. 2020, 117, 15182−15192.
(16) Nitiss, J. L. Targeting DNA Topoisomerase II in Cancer
Chemotherapy. Nat. Rev. Cancer 2009, 9, 338−350.
(17) Pang, B.; Qiao, X.; Janssen, L.; Velds, A.; Groothuis, T.;
Kerkhoven, R.; Nieuwland, M.; Ovaa, H.; Rottenberg, S.; van Tellingen,
O.; Janssen, J.; Huijgens, P.; Zwart, W.; Neefjes, J. Drug-Induced
Histone Eviction from Open Chromatin Contributes to the Chemo-
therapeutic Effects of Doxorubicin. Nat. Commun. 2013, 4, No. 1908.
(18) Pang, B.; de Jong, J.; Qiao, X.; Wessels, L. F. A.; Neefjes, J.
Chemical Profiling of the Genome with Anti-Cancer Drugs Defines
Target Specificities. Nat. Chem. Biol. 2015, 11, 472−480.
́
G. A.; Martens, J.; Oomens, J.; Codee, J. D. C.; Boltje, T. J.
Characterization of Glycosyl Dioxolenium Ions and Their Role in
Glycosylation Reactions. Nat. Commun. 2020, 11, No. 2664.
(37) Komarova, B. S.; Tsvetkov, Y. E.; Nifantiev, N. E. Design of α-
Selective Glycopyranosyl Donors Relying on Remote Anchimeric
Assistance. Chem. Rec. 2016, 16, 488−506.
(38) Hansen, T.; Lebedel, L.; Remmerswaal, W. A.; van der Vorm, S.;
Wander, D. P. A.; Somers, M.; Overkleeft, H. S.; Filippov, D. V.; Desire,
J.; Mingot, A.; Bleriot, Y.; van der Marel, G. A.; Thibaudeau, S.; Codee,
́
́
(19) Yang, F.; Kemp, C. J.; Henikoff, S. Doxorubicin Enhances
Nucleosome Turnover around Promoters. Curr. Biol. 2013, 23, 782−
787.
́
J. D. C. Defining the SN1 Side of Glycosylation Reactions:
Stereoselectivity of Glycopyranosyl Cations. ACS Cent. Sci. 2019, 5,
781−788.
(20) Iihoshi, H.; Ishihara, T.; Kuroda, S.; Ishihara, N.; Saitoh, H.
Aclarubicin, an Anthracycline Anti-Cancer Drug, Fluorescently
Contrasts Mitochondria and Reduces the Oxygen Consumption Rate
in Living Human Cells. Toxicol. Lett. 2017, 277, 109−114.
(21) Wijdeven, R. H.; Pang, B.; van der Zanden, S. Y.; Qiao, X.;
Blomen, V.; Hoogstraat, M.; Lips, E. H.; Janssen, L.; Wessels, L.;
Brummelkamp, T. R.; Neefjes, J. Genome-Wide Identification and
Characterization of Novel Factors Conferring Resistance to Topo-
isomerase II Poisons in Cancer. Cancer Res. 2015, 75, 4176−4187.
(22) Gottesman, M. M.; Ling, V. The Molecular Basis of Multidrug
Resistance in Cancer: The Early Years of P-Glycoprotein Research.
FEBS Lett. 2006, 580, 998−1009.
(23) Mechetner, E.; Kyshtoobayeva, A.; Zonis, S.; Kim, H.; Stroup, R.;
Garcia, R.; Parker, R. J.; Fruehauf, J. P. Levels of Multidrug Resistance
(MDR1) P-Glycoprotein Expression by Human Breast Cancer
Correlate with in Vitro Resistance to Taxol and Doxorubicin. Clin.
Cancer Res. 1998, 4, 389−398.
(24) Cox, J.; Weinman, S. Mechanisms of Doxorubicin Resistance in
Hepatocellular Carcinoma. Hepatic Oncol. 2016, 3, 57−59.
(25) Pajic, M.; Iyer, J. K.; Kersbergen, A.; Van Der Burg, E.; Nygren, A.
O. H.; Jonkers, J.; Borst, P.; Rottenberg, S. Moderate Increase in
Mdr1a/1b Expression Causes in Vivo Resistance to Doxorubicin in a
Mouse Model for Hereditary Breast Cancer. Cancer Res. 2009, 69,
6396−6404.
(39) Pearlman, B. A.; McNamara, J. M.; Hasan, I.; Hatakeyama, S.;
Sekizaki, H.; Kishi, Y. Practical Total Synthesis of ( )-Aklavinone and
Total Synthesis of Aklavin. J. Am. Chem. Soc. 1981, 103, 4248−4251.
́
(40) Garro-Helion, F.; Merzouk, A.; Guibe, F. Mild and Selective
Palladium(0)-Catalyzed Deallylation of Allylic Amines. Allylamine and
Diallylamine as Very Convenient Ammonia Equivalents for the
Synthesis of Primary Amines. J. Org. Chem. 1993, 58, 6109−6113.
(41) Johdo, O.; Nishida, H.; Okamoto, R.; Yoshimoto, A.; Takeuchi,
T. New Anthracycline Antibiotics 10-Epi-Oxaunomycin and 10-Epi-11-
Deoxyoxaunomycin. J. Antibiot. 1995, 48, 1153−1158.
(42) Veeneman, G. H.; Van Leeuwen, S. H.; Zuurmond, H.; Van
Boom, J. H. Synthesis of Carbohydrate-Antigenic Structures of
Mycobacterium tuberculosis Using an Iodonium Ion Promoted
Glycosidation Approach. J. Carbohydr. Chem. 1990, 9, 783−796.
(43) Cipollone, A.; Berettoni, M.; Bigioni, M.; Binaschi, M.; Cermele,
C.; Monteagudo, E.; Olivieri, L.; Palomba, D.; Animati, F.; Goso, C.;
Maggi, C. Novel Anthracycline Oligosaccharides: Influence of
Chemical Modifications of the Carbohydrate Moiety on Biological
Activity. Bioorg. Med. Chem. 2002, 10, 1459−1470.
(44) Craine, L.; Raban, M. The Chemistry of Sulfenamides. Chem.
Rev. 1989, 89, 689−712.
(45) Noshita, T.; Sugiyama, T.; Kitazumi, Y.; Oritani, T. Phenolic
Ferrier Reaction and Its Application to the Natural Product Synthesis.
Tetrahedron Lett. 1994, 35, 8259−8262.
(26) Krohn, K., Ed. Anthracycline Chemistry and Biology II; Topics in
Current Chemistry; Springer: Berlin, Heidelberg, 2008; Vol. 283.
(27) Yoshimoto, A.; Johdo, O.; Nishida, H.; Okamoto, R.; Takeuchi,
T. Anthracycline Metabolites from Baumycin-Producing Streptomyces
Sp. D788. III. New Anthracycline Metabolites Produced by Blocked
Mutants 4L-660 and YDK-18. J. Antibiot. 1993, 46, 1758−1761.
(28) Tong, G. L.; Wu, H. Y.; Smith, T. H.; Henry, D. W. Adriamycin
Analogs. 3. Synthesis of N-Alkylated Anthracyclines with Enhanced
Efficacy and Reduced Cardiotoxicity. J. Med. Chem. 1979, 22, 912−918.
(46) Zhang, X.; Zhou, Y.; Zuo, J.; Yu, B. Total Synthesis of Periploside
A, a Unique Pregnane Hexasaccharide with Potent Immunosuppressive
Effects. Nat. Commun. 2015, 6, No. 5879.
(47) Ma, Y.; Li, Z.; Shi, H.; Zhang, J.; Yu, B. Assembly of Digitoxin by
Gold(I)-Catalyzed Glycosidation of Glycosyl o-Alkynylbenzoates. J.
Org. Chem. 2011, 76, 9748−9756.
(48) Soubeyrand, S.; Pope, L.; Hache, R. J. G. Topoisomerase IIα-
Dependent Induction of a Persistent DNA Damage Response in
́
̈
̈
̈
(29) Kunnari, T.; Niemi, J.; Ylihonko, K.; Mantsala, P.; Hakala, J.
Hybrid Anthracyclines by a Genetically Engineered Streptomyces
Galilaeus Mutant. Bioorg. Med. Chem. Lett. 1997, 7, 725−726.
Response to Transient Etoposide Exposure. Mol. Oncol. 2010, 4, 38−
51.
O
https://dx.doi.org/10.1021/acs.jmedchem.0c01191
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(49) van Maanen, J. M. S.; Retel, J.; de Vries, J.; Pinedo, H. M.
Mechanism of Action of Antitumor Drug Etoposide: A Review. JNCI, J.
Natl. Cancer Inst. 1988, 80, 1526−1533.
(50) Speth, P. A. J.; van Hoesel, Q. G. C. M.; Haanen, C. Clinical
Pharmacokinetics of Doxorubicin. Clin. Pharmacokinet. 1988, 15, 15−
31.
́
(51) Olive, P. L.; Wlodek, D.; Banath, J. P. DNA Double-Strand
Breaks Measured in Individual Cells Subjected to Gel Electrophoresis.
Cancer Res. 1991, 51, 4671−4676.
(52) Dordal, M. S.; Ho, A. C.; Jackson-Stone, M.; Fu, Y. F.; Goolsby,
C. L.; Winter, J. N. Flow Cytometric Assessment of the Cellular
Pharmacokinetics of Fluorescent Drugs. Cytometry 1995, 20, 307−314.
(53) Capranico, G.; De Isabella, P.; Penco, S.; Tinelli, S.; Zunino, F.
Role of DNA Breakage in Cytotoxicity of Doxorubicin, 9-Deoxydox-
orubicin, and 4-Demethyl-6-Deoxydoxorubicin in Murine Leukemia
P388 Cells. Cancer Res. 1989, 49, 2022−2027.
(54) Gate, L.; Couvreur, P.; Nguyen-Ba, G.; Tapiero, H. N-
Methylation of Anthracyclines Modulates Their Cytotoxicity and
Pharmacokinetic in Wild Type and Multidrug Resistant Cells. Biomed.
Pharmacother. 2003, 57, 301−308.
(55) Gianni, L. Anthracycline Resistance: The Problem and Its
Current Definition. Semin. Oncol. 1997, 24, S10−S17.
(56) Mansour, O. C.; Evison, B. J.; Sleebs, B. E.; Watson, K. G.;
Nudelman, A.; Rephaeli, A.; Buck, D. P.; Collins, J. G.; Bilardi, R. A.;
Phillips, D. R.; Cutts, S. M. New Anthracenedione Derivatives with
Improved Biological Activity by Virtue of Stable Drug-DNA Adduct
Formation. J. Med. Chem. 2010, 53, 6851−6866.
(57) Liaw, Y. C.; Gao, Y. G.; Robinson, H.; Van der Marel, G. A.; Van
Boom, J. H.; Wang, A. H. J. Antitumor Drug Nogalamycin Binds DNA
in Both Grooves Simultaneously: Molecular Structure of Nogalamycin-
DNA Complex. Biochemistry 1989, 28, 9913−9918.
(58) Frederick, C. A.; Williams, L. D.; Ughetto, G.; van der Marel, G.
A.; Van Boom, J. H.; Rich, A.; Wang, A. H. J. Structural Comparison of
Anticancer Drug-DNA Complexes: Adriamycin and Daunomycin.
Biochemistry 1990, 29, 2538−2549.
(59) Zeman, S. M.; Phillips, D. R.; Crothers, D. M. Characterization of
Covalent Adriamycin-DNA Adducts. Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 11561−11565.
(60) Kloc, K.; Mlochowski, J.; Syper, L. Synthesis of Novel Quinones
with Silver (II) Dipicolinate as a New Selective Oxidant. Chem. Lett.
1980, 9, 725−728.
P
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J. Med. Chem. XXXX, XXX, XXX−XXX