Exploring the Effects of Glycosylation and Etherification of the Side Chains of the Anticancer Drug Mitoxantrone

Article abstract of DOI:10.1002/cmdc.201500274

Herein we report the synthesis and biological evaluation of symmetric and asymmetric analogues of the DNA intercalating drug mitoxantrone (MTX) in which the side chains of the parent drug were modified through glycosylation or methyl etherification. Several analogues with glycosylated side chains exhibited higher DNA affinity than the parent MTX. The most potent in vitro cytotoxicity was observed for MTX analogue 8 (1,4-dimethoxy-5,8-bis[2-(2-methoxyethylamino)ethylamino]anthracene-9,10-dione) with methoxy ether containing side chains. Treatment of melanoma-bearing mice with MTX or analogue 8 decreased the intraperitoneal tumor burden relative to untreated mice; the effect of 8 was less pronounced than that of MTX. In vitro metabolism assays of MTX with rabbit liver S9 fraction gave rise to several metabolites; almost no metabolites were detected for MTX analogue 8. The results presented indicate that derivatization of the MTX side chain primary hydroxy groups may result in a significant improvement in DNA affinity and lower susceptibility to the formation of potentially toxic metabolites.

Full text of DOI:10.1002/cmdc.201500274

DOI: 10.1002/cmdc.201500274
Full Papers
Exploring the Effects of Glycosylation and Etherification of
the Side Chains of the Anticancer Drug Mitoxantrone
Pazit Shaul,[a] Kfir B. Steinbuch,[a] Eran Blacher,[b] Reuven Stein,*[b] and Micha Fridman*[a]
Herein we report the synthesis and biological evaluation of
symmetric and asymmetric analogues of the DNA intercalating
drug mitoxantrone (MTX) in which the side chains of the
parent drug were modified through glycosylation or methyl
etherification. Several analogues with glycosylated side chains
exhibited higher DNA affinity than the parent MTX. The most
potent in vitro cytotoxicity was observed for MTX analogue 8
(1,4-dimethoxy-5,8-bis[2-(2-methoxyethylamino)ethylamino]an-
thracene-9,10-dione) with methoxy ether containing side
chains. Treatment of melanoma-bearing mice with MTX or ana-
logue 8 decreased the intraperitoneal tumor burden relative to
untreated mice; the effect of 8 was less pronounced than that
of MTX. In vitro metabolism assays of MTX with rabbit liver S9
fraction gave rise to several metabolites; almost no metabo-
lites were detected for MTX analogue 8. The results presented
indicate that derivatization of the MTX side chain primary hy-
droxy groups may result in a significant improvement in DNA
affinity and lower susceptibility to the formation of potentially
toxic metabolites.
Introduction
Developed in the late 1970s, the synthetic anthracenedione
mitoxantrone (MTX, Figure 1A) is a chemotherapeutic drug
used for the treatment of several cancers, including prostate
tumors that have not responded to hormone treatment, sever-
al types of breast tumors, acute myelogenous leukemia, and
non-Hodgkin’s lymphoma.[1,2] MTX is closely related to the an-
thracycline family of chemotherapeutic agents, which also in-
cludes doxorubicin (Figure 1A). MTX inhibits DNA topoisomer-
ase II by stabilizing a ternary complex between the enzyme
and double-stranded DNA, leading to the inhibition of cell divi-
sion.[3] Recent evidence suggests that MTX may also act by in-
hibiting PIM1 kinase activity,[4] inducing the formation of free
radical oxygen species, and affecting iron metabolism.[5]
in urine, bile or liver of mammals, including humans, that were
treated with the drug, confirming their formation in vivo.[8,12–14]
Thioether-tethered glutathione (GSH)-derived metabolites
formed by nucleophilic attack by the GSH thiol group on the
aromatic system of MTX resulted in compounds 1a and 1b
(Figure 1B); these metabolites were detected after incubation
of the drug with horseradish peroxidase and GSH or with cul-
tures of rat or human hepatocytes.[15] GSH thioether metabo-
lites were detected in the bile of MTX-treated minipigs, there-
by demonstrating that this metabolic pathway occurs in vivo
as well.[16] LC–MS analysis of samples of MTX that were pre-in-
cubated with rat liver S9 fraction revealed the formation of an
acetoxy metabolite of the drug (general structure 1c); the
exact structure of this metabolite is not yet known.[17] Horse-
radish peroxidase was found to catalyze the intramolecular oxi-
dative cyclization of one or both of the side chains of MTX
(Figure 1B, metabolites 1d and 1e).[11] These cyclization me-
tabolites are formed by intramolecular Michael addition of the
secondary amine of one or both of the side chains of the
parent drug. Metabolite 1d was detected in urine samples ob-
tained from MTX-treated human cancer patients.[18] Finally, glu-
curonidation of one of the phenol groups of MTX or oxidation
of the primary hydroxy group of one or both side chains of
MTX to the corresponding mono- or dicarboxylic acids results
in 1 f, 1g, 1h (Figure 1B), which were characterized by HPLC,
LC–MS, and NMR methods. Mono- or dicarboxylic acid–MTX
conjugates were isolated from urine samples of MTX-treated
patients, and a glucoronic acid–MTX conjugate was isolated
from urine samples of minipigs.[8,13,19]
The most severe side effect that accompanies treatment
with MTX is cardiomyopathy. This side effect occurs in approxi-
mately one fifth of patients treated with the drug, limits its life-
time maximum dose, and prevents its use in patients who also
suffer from cardiovascular diseases.[6,7] The profile of MTX me-
tabolites varies in different mammals,[8] and oxidoreductases
such as CYP2E1, CYP450, and horseradish peroxidase[9–11]
modify this anthracenedione. Metabolites of MTX were identi-
fied after incubation of the drug with liver cell cultures or liver
tissue extracts.[12,13] Some of these metabolites were detected
[a] P. Shaul, K. B. Steinbuch, Dr. M. Fridman
School of Chemistry, Beverly and Raymond Sackler Faculty of Exact
Sciences, Tel Aviv University, Tel Aviv 6997801 (Israel)
E-mail: mfridman@post.tau.ac.il
[b] E. Blacher, Prof. R. Stein
Department of Neurobiology, George S. Wise Faculty of Life Sciences,
Tel Aviv University, Tel Aviv 6997801 (Israel)
E-mail: reuvens@tauex.tau.ac.il
In humans, the main metabolites of MTX that were identi-
fied are the mono- and dicarboxylic acid derivatives 1g and
1h, respectively. Compounds 1g and 1h were also identified
in primary cultures of hepatocytes isolated from rabbits that
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500274.
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this drug.[17] Moreover, MTX cytotoxicity against H9c2
cells was decreased in the presence of CYP450 and
CYP2E1 inhibitors, further supporting the hypothesis
that MTX metabolites may be involved in both its an-
titumor activity and in undesired side effects caused
by this drug.[17]
In this study, we synthesized a collection of sym-
metric and asymmetric MTX analogues in which the
primary hydroxy group of one or both side chains
was modified by O-glycosylation or methyl etherifica-
tion. We studied the impact of these modifications
on target DNA affinity, cytotoxic activity, and metabo-
lism and evaluated the potential of these chemical
modifications to lead to the development of novel
MTX-derived anticancer drugs.
Results and Discussion
Design and synthesis of MTX analogues
The syntheses of numerous MTX analogues have
been reported over the last decades. These ana-
logues differ from the parent MTX in the substitution
pattern around the anthracenedione segment of the
drug or in the side chains.[20–23] Amongst the reported
MTX analogues were symmetrical compounds with
two identical side chains, and asymmetric com-
pounds with two different side chains. Recently, an
engineered glycosyltransferase with relaxed substrate
specificity (OleD SP) was used for the preparation of
an MTX derivative in which the primary hydroxy
group of only one of the side chains was b-glucosy-
lated.[24]
We prepared MTX analogues in which the primary
hydroxy group of the side chain was either O-glyco-
sylated or converted into the corresponding methyl
ether. For the glycosylated side chain analogues we
focused on three sugar scaffolds: the aldohexose d-
glucose, the ketohexose d-fructose, and the less hin-
dered aldopentose d-ribose. These sugars were
chosen to allow us to determine if and how the size
and stereochemistry of the sugar group influences
the biological activity of the resultant MTX ana-
logues. The synthetic routes are shown in Scheme 1.
Figure 1. Chemical structures of A) MTX and the anthracycline doxorubicin, and B) MTX
metabolites.
were treated with the drug.[12] Carboxylic acid metabolites 1g
and 1h have been chemically synthesized, and their cytotoxic
activity has been evaluated in cells susceptible to the parent
MTX.[19] Neither of the carboxylic acid metabolites had signifi-
cant cytotoxic activity against MTX-sensitive cancer cells, sug-
gesting that this metabolic pathway serves to detoxify MTX.
On the other hand, there is evidence that the formation of
some MTX metabolites may lead to enhanced cytotoxicity
against cardiomyoblasts. A mixture of metabolized MTX in
which metabolites 1a–1e were identified by LC–MS displayed
higher cytotoxic activity against H9c2 cardiomyoblast cells
than did unmetabolized MTX, suggesting that MTX metabo-
lism may also be involved in the undesired cardiotoxicity of
The amine groups of 2-(2-hydroxyethylamino)ethylamine, the
precursor for the side chains of MTX, were converted into the
corresponding benzyl carbamates to yield N3,N5-bis(benzyloxy-
carbonyl)-5-amino-3-azapentan-1-ol following a previously re-
ported protocol.[25] Lewis acid catalyzed glycosylation of the
Cbz-protected side chain with 1,2,3,4,6-penta-O-acetyl-b-d-glu-
copyranose or with 1,2,3,5-tetra-O-acetyl-b-d-ribofuranose gave
compounds 2a and 3a, respectively. Deacetylation of 2a and
3a resulted in 2b and 3b, which were subjected to palladium-
catalyzed catalytic hydrogenation of the Cbz groups to yield
compounds 2c and 3c, respectively (Scheme 1A). The fructo-
pyranose-substituted side chain 4d was prepared by glycosyla-
tion of the Cbz-protected side chain with the previously re-
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Scheme 1. Synthesis of glycosylated and methyl ether MTX analogues. Reagents and conditions: a) sugar acetate, BF3OEt2, CH2Cl2, 4 Š MS, RT, overnight;
b) NaOMe (0.5m in MeOH), MeOH, RT, 3 h; c) H2, Pd/C, MeOH, RT, overnight; d) N3,N5-bis(benzyloxycarbonyl)-5-amino-3-azapentan-1-ol, TMSOTf, CH2Cl2, 4 Š
MS, À208C, 1 h, then RT, overnight; e) 2c, 3c, or 4d or N-(2-methoxyethyl)-1,2-diaminoethane, DMSO, 408C (or 358C for 8), overnight; f) 2c, 3c, or 4d
(3 equiv), pyridine, 408C, overnight; g) N-(2-hydroxyethyl)ethylenediamine, DMSO, RT, overnight.
ported N-phenyltrifluoroacetimidate fructopyranose glycosyl
donor 4a[26] to yield compound 4b. Removal of the benzoyl
esters followed by catalytic hydrogenation of the Cbz groups
of 4b gave compounds 4c and 4d, respectively (Scheme 1B).
Finally, N-(2-methoxyethyl)-1,2-diaminoethane[27] was used as
a precursor for the preparation of the MTX analogue with O-
methyl ether side chains. This compound was prepared to
compare the effect of the hindered sugar acetals with a signifi-
cantly smaller methyl ether group. The glycosylated side chain
derivatives 2c, 3c, or 4d and N-(2-methoxyethyl)-1,2-diamino-
ethane were reacted with the 1,4-difluoroanthracene-9,10-
dione prepared via a Friedel–Crafts bis-cycloacylation as previ-
ously reported.[20]
metric disubstituted anthracenediones 5–7 were obtained as
the minor products when 3 equivalents of the O-glycosylated
side chains were used and the reaction was performed over-
night in pyridine at 408C. Less than 3 equivalents resulted in
decreased yields and a significant amount of the unreacted
1,4-difluoroanthracene-9,10-dione starting material. Overnight
reaction of the monosubstituted fluoroanthracenediones 9a,
10a, and 11 a with 1.2–1.5 equivalents of N-(2-hydroxyethyl)e-
thylenediamine at room temperature in DMSO gave the
hetero-disubstituted MTX analogues 9–11 (Scheme 1C). The
parent drug MTX was prepared by reaction of 1,4-difluoro-5,8-
dihydroxyanthracene-9,10-dione with 3 equivalents of N-(2-hy-
droxyethyl)ethylenediamine in pyridine, as previously reported,
in 38% isolated yield.[22]
The symmetric disubstituted anthracenediones 5–8 were ob-
tained as the major product when 4 equivalents of the side
chain were reacted overnight with 1,4-difluoro-5,8-dihydrox-
yanthracene-9,10-dione in DMSO at 35–408C (Scheme 1C). The
use of more equivalents of the side chain or elevated tempera-
tures resulted in undesired Michael-type intramolecular cycliza-
tion.[22] The monosubstituted fluoroanthracenediones 9a, 10a,
and 11 a were obtained as the major products, and the sym-
Effect of side chain OH group substitution on DNA affinity
Previous structural information obtained from crystals of the
complex composed of topoisomerase II, double-stranded DNA,
and MTX provided evidence for the crucial role of the interac-
tions between MTX and DNA in facilitating its mode of
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action.[28] We therefore first evaluated the DNA affinity of the
collection of MTX analogues. DNA affinity was evaluated using
a fluorescent DNA intercalator displacement assay.[29] The con-
centration of MTX analogue at which half (IC50) of the minor
groove binding fluorescent dye SYBRꢁ safe (SYBR) was dis-
placed from the DNA was determined for each analogue; the
results are summarized in Table 1.
Effect of side chain OH group substitution on in vitro
cytotoxicity
The effect of the MTX analogues on cell viability was evaluated
by MTT assay with four human cancer cell lines: human hepa-
toma-derived cells (HepG2), human lung carcinoma-derived
cells (A549), human breast carcinoma-derived cells (MCF7), and
colorectal adenocarcinoma-derived cells (HT-29). EC50 values
were determined using the Hill equation, and the results are
summarized in Table 2.
Table 1. Displacement of a fluorescent DNA intercalator by MTX ana-
logues.
In all four cell lines, the parent MTX and its dimethoxy ana-
logue 8 had the most potent cytotoxic effects (Ymax ꢀ90%). All
of the MTX analogues with one glycosylated side chain (com-
pounds 9–11) were more potent than the corresponding digly-
cosylated analogues (compounds 5–7). Of the three tested
sugar scaffolds, the most cytotoxic compounds were those in
which the primary hydroxy group of one or both side chains
was O-glycosylated with a b-d-glucopyranose (compounds 9
and 5, respectively). The compounds that contained the small-
est glycoside scaffold, mono- and di-b-d-ribofuranose ana-
logues 10 and 6, respectively, were the least cytotoxic. Hence,
no correlation could be made between the size of the sugar
scaffold and the cytotoxicity of the glycosylated MTX ana-
logues.
Compound
IC50 [mm][a]
MTX
5
6
7
8
9
10
11
1.83Æ0.15
1.06Æ0.09
1.45Æ0.11
0.83Æ0.04
2.66Æ0.39
0.77Æ0.01
0.57Æ0.09
1.68Æ0.13
[a] Experiments were performed in 96-well plates at 378C; each well con-
tained 500 ng of phage l DNA (48 kb), SYBR at final dilution of 1:10000,
and test compound.
Relative to MTX, both the monoglucopyranosyl analogue 9
and the monoribofuranosyl analogue 10 displayed higher DNA
affinity; these two analogues displaced SYBR at significantly
lower IC50 concentrations (~2.4-fold and ~3.2-fold, respectively)
than MTX. Of all MTX analogues tested, the dimethoxy ana-
logue 8 demonstrated the weakest DNA affinity. The results of
the fluorescent DNA intercalator displacement assay indicated
that the glycosylation of MTX side chain primary hydroxy
groups offers a useful strategy to likely enhance the DNA affin-
ity of the resultant MTX analogue.
The results of the DNA intercalator displacement experi-
ments (Table 1) did not correlate with the cytotoxic potency of
the MTX analogues (Table 2). For example, MTX and the me-
thoxy ether side chain analogue 8 were the most cytotoxic of
the compounds tested, but had lower affinity for DNA than
the glycosylated side chain analogues. The mono-b-d-ribofura-
nose MTX analogue 10, which had the highest DNA affinity
(IC50 >3-fold lower than that of MTX, Table 1), demonstrated
Table 2. In vitro cytotoxic activity of MTX analogues.[a,b]
Compound
EC50 [mm]
HepG2 cells
nH
Ymax [%]
Compound
EC50 [mm]
A549 cells
nH
Ymax [%]
MTX
5
6
7
8
9
10
11
0.10Æ0.01
0.15Æ0.03
ND
0.86Æ0.15
0.28Æ0.09
0.11Æ0.02
0.98Æ0.38
0.29Æ0.09
0.74Æ0.08
1.27Æ0.32
ND
1.10Æ0.19
0.52Æ0.09
0.73Æ0.10
0.75Æ0.20
0.89Æ0.22
100Æ3
70Æ4
ND
80Æ5
100Æ7
90Æ3
89Æ9
86Æ7
MTX
5
6
7
8
9
10
11
0.18Æ0.09
0.06Æ0.01
3.00Æ0.90
0.65Æ0.08
0.59Æ0.16
0.05Æ0.01
0.33Æ0.09
0.53Æ0.11
0.45Æ0.09
0.93Æ0.14
1.34Æ0.39
1.77Æ0.45
0.60Æ0.09
0.48Æ0.08
1.09Æ0.26
0.75Æ0.13
98Æ9
58Æ2
47Æ4
57Æ2
100Æ6
68Æ3
63Æ5
89Æ5
MCF7 cells
HT-29 cells
MTX
5
6
0.03Æ0.00
0.12Æ0.01
ND
2.23Æ0.38
2.78Æ0.54
ND
89Æ2
86Æ3
ND
MTX
5
6
1.62Æ0.46
0.52Æ0.11
ND
0.76Æ0.13
2.59Æ1.31
ND
95Æ7
30Æ3
ND
7
8
9
10
11
0.86Æ0.09
0.15Æ0.03
0.06Æ0.00
0.45Æ0.04
0.38Æ0.05
4.55Æ2.01
1.13Æ0.24
1.44Æ0.10
3.54Æ0.94
3.77Æ1.16
77Æ7
92Æ6
88Æ1
76Æ4
83Æ6
7
8
9
10
11
ND
ND
ND
3.92Æ1.11
0.66Æ0.23
ND
0.97Æ0.18
1.15Æ0.51
ND
93Æ8
55Æ6
ND
ND
ND
ND
[a] Cells (6”103 per well) were plated in 96-well microtiter plates. After 24 h, the tested compounds (concentrations up to 50 mm) were added. After 72 h,
cell viability was determined by MTT assay. The percentage of cell death, along with Ymax values, were determined as a decrease in MTT values relative to
values from vehicle-treated cells. Experiments were performed in triplicate, and the results were obtained from three independent experiments. [b] ND:
not determined, as EC50 was >50 mm. EC50: concentration that gives half-maximal response; Ymax: percentage of the maximal response; nH: Hill coefficient
describes the slope of the response.
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poor cytotoxicity. The inconsistency between cytotoxicity and
DNA affinity further implies that, like the parent MTX,[22,30] the
cytotoxic effect of the examined MTX analogues is likely to
result from perturbation of several cellular activities or from
possible reduced cell permeability of these synthetic ana-
logues.
Antitumor activity of MTX and analogue 8 in a mouse
melanoma model
To assess the effect of MTX analogue 8 in vivo, we used
a mouse model of melanoma that was previously used to
demonstrate that MTX inhibits melanoma progression.[31]
B16F10 melanoma cells that express the fluorescent marker
mCherry[32,33] were injected intraperitoneally (i.p.) into mice
treated with vehicle, MTX, or MTX analogue 8. Tumor progres-
sion was monitored 27 days after injection of tumor cells. We
first verified that these cells were sensitive to MTX and com-
pound 8 in vitro using the MTT assay. Both MTX and com-
pound 8 were toxic to mCherry-B16F10 cells, with respective
EC50 values of 0.137 and 0.199 mm (Ymax ~100%).
Next, the effect of MTX and analogue 8 on melanoma pro-
gression in vivo was examined. The experiment was conducted
on three groups of C57BL/6J mice, each injected with mCher-
ry-B16F10 cells and treated with 1) vehicle (control), 2) MTX,
and 3) compound 8. The drugs (3.2 mgkgÀ1) were given by i.p.
injection 1, 5, and 9 days following tumor cells injection. The
dose of 3.2 mgkgÀ1 was chosen, as this dose of MTX was previ-
ously demonstrated to yield an optimal survival effect in the
B16 in vivo melanoma progression model.[34] The experiment
was terminated after 27 days, the internal organs were re-
moved, and mCherry expression in the abdominal cavity was
imaged and quantified (Figure 2). Multispectral image cubes
were acquired through the l 550–800 nm spectral range in
10 nm steps using excitation (575–605 nm) and emission
(645 nm long-pass) filter set. Vehicle-treated mice (control) had
large tumor masses in abdominal cavities (Figure 2A–C). In
contrast, a significant decrease in tumor mass was observed in
compound-8-treated mice (Figure 2D–F) and in MTX-treated
mice (Figure 2G–I). However, quantification of the fluorescent
signal in these samples using a Cri Maestro fluorescence imag-
ing system (Figure 2 J) revealed that in MTX-treated mice there
was a decrease of 92% in tumor mass relative to the vehicle-
treated group, whereas mice treated with MTX analogue 8 had
44% less tumor mass (p=0.05, Student t test).
Figure 2. mCherry-B16F10 melanoma cells (5”104 per 100 mL) were injected
i.p. into 8-week-old C57BL/6J female mice. A–C) Vehicle (saline), D–F) MTX
analogue 8, or G–I) MTX (3.2 mgkgÀ1, equivalent for 0.4 mgmLÀ1) were ad-
ministered i.p. 1, 5, and 9 days after implantation of tumor cells. After 27
days, mice were euthanized with CO2, the abdominal cavities were dissected,
and the fluorescence signals of mCherry-expressing tumors were visualized
(A–I) and quantified (J) using a Cri MaestroTM in vivo fluorescence imaging
system.
The observed differences between the in vitro and in vivo
activities of MTX analogue 8 may be explained by the differen-
ces between the pharmacokinetic properties of this compound
and of the parent MTX. Analysis of urine samples collected two
hours after each drug administration revealed that the urine of
MTX-treated mice was colored with the characteristic blue
shade of this drug; no such color change was observed in
urine samples collected from mice treated with analogue 8. In
LC–ESIMS analysis of the urine samples, a compound with
a molecular weight of 445.2, which corresponds to [M+H]+ of
MTX, was detected in the urine from MTX-treated mice. In con-
trast, no traces of analogue 8 were detected in the urine sam-
ples that were collected from mice treated with compound 8,
indicating that this MTX analogue differs from the parent drug
in body distribution and clearance.
Effect of side chain OH group substitution on rabbit liver
S9-fraction-induced metabolism
Previous studies have demonstrated that the liver is a major
organ in which metabolism of MTX takes place.[35] Moreover,
the metabolic profiles of MTX in primary cultures of human
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and rabbit hepatocytes were similar.[8] We therefore evaluated
the metabolism of compound 8 and MTX in the rabbit liver S9
fraction. The S9 fraction was supplemented with two cofac-
tors: GSH and NADPH (4 and 1 mm, respectively). As controls,
MTX and compound 8 were incubated with the S9 fraction in
the absence of exogenous GSH and NADPH or with a buffer
solution containing GSH and NADPH without the S9 fraction.
Aliquots of reactions were subjected to analytical RP-HPLC.
Neither MTX nor its analogue 8 were affected by any of the
control conditions.
A metabolite with an identical molecular weight was previ-
ously detected amongst the metabolites that were formed
when MTX was incubated in rat liver S9 fraction.[17] A previous
study demonstrated that carboxylic acid metabolites 1g and
1h were formed by incubating primary rabbit hepatocytes cul-
tures with MTX;[12] these metabolites were not detected when
MTX was incubated with the rabbit liver S9 fraction. None of
the molecular weights measured for the less polar fractions
matched the molecular weight of any previously reported MTX
metabolite.
Comparison of the analytical RP-HPLC chromatograms of
pure MTX, pure compound 8, and samples of MTX or analogue
8 that were incubated for 4 h in rabbit S9 fraction containing
the two co-factors revealed that metabolites that are less polar
than those of the parent drug were formed (Figure 3).
In contrast to the parent MTX, incubation of MTX analogue
8 with rabbit liver S9 fraction supplemented with the two co-
factors resulted in very low (nearly undetectable) amount of
metabolites (Figure 3D); integration of its HPLC spectrum re-
vealed 98.15% unmetabolized compound 8 and 1.85% metab-
olites. The major metabolite (0.77%) had a retention time of
4.11 min (Figure 3D). This metabolite had a mass of 471.2243
corresponding to [M+H]+ of an intramolecular oxidative cycli-
zation of one side chain (similar to MTX metabolite 1d,
Figure 1).
Integration of the HPLC spectrum of metabolized MTX re-
vealed that 87.81% of the drug was not metabolized; all of
the metabolites were detected at l=598 and 650 nm. ESIMS
analysis of metabolized MTX indicated that the most signifi-
cant metabolite (6.64%, tR =2.50 min, Figure 3C) had a molecu-
lar weight of 503.2128 corresponding to [M+H]+ of an ace-
toxy metabolite with the general structure 1c (Figure 1B).
Results of the in vitro metabolism experiments indicate that
conversion of the primary hydroxy group in each of the two
side chains of MTX into the cor-
responding
methoxy
ether
groups had a general stabilizing
effect. Chemical modification of
the primary hydroxy group in
each of the two side chains of
MTX resulted in an analogue
with high resistance to rabbit
liver S9-fraction- induced metab-
olism.
Conclusions
A
synthetic pathway for the
preparation of asymmetric and
symmetric analogues of the DNA
intercalating chemotherapeutic
drug MTX was reported. In these
analogues, one or both of the N-
(2-hydroxyethyl)ethylenediamine
side chains of MTX were dis-
placed by a side chain in which
the primary hydroxy group was
either O-glycosylated or convert-
ed into methoxy ether.
The affinities of the synthe-
sized MTX analogues for double-
stranded DNA were evaluated
using a fluorescent DNA interca-
lator displacement assay. Cyto-
toxicity was evaluated in four
different cancer cell types. No
correlation between DNA affinity
and in vitro cytotoxicity was ob-
served. For example, the dime-
Figure 3. Reversed-phase C18 analytical HPLC chromatograms of MTX and analogue 8 detected at l=598 nm.
A) MTX prior to incubation; B) analogue 8 prior to incubation; C) MTX after 4 h incubation with rabbit liver S9
fraction supplemented with GSH and NADPH; D) compound 8 after 4 h incubation with rabbit liver S9 fraction
supplemented with GSH and NADPH.
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lowed by the addition of 1,2,3,4,6-penta-O-acetyl-b-d-glucopyra-
nose (3 g, 7.7 mmol) and N3,N5-bis(benzyloxycarbonyl)-5-amino-3-
azapentan-1-ol (8.6 g, 23 mmol). After stirring for 10 min at room
temperature, BF3OEt2 (2.9 mL, 23 mmol) was added. The reaction
mixture was stirred at room temperature overnight. The reaction
progress was monitored by TLC (60% EtOAc, 40% PE). The reaction
was diluted with EtOAc and filtered through Celite. The filtrate was
washed with saturated (aq.) NaHCO3, followed by a wash with
brine, dried over MgSO4 and concentrated. The crude was purified
by chromatography (silica gel, PE/EtOAc) to yield 2a as a white
thoxy MTX analogue 8 had the weakest DNA affinity, but was
the most cytotoxic.
Both the parent MTX and its analogue 8 exhibited potent
cytotoxic activity against mouse B16 melanoma cells in vitro,
and were therefore tested in a mouse model. Relative to vehi-
cle-treated mice, MTX- or 8-treated mice had a significantly
lower tumor burden, with the more pronounced effect being
observed in MTX-treated mice. Analyses of the metabolic pro-
files of rabbit liver S9-fraction-treated MTX and analogue 8 in-
dicated that ~13% of the parent MTX was converted into me-
tabolites, whereas <2% of the dimethoxy analogue 8 was de-
graded. The measured molecular weight of the major metabo-
lite of MTX matched that of a previously characterized oxida-
tive acetoxylation metabolite. The major metabolite formed
from analogue 8 is likely an oxidative cyclization product
formed by intramolecular attack of a side chain secondary
amine on the anthracenedione core of this analogue.
1
powder (2 g, 37%) as a mixture of conformers. H NMR (400 MHz,
CDCl3): d=7.32 (bs), 4.95–5.15 (m), 4.47 (d, J=6.2 Hz), 4.40 (d, J=
6.2 Hz), 4.18–4.21 (m), 4.08–4.10 (m), 3.92–3.99 (m), 3.84–3.91 (m),
3.70–3.77 (m), 3.59–3.67 (m), 3.47–3.56 (m), 3.38–3.47 (m), 3.31–
3.35 (m), 1.98–2.04 ppm (m); 13C NMR (100 MHz, CDCl3): d=170.6,
170.2, 169.3, 156.6, 156.5, 156.4, 156.1, 136.6, 136.4, 136.3, 128.5,
128.4, 128.0, 127.9, 100.7, 100.6, 72.6, 71.8, 71.2, 71.0, 68.3, 68.2,
67.8, 67.3, 67.0, 66.6, 61.8, 61.7, 48.5, 48.2, 47.4, 39.3, 39.7, 20.6,
20.5 ppm; ESIMS(+), m/z calcd 702.26 for C34H43N2O14, found
703.38 [M+H]+.
Relatively small changes of the primary hydroxy group in
each of the MTX side chains resulted in a profound effect on
the pharmacokinetic properties; whereas measureable
amounts of MTX were found in urine samples collected from
mice two hours after i.p. administration of the drug, no trace
of compound 8 was detected in the urine samples collected
from mice treated with this MTX analogue.
[N3,N5-Bis(benzyloxycarbonyl)-5’-amino-3’-azapentyl]-b-d-gluco-
pyranoside (2b): A catalytic amount of NaOMe (0.5m solution in
MeOH) was added to
a suspension of compound 2a (2 g,
2.8 mmol) in MeOH (15 mL). The reaction mixture was stirred at
room temperature for 3 h; progress was monitored by TLC (10%
MeOH, 90% CH2Cl2). The reaction mixture was evaporated to dry-
ness, and the residue was purified by flash chromatography (silica
gel, MeOH/CH2Cl2) to yield 2b as a white powder (1.5 g, quant) as
a mixture of conformers. 1H NMR (400 MHz, CDCl3): d=7.33 (bs),
5.00–5.09 (m), 4.26 (d, J=7.7 Hz), 4.20 (d, J=7.6 Hz), 3.96–4.03 (m),
3.90–3.95 (m), 3.82 (t, J=10.0), 3.67–3.74 (m), 3.61–3.66 (m), 3.44–
3.54 (m), 3.16–3.19 (m); 13C NMR (100 MHz, CDCl3): d=157.5, 156.8,
136.9, 136.6, 128.1, 128.0, 127.7, 127.6, 127.5, 127.4, 103.2, 76.6,
73.6, 70.2, 70.1, 68.0, 67.3, 66.9, 66.5, 66.0, 61.3, 61.2 ppm;
ESIMS(À), m/z calcd 534.22 for C26H33N2O10, found 533.32 [MÀH]À.
To conclude, despite the anticancer potency of MTX, its clini-
cal use is limited due to its toxic side effects. Thus there is an
urgent need for potent anticancer MTX analogues that cause
fewer and less severe side effects. The results presented herein
suggest that MTX analogues with modified side chain primary
hydroxy groups offer a promising direction for the develop-
ment of such antitumor agents.
[2-(2-Aminoethylamino)ethyl]-b-d-glucopyranoside (2c): Com-
pound 2b (1.5 g, 2.8 mmol) dissolved in MeOH (8 mL) was added
to 10% palladium on carbon (catalytic amount) and stirred at
room temperature under a hydrogen balloon overnight. The reac-
tion was monitored by TLC (10% MeOH, 90% CH2Cl2). The reaction
mixture was filtered over a Celite bed. Concentration of the filtrate
gave 2c as a white powder (746 mg, quant). 1H NMR (500 MHz,
CD3OD): d=4.35 (d, J=7.8 Hz, 1H), 4.03–4.08 (m, 1H),3.92 (dd, J1 =
1.9, J2 =11.8 Hz, 1H), 3.83–3.88 (m, 1H), 3.69 (dd, J1 =5.6, J2 =
11.8 Hz, 1H), 3.38–3.42 (m, 1H), 3.29–3.33 (m, 2H), 3.24 ppm (dd,
J1 =7.8, J2 =9.2 Hz, 1H), 3.06–3.16 (m, 6H); 13C NMR (125.7 MHz,
CD3OD): d=105.2, 78.9, 78.8, 75.8, 72.3, 69.2, 63.4, 47.5, 39.7 ppm;
ESIMS(+), m/z calcd 266.15 for C10H23N2O6, found 267.19 [M+H]+.
Experimental Section
Chemistry
General methods. Reactions were monitored by TLC (Merck, silica
gel 60 F254
) and visualized using a cerium molybdate stain
[(NH4)2Ce(NO3)6 (5 g), (NH4)6Mo7O24·4H2O (120 g), H2SO4 (80 mL),
H2O (720 mL)]. Compounds were purified by SiO2 flash chromatog-
raphy (Merck Kieselgel 60) or by an ECOM preparative HPLC
system using
a Phenomenex Luna axia 5 mm C18 column
1
(250 mm”21.2 mm). H (including 1D TOCSY) and 13C NMR spectra
were recorded on Bruker Avance 400 and 500 spectrometers. Mul-
tiplicities are reported with the following abbreviations: b, broad;
s, singlet; d, doublet; t, triplet; dt, doublet of triplet; dd, doublet of
doublet; m, multiplet. Low-resolution electrospray ionization mass
spectra were recorded on a Waters 3100 mass detector. High-reso-
lution electrospray mass spectra were recorded on a Waters Synapt
instrument. The purity of the new compounds was determined by
reversed-phase HPLC using a C18 column (5 mm, 250 mm”4.6 mm)
and a gradient of 1:9!9:1 CH3CN/H2O (0.1% TFA) over 30 min at
1 mLminÀ1. The identities of the compounds were confirmed by
mass spectrometry. The purity of all of MTX derivatives tested was
ꢀ95%. All reagents were used without further purification and
were purchased from Sigma–Aldrich, Alfa Aesar, and TCI.
[N3,N5-Bis(benzyloxycarbonyl)-5’-amino-3’-azapentyl]-2,3,5-triace-
tyl-b-d-ribofuranoside (3a): To powdered, flame-dried 4 Š molecu-
lar sieves (300 mg) was added CH2Cl2 (20 mL), followed by the ad-
dition of 1,2,3,5-tetra-O-acetyl-b-d-ribofuranose (1.6 g, 5.1 mmol)
and N3,N5-bis(benzyloxycarbonyl)-5-amino-3-azapentan-1-ol (6.6 g,
17.7 mmol). After stirring for 10 min at room temperature, BF3OEt2
(1.9 mL, 15.2 mmol) was added. The reaction mixture was stirred at
room temperature overnight with progress monitored by TLC
(60% EtOAc, 40% PE). The reaction was diluted with EtOAc, and fil-
tered through Celite. The filtrate was washed with saturated (aq.)
NaHCO3 and then with brine, and was dried over MgSO4 and con-
centrated. The crude was purified by chromatography (silica gel,
PE/EtOAc) to yield 3a as a white powder (2.1 g, 66%) as a mixture
of conformers. 1H NMR (400 MHz, CDCl3): d=7.31 (bs), 4.94–5.27
[N3,N5-Bis(benzyloxycarbonyl)-5’-amino-3’-azapentyl]-2,3,4,6-
tetra-O-acetyl-b-d-glucopyranoside (2a): To powdered, flame-
dried 4 Š molecular sieves (1 g) was added CH2Cl2 (25 mL), fol-
ChemMedChem 2015, 10, 1528 – 1538
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
(m), 4.23–4.26 (m), 4.08–4.11 (m), 3.79–3.84 (m), 3.37–3.49 (m),
2.02–2.07 ppm (m); 13C NMR (100 MHz, CDCl3): d=170.5, 169.6,
156.7, 156.5, 156.4, 156.1, 136.6, 136.4, 128.5, 128.4, 128.3, 128.0,
127.9, 105.2, 78.7, 74.6, 71.4, 67.4, 66.6, 66.4, 64.4, 48.0, 47.3, 40.0,
20.7, 20.5 ppm; ESIMS(+), m/z calcd 630.64 for C31H38N2O12, found
653.26 [M+Na]+.
ness, and the residue was purified by flash chromatography (silica
gel, MeOH/CH2Cl2) to yield 4c as a white powder (550 mg, 98%) as
a mixture of conformers. 1H NMR (400 MHz, CDCl3): d=7.34 (bs),
5.47–5.53 (m), 5.10 (d, J=9.4 Hz), 5.07 (d, J=9.4 Hz), 3.38–
3.96 ppm (m); 13C NMR (100 MHz, CDCl3): d=157.0, 156.6, 136.4,
136.2, 128.6, 128.5, 128.2, 128.0, 99.9, 70.8, 70.3, 69.1, 67.5, 67.0,
66.8, 63.5, 63.2, 60.0, 59.4, 48.8, 48.4, 47.8, 39.8 ppm; ESIMS(+), m/z
calcd 534.22 for C26H33N2O10, found 557.23 [M+Na]+.
[N3,N5-Bis(benzyloxycarbonyl)-5’-amino-3’-azapentyl]-b-d-ribofur-
anoside (3b): A catalytic amount of NaOMe (0.5m solution in
MeOH) was added to a suspension of compound 3a (2.1 g,
3.33 mmol) in MeOH (10 mL). The reaction mixture was stirred at
room temperature for 3 h, and reaction progress was monitored
by TLC (10% MeOH, 90% CH2Cl2). The reaction mixture was evapo-
rated to dryness, and the residue was purified by flash chromatog-
raphy (silica gel, MeOH/CH2Cl2) to yield 3b as a white powder
[2-(2-Aminoethylamino)ethyl]-b-d-fructopyranoside (4d): Com-
pound 4c (290 mg, 0.54 mmol) dissolved in MeOH (5 mL) was
added to 10% palladium on carbon (catalytic amount) and stirred
at room temperature under a hydrogen balloon overnight. The re-
action was monitored by TLC (10% MeOH, 90% CH2Cl2). The reac-
tion mixture was filtered over a Celite bed. Concentration of the fil-
a
mixture of conformers. 1H NMR (400 MHz,
trate gave 4d as
a
white powder (130 mg, 95%). 1H NMR
(1.2 g, 73%) as
(400 MHz, CD3OD): d=3.93 (d, J=9.9 Hz, 1H), 3.88–3.89 (m, 1H),
3.82 (dd, J1 =3.4, J2 =9.9 Hz, 1H), 3.75–3.77 (m, 3H), 3.64–3.73 (m,
3H), 2.74–2.86 ppm (m, 6H); 13C NMR (125.7 MHz, CD3OD): d=
102.6, 72.3, 71.8, 71.7, 66.2, 64.4, 61.5, 52.5, 51.0, 42.1 ppm;
ESIMS(+), m/z calcd 266.15 for C10H23N2O6, found 267.23 [M+H]+.
CD3OD): d=7.30–7.37 (m), 5.04–5.12 (m), 4.01–4.07 (m), 3.92–3.96
(m), 3.81–3.91 (m) 3.69–3.72 (m), 3.45–3.56 (m), 3.29–3.30 ppm (m);
13C NMR (100 MHz, CD3OD): d=157.5, 156.7, 156.6, 136.9, 136.6,
128.1, 128.0, 127.7, 127.6, 127.5, 127.4, 107.3, 83.5, 74.8, 71.2, 66.9,
66.0, 65.6, 65.2, 63.5, 39.0, 38.7 ppm; ESIMS(+), m/z calcd 504.53
for C25H32N2O9, found 527.15 [M+Na]+.
1,4-Bis[[2-[(2-(b-d-glucopyranoside)ethyl)amino]ethyl]amino]-
5,8-dihydroxyanthracene-9,10-dione (5):
A mixture of 1,4-di-
[2-(2-Aminoethylamino)ethyl]-b-d-ribofuranoside (3c): Com-
pound 3b (500 mg, 0.99 mmol) dissolved in MeOH (5 mL) was
added to 10% palladium on carbon (catalytic amount) and stirred
at room temperature under a hydrogen balloon overnight. Reac-
tion progress was monitored by TLC (10% MeOH, 90% CH2Cl2).
The reaction mixture was filtered over a Celite bed. Concentration
fluoro-5,8-dihydroxyanthracene-9,10-dione (30 mg, 0.11 mmol) and
compound 2c (116 mg, 0.44 mmol) in DMSO (0.8 mL) was stirred
at 408C overnight. Monitoring of the reaction by ESIMS indicated
the formation of the disubstituted product, compound 5 ([MÀH]+,
m/z 769.8) The reaction mixture was lyophilized, and the residue
was purified by HPLC using a Phenomenex Luna axia 5 mm C18
column (250 mm”21.20 mm) at a flow rate of 20 mLminÀ1. HPLC
solvent A was H2O (0.1% TFA), and B was MeOH (0.1% TFA). The
elution gradient was 30% B for 2 min followed by 30!100% B
over 14 min. Product elution was monitored at l=256 nm. Com-
pound 5 was detected at 6.2 min. Fractions containing the pure
product were concentrated under vacuum, dissolved in H2O, and
freeze-dried to yield compound 5 as a blue powder (26 mg, 31%).
1H NMR (500 MHz, CD3OD): d=7.30 (s, 2H), 7.04 (s, 2H), 4.39 (d, J=
7.8 Hz, 2H), 4.15 (dt, J1 =12.8, J2 =5.0 Hz, 2H), 4.02 (dt, J1 =12.7,
J2 =5.0 Hz, 2H), 3.93 (dd, J1 =2.3, J2 =11.8 Hz, 2H), 3.86 (t, J1 =
6.3 Hz, 4H), 3.67 (dd, J1 =6.4, J2 =11.9 Hz, 2H), 3.35–3.47 (m, 12H),
3.24–3.29 ppm (m, 4H); 13C NMR (125.7 MHz, CD3OD): d=188.1,
162.8, 162.4, 162.1, 161.8, 157.4, 147.8, 127.5, 125.8, 121.7, 119.4,
117.0, 116.6, 112.1, 105.1, 79.0, 78.7, 75.7, 72.4, 66.8, 63.4, 48.8,
40.7 ppm; HR ESIMS(+), m/z calcd 769.3144 for C34H49N4O16, found
769.3149 [M+H]+.
1
of the filtrate gave 3c as a white powder (233 mg, quant). H NMR
(400 MHz, CD3OD): d=4.88 (bs, 1H), 4.18 (dd, J1 =4.8, J2 =7.1 Hz,
1H), 3.92–3.96 (m, 2H), 3.75–3.84 (m, 2H), 3.56–3.66 (m, 2H), 2.70–
2.85 ppm (m, 6H); 13C NMR (125.7 MHz, CD3OD): d=107.7, 83.4,
75.0, 70.3, 66.9, 62.2, 50.3, 48.5, 39.9 ppm; ESIMS(+), m/z calcd
236.27 for C9H20N2O5, found 259.09 [M+Na]+.
[N3,N5-Bis(benzyloxycarbonyl)-5’-amino-3’-azapentyl]-1,3,4,5-
tetra-O-benzoyl-b-d-fructopyranoside (4b): To powdered, flame-
dried 4 Š molecular sieves (200 mg) was added CH2Cl2 (10 mL), fol-
lowed by the addition of compound 4a (1.1 g, 1.4 mmol) and
N3,N5-bis(benzyloxycarbonyl)-5-amino-3-azapentan-1-ol
(2.0 g,
5.6 mmol). After stirring for 10 min at À408C, TMSOTf (150 mL) was
added. The reaction mixture was stirred at À208C for 1 h and then
at room temperature overnight; progress of the reaction was
monitored by TLC (60% EtOAc, 40% PE). The reaction was diluted
with EtOAc and filtered through Celite. The filtrate was washed
with saturated (aq.) NaHCO3 then with brine and was dried over
MgSO4 and concentrated. The crude was purified by chromatogra-
phy (silica gel, PE/EtOAc) to yield as a white powder 4b (1.1 g,
81%) as a mixture of conformers. 1H NMR (400 MHz, CDCl3): d=
8.02 (bs), 7.86 (bs), 7.78 (d, J=7.5), 7.50–7.58 (m), 7.20–7.42 (m,
20H), 6.28 (d, J=10.3), 5.93 (d, J=10.3 Hz), 5.85 (d, J=10.8 Hz),
5.71 (s), 5.65 (s), 5.00–5.22 (m), 4.73 (dd, J1 =4.7, J2 =11.5 Hz), 4.27
(m), 3.48–4.01 ppm (m); 13C NMR (100 MHz, CDCl3): d=165.6, 156.6,
156.1, 136.5, 136.4, 136.2, 133.2, 133.1, 133.0, 129.7, 129.0, 128.9,
128.6, 128.5, 128.4, 128.2, 128.1, 99.5, 70.0, 69.9, 69.0, 68.4, 67.9,
67.5, 66.6, 62.6, 62.2, 62.0, 60.4, 60.3, 48.4, 47.5, 39.8 ppm;
ESIMS(+), m/z calcd 950.9 for C54H50N2O14, found 973.7 [M+Na]+.
1,4-Bis[[2-[(2-(b-d-ribofuranoside)ethyl)amino]ethyl]amino]-5,8-
dihydroxyanthracene-9,10-dione (6): A mixture of 1,4-difluoro-5,8-
dihydroxyanthracene-9,10-dione (22 mg, 0.08 mmol) and com-
pound 3c (75 mg, 0.32 mmol) in DMSO (0.5 mL) was stirred at
408C overnight. ESIMS indicated the formation of the disubstituted
product, compound 6 ([MÀH]+, m/z 709.7). The reaction mixture
was lyophilized, and the residue was purified by HPLC using a Phe-
nomenex Luna axia 5 mm C18 column (250 mm”21.20 mm) at
a flow rate of 20 mLminÀ1. HPLC solvent A was H2O (0.1% TFA)
and B was MeOH (0.1% TFA). The elution gradient was 30% B for
2 min followed by 30!100% B over 14 min. Product elution was
monitored at l=256 nm. Compound 6 was detected at 7.0 min.
Fractions containing the pure product were concentrated under
vacuum, dissolved in H2O, and freeze-dried to yield compound 6
as a blue powder (16 mg, 43%). 1H NMR (400 MHz, CD3OD): d=
7.44 (s, 2H), 7.16 (s, 2H), 4.95 (s, 2H), 4.29 (dd, J1 =4.8, J2 =7.5 Hz,
2H), 3.94–4.00 (m, 9H), 3.84–3.89 (m, 6H), 3.74 (dd, J1 =3.3, J2 =
12.4 Hz), 3.36–3.41 ppm (m, 7H); 13C NMR (100.6 MHz, CD3OD): d=
[N3,N5-Bis(benzyloxycarbonyl)-5’-amino-3’-azapentyl]-b-d-fructo-
pyranoside (4c): A catalytic amount of NaOMe (0.5m solution in
MeOH) was added to
a suspension of compound 3b (1 g,
1.05 mmol) in MeOH (10 mL). The reaction mixture was stirred at
room temperature for 3 h with progress monitored by TLC (10%
MeOH, 90% CH2Cl2). The reaction mixture was evaporated to dry-
ChemMedChem 2015, 10, 1528 – 1538
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
186.4, 155.4, 145.6, 125.4, 123.6, 114.5, 110.2, 108.2, 83.0, 75.0, 69.2,
64.0, 60.0, 46.3, 38.4 ppm; HR ESIMS(À), m/z calcd 707.2776 for
C32H43N4O14, found 707.2774 [MÀH]À.
(d, J=8.0 Hz, 1H), 4.09–4.13 (m, 1H), 3.96–4.00 (m, 1H), 3.90 (m,
1H), 3.78 (t, J=5.8 Hz, 2H), 3.64 (dd, J1 =6.2, J2 =11.3 Hz, 1H),
3.23–3.38 ppm (m, 8H); ESIMS(À), m/z calcd 522.48 for
C24H27FN2O10, found 521.30 [MÀH]À.
1,4-Bis[[2-[(2-(b-d-fructopyranoside)ethyl)amino]ethyl]amino]-
5,8-dihydroxyanthracene-9,10-dione (7):
A
mixture of 1,4-di-
1-[[2-[(2-(b-d-Glucopyranoside)ethyl)amino]ethyl]amino]-4-[[2-
[(2-hydroxyethyl)amino]ethyl]amino]-5,8-dihydroxyanthracene-
9,10-dione (9): To a solution of compound 9a (15 mg, 0.03 mmol)
in DMSO (0.5 mL), N-(2-hydroxyethyl)ethylenediamine (5 mL,
0.05 mmol) was added. The reaction mixture was stirred at RT over-
night. The reaction was monitored by ESIMS ([MÀH]+, m/z 607.6).
The reaction mixture was lyophilized, and the residue was purified
fluoro-5,8-dihydroxyanthracene-9,10-dione (30 mg, 0.11 mmol) and
compound 4d (116 mg, 0.44 mmol) in DMSO (0.5 mL) was stirred
at 408C overnight. ESIMS indicated the formation of the disubsti-
tuted product, compound 7 ([MÀH]+, m/z 769.8). The reaction
mixture was lyophilized, and the residue was purified by HPLC
using a Phenomenex Luna axia 5 mm C18 column (250 mm”
21.20 mm) at a flow rate of 20 mLminÀ1. HPLC solvent A was H2O
(0.1% TFA) and B was MeOH (0.1% TFA). The elution gradient was
30% B for 2 min followed by 30!100% B over 14 min. Product
elution was monitored at l=256 nm. Compound 7 was detected
at 5.7 min. Fractions containing the pure product were concentrat-
ed under vacuum, dissolved in H2O, and freeze-dried to yield com-
pound 7 as a blue powder (27 mg, 35%). 1H NMR (500 MHz
CD3OD): d=7.44 (s, 2H), 7.14 (s, 2H), 3.92–3.94 (m, 4H), 3.84–3.88
(m, 7H), 3.75–3.78 (m, 4H), 3.70–3.73 (m, 8H), 3.31–3.34 ppm (m,
7H); 13C NMR (125.7 MHz, CD3OD): d=186.4, 160.7, 155.4, 145.6,
125.5, 123.6, 114.4, 110.2, 100.5, 69.7, 69.2, 64.4, 62.5, 56.0, 46.2,
38.6 ppm; HR ESIMS(+), m/z calcd 769.3144 for C34H49N4O16, found
769.3151 [M+H]+.
by HPLC using
a Phenomenex Luna axia 5 mm C18 column
(250 mm”21.20 mm) at a flow rate of 20 mLminÀ1. HPLC solvent A
was H2O (0.1% TFA), and B was MeOH (0.1% TFA). The elution gra-
dient was 30% B for 2 min followed by 30!100% B over 14 min.
Product elution was monitored at l=256 nm. Compound 9 was
detected at 8.2 min. Fractions containing the pure product were
concentrated under vacuum, dissolved in H2O, and freeze-dried to
yield compound 9 as a blue powder (6 mg, 35%). 1H NMR
(500 MHz, CD3OD): d=7.38 (s, 2H), 7.11 (s, 2H), 4.39 (d, J=7.8 Hz,
1H), 4.15 (dt, J1 =12.8, J2 =4.9 Hz, 1H), 4.02 (dt, J1 =12.8, J2 =
5.0 Hz, 1H), 3.93 (dd, J1 =2.3, J2 =11.8 Hz, 1H), 3.87 (m, 6H), 3.66
(dd, J1 =6.4, J2 =11.8 Hz, 1H), 3.35–3.47 (m, 8H), 3.23–3.28 ppm (m,
4H); 13C NMR (125.7 MHz, CD3OD): d=188.4, 162.9, 162.5, 162.3,
161.9, 157.5, 147.9, 127.6, 125.9,125.8, 119.4, 117.1, 116.7, 112.3,
112.2, 105.1, 79.0, 78.7, 75.7, 72.4, 66.8, 63.4, 58.6, 51.8, 48.8, 48.6,
40.8 ppm; HR ESIMS(+), m/z calcd 607.2615 for C28H38N4O11, found
607.2611 [M+H]+.
1,4-Bis[[2-[(2-methoxyethyl)amino]ethyl]amino]-5,8-dihydrox-
yanthracene-9,10-dione (8): A mixture of 1,4-difluoro-5,8-dihy-
droxyanthracene-9,10-dione (50 mg, 0.18 mmol) and N-(2-methox-
yethyl)-1,2-diaminoethane (63 mg, 0.54 mmol) in pyridine (0.7 mL)
was stirred at 358C overnight. ESIMS indicated the formation of
the disubstituted product, compound 8 ([MÀH]+, m/z 473.5). The
reaction mixture was evaporated to dryness, and the residue was
purified by HPLC using a Phenomenex Luna axia 5 mm C18 column
(250 mm”21.20 mm) at a flow rate of 20 mLminÀ1. HPLC solvent A
was H2O (0.1% TFA) and B was MeOH (0.1% TFA). The elution gra-
dient was 30% B for 2 min followed by 30!100% B over 14 min.
Product elution was monitored at l=256 nm. Compound 8 was
detected at 8.7 min. Fractions containing the pure product were
concentrated under vacuum, dissolved in H2O, and freeze-dried to
yield compound 8 as a blue powder (39 mg, 49%). 1H NMR
(400 MHz, CD3OD): d=7.35 (s, 2H), 7.11 (s, 2H), 3.83 (t, J=6.4 Hz,
4H), 3.69 (t, J=5.1 Hz, 4H), 3.44 (s, 6H), 3.37 (t, J=6.2 Hz, 4H),
3.32–3.34 ppm (m, 4H); 13C NMR (100.6 MHz, CD3OD): d=186.3,
155.4, 145.6, 125.4, 123.5, 114.5, 110.1, 66.7, 57.9, 46.4, 38.5 ppm;
HR ESIMS(À), m/z calcd 471.2244 for C24H31N4O6, found 471.2241
[MÀH]À.
1-[[2-[(2-(b-d-Ribofuranoside)ethyl)amino]ethyl]amino]-4-fluoro-
5,8-dihydroxyanthracene-9,10-dione (10a): A mixture of 1,4-di-
fluoro-5,8-dihydroxyanthracene-9,10-dione (30 mg, 0.11 mmol) and
compound 3c (77 mg, 0.33 mmol) in pyridine (0.5 mL) was stirred
at 408C overnight. ESIMS indicated the formation of the monosub-
stituted product, compound 10a ([MÀH]+, m/z 492.3). The reac-
tion mixture was evaporated to dryness, and the residue was puri-
fied by HPLC using a Phenomenex Luna axia 5 mm C18 column
(250 mm”21.20 mm) at a flow rate of 20 mLminÀ1. HPLC solvent A
was H2O (0.1% TFA) and B was MeOH (0.1% TFA). The elution gra-
dient was 30% B for 2 min followed by 30!100% B over 14 min.
Product elution was monitored at l=256 nm. Compound 10a was
detected at 10.0 min. Fractions containing the pure product were
concentrated under vacuum, dissolved in H2O, and freeze-dried to
yield compound 10a as a purple powder (10 mg, 25%). 1H NMR
(400 MHz, CD3OD): d=7.42 (dd, J1 =J2 =10.1 Hz, 1H), 7.31 (dd, J1 =
4.0, J2 =10.1 Hz, 2H), 7.22 (s, 2H), 4.96 (S, 1H), 4.30 (dd, J1 =4.8,
J2 =7.9 Hz, 1H), 3.95–4.01 (m, 4H), 3.88 (dd, J1 =2.7, J2 =12.2 Hz,
1H), 3.81 (t, J2 =6.2 Hz, 2H), 3.76 (dd, J1 =2.7, J2 =12.2 Hz, 1H),
3.35–3.45 ppm (m, 4H); ESIMS(+), m/z calcd 492.45 for
C23H25FN2O9, found 491.28 [MÀH]À.
1-[[2-[(2-(b-d-Glucopyranoside)ethyl)amino]ethyl]amino]-4-
fluoro-5,8-dihydroxyanthracene-9,10-dione (9a): A mixture of
1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione
(30 mg,
0.11 mmol) and compound 2c (88 mg, 0.33 mmol) in pyridine
(0.6 mL) was stirred at 408C overnight. Monitoring of the reaction
by ESIMS indicated the formation of the monosubstituted product,
compound 9a ([MÀH]À, m/z 521.3). The reaction mixture was
evaporated to dryness, and the residue was purified by HPLC using
a Phenomenex Luna axia 5 mm C18 column (250 mm”21.20 mm) at
a flow rate of 20 mLminÀ1. HPLC solvent A was H2O (0.1% TFA),
and B was MeOH (0.1% TFA). The elution gradient was 30% B for
2 min followed by 30!100% B over 14 min. Product elution was
monitored at l=256 nm. Compound 9a was detected at 11.6 min.
Fractions containing the pure product were concentrated under
vacuum, dissolved in H2O, and freeze-dried to yield compound 9a
1-[[2-[(2-(b-d-Ribofuranoside)ethyl)amino]ethyl]amino]-4-[[2-[(2-
hydroxyethyl)amino]ethyl]amino]-5,8-dihydroxyanthracene-
9,10-dione (10): To
a solution of compound 10a (10 mg,
0.02 mmol) in DMSO (0.3 mL), N-(2-hydroxyethyl)ethylenediamine
(3 mL, 0.03 mmol) was added. The reaction mixture was stirred at
RT overnight. Completion of the reaction was confirmed by ESIMS
([MÀH]+, m/z 576.6). The reaction mixture was lyophilized, and the
residue was purified by HPLC using a Phenomenex Luna axia 5 mm
C18 column (250 mm”21.20 mm) at a flow rate of 20 mLminÀ1
.
HPLC solvent A was H2O (0.1% TFA) and B was MeOH (0.1% TFA).
The elution gradient was 30% B for 2 min followed by 30!100%
B over 14 min. Product elution was monitored at l=256 nm. Com-
pound 10 was detected at 8.8 min. Fractions containing the pure
1
as a purple powder (22 mg, 35%). H NMR (400 MHz, CD3OD): d=
7.36 (dd, J1 =J2 =10.0 Hz, 1H), 7.25–7.28 (m, 1H), 7.16 (s, 2H), 4.37
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product were concentrated under vacuum, dissolved in H2O, and
freeze-dried to yield compound 10 as a blue powder (6 mg, 53%).
1H NMR (400 MHz, CD3OD): d=7.44 (s, 2H), 7.16 (s, 2H), 4.95 (s,
1H), 4.29 (dd, J1 =4.6, J2 =7.7 Hz, 1H), 3.94–4.00 (m, 4H), 3.84–3.89
(m, 7H), 3.74 (dd, J1 =3.2, J2 =12.3 Hz, 1H), 3.36–3.42 (m, 5H),
3.24–3.26 ppm (m, 3H); 13C NMR (100 MHz, CD3OD): d=186.4,
155.4, 145.6, 125.4, 123.6, 114.5, 110.2, 108.2, 83.0, 75.0, 69.2, 64.0,
60.0, 56.3, 49.6, 46.3, 38.5, 38.4 ppm; HR ESIMS(À), m/z calcd
575.2254 for C29H35N4O10, found 575.2291 [MÀH]À.
3 mgmLÀ1 puromycin. MCF7 and HepG2 cell lines were grown in
DMEM supplemented with 10% FCS and 1% penicillin–streptomy-
cin. HT-29 cells were grown in RPMI, supplemented with 10% FCS
and 1% penicillin–streptomycin. A549 cells were maintained in
DMEM/F12, supplemented with 10% FCS, 1% penicillin–streptomy-
cin, 1% l-glutamine, and 5% NaHCO3. All cells were grown in a hu-
midified chamber of 95% air, 5% CO2 at 378C.
MTT assays: Cells (6”103 per well) were plated into 96-well micro-
titer plates and allowed to adhere for 24 h, after which 10 mL of so-
lution containing the test compounds at various concentrations up
to 50 mm were added. The treated cells were incubated for an ad-
ditional 72 h. Cell viability was determined by the MTT assay as fol-
lows: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) was added to the medium at a final concentration of
0.5 mgmLÀ1, followed by incubation at 378C in a CO2 incubator.
After 2 h, the medium was aspirated and DMSO (200 mL) was
added to the wells. The plates were read on a Synergy HT MultiDe-
tection Microplate Reader using a test wavelength of 490 nm and
a reference wavelength of 690 nm. Each compound was tested in
triplicate, and the results reported are averages of at least three in-
dependent experiments.
1-[[2-[(2-(b-d-Fructopyranoside)ethyl)amino]ethyl]amino]-4-
fluoro-5,8-dihydroxyanthracene-9,10-dione (11a): A mixture of
1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione
(33 mg,
0.12 mmol) and compound 4d (95 mg, 0.36 mmol) in pyridine
(0.6 mL) was stirred at 408C overnight. ESIMS indicated the forma-
tion of the monosubstituted product, compound 11 a ([MÀH]+, m/
z 523.5). The reaction mixture was evaporated to dryness, and the
residue was purified by HPLC using a Phenomenex Luna axia 5 mm
C18 column (250 mm”21.20 mm) at a flow rate of 20 mLminÀ1
.
HPLC solvent A was H2O (0.1% TFA) and B was MeOH (0.1% TFA).
The elution gradient was 30% B for 2 min followed by 30!100%
B over 14 min. Product elution was monitored at l=256 nm. Com-
pound 11 a was detected at 10.5 min. Fractions containing the
pure product were concentrated under vacuum, dissolved in H2O,
and freeze-dried to yield compound 11 a as a purple powder
(15 mg, 24%). 1H NMR (500 MHz, CD3OD): d=7.47 (dd, J1 =J2 =
9.5 Hz, 1H), 7.36 (dd, J1 =3.9, J2 =9.6 Hz, 1H), 7.25 (s, 2H), 3.97 (d,
J=9.9 Hz, 1H), 3.88–3.89 (m, 1H), 3.73–3.85 (m, 8H), 3.35–
3.46 ppm (m, 5H); ESIMS(+), m/z calcd 522.48 for C24H27FN2O10,
found 523.30 [M+H]+.
In vivo antitumor activity: mCherry B16F10 melanoma cells (5”104
per 100 mL) were injected i.p. into 8-week-old C57BL/6J female
mice. On days 1, 5, and 9 after implantation of the tumor cells,
MTX, compound 8 (3.2 mgkgÀ1, equivalent for 0.4 mgmLÀ1) or ve-
hicle (saline) were administered i.p. to the mice (n=13, 14, and 14
respectively). The mice were weighed daily. After 27 days, mice
were sacrificed using CO2, the abdominal cavities were dissected,
and mCherry expression was imaged and quantified using the Cri
Maestro in vivo fluorescence imaging system. Multispectral image
cubes were acquired through the l 550–800 nm spectral range in
10 nm steps using excitation (l 575–605 nm) and emission
(l 645 nm long-pass) filters. Autofluorescence and undesired back-
ground signals were eliminated by use of spectral analysis and
a linear unmixing algorithm.
1-[[2-[(2-(b-d-Fructopyranoside)ethyl)amino]ethyl]amino]-4-[[2-
[(2-hydroxyethyl)amino]ethyl]amino]-5,8-dihydroxyanthracene-
9,10-dione (11): To
a solution of compound 11 a (13 mg,
0.02 mmol) in DMSO (0.3 mL), N-(2-hydroxyethyl)ethylenediamine
(3 mL, 0.03 mmol) was added. The reaction mixture was stirred at
RT overnight. Completion was confirmed by ESIMS ([MÀH]+, m/z
607.6). The reaction mixture was lyophilized, and the residue was
purified by HPLC using a Phenomenex Luna axia 5 mm C18 column
(250 mm”21.20 mm) at a flow rate of 20 mLminÀ1. HPLC solvent A
was H2O (0.1% TFA) and B was MeOH (0.1% TFA). The elution gra-
dient was 30% B for 2 min followed by 30!100% B over 14 min.
Product elution was monitored at l=256 nm. Compound 11 was
detected at 8.3 min. Fractions containing the pure product were
concentrated under vacuum, dissolved in H2O, and freeze-dried to
yield compound 11 as a blue powder (6 mg, 40%). 1H NMR
(400 MHz, CD3OD): d=7.38 (s, 2H), 7.11 (s, 2H), 3.92–3.96 (m, 2H),
3.69–3.84 (m, 13H), 3.31–3.42 (m, 6H), 3.22 ppm (t, J=5.1 Hz, 2H);
13C NMR (100 MHz, CD3OD): d=186.3, 155.4, 145.6, 125.4, 123.6,
114.5, 110.1, 100.5, 69.7, 69.2, 64.4, 62.5, 56.3, 56.0, 49.6, 46.3, 46.2,
38.6, 38.5 ppm; HR ESIMS(+), m/z calcd 607.2615 for C28H39N4O11,
found 607.2617 [M+H]+.
Isolation of rabbit liver S9 fraction and evaluation of the metabolic
profiles of MTX and compound 8: New Zealand White rabbits (4
months of age) were euthanized by intracardiac injection of
sodium pentobarbital, and 2 g pieces of the liver were harvested.
Isolation of the S9 fraction and evaluation of the metabolic profile
of MTX and compound 8 were carried out as previously de-
scribed.[17] Briefly, the liver tissue was dissociated in phosphate
buffer (100 mm, pH 7.4) at a concentration of 0.25 gmLÀ1 using
GentleMACS dissociator (Miltenyi Biotec). Liver homogenate was
centrifuged at 9000”g for 20 min at 48C, and the supernatant was
collected (liver S9 fraction). The protein concentration was deter-
mined using the Bradford method. For assessing MTX or com-
pound 8 metabolism, these compounds (at 1 mm) were incubated
with 4 mgmLÀ1 S9 fraction supplemented with 1 mm NADPH and
4 mm GSH at 378C. After 4 h, MeOH was added at a ratio of 1:4.
Samples were centrifuged at 16000”g for 5 min at 48C. The super-
natant was collected and evaporated, and the residues were ana-
lyzed by LC–MS. The experiments were performed twice on two
different S9 fractions, each prepared from a different animal.
Biology
Animals: C57BL/6J female mice and New Zealand White male rab-
bits were purchased from Harlan (catalog numbers 2BL/6R24 and
1NZWR05, respectively). Animals were maintained and treated in
accordance with all applicable rules and guidelines of the Animal
Care and Use Committee of Tel Aviv University.
Fluorescent DNA intercalator displacement assay: Displacement reac-
tions were performed in 96-well plates (Greiner Bio-One). Each well
contained 500 ng of l DNA (48 kb, New England Biolabs) and SYBR
safe (Life Technologies) at a final dilution of 1:10000 (according to
the manufacturer’s protocol). The compounds were tested over
a range of concentrations, and the final reaction volume was 60 mL
[Tris·EDTA buffer containing 5% DMSO (MERCK)]. Reactions were
Cell culture: mCherry-B16F10 melanoma cells were a gift from Prof.
Satchi-Fainaro, Tel Aviv University.[33] The cells were maintained in
DMEM (Invitrogen), supplemented with 10% fetal calf serum (FCS),
1% penicillin–streptomycin, 4 mm l-glutamine (Invitrogen), and
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Acknowledgements
This work was supported by a grant from the Israel Science Foun-
dation (ISF) (grant 58/10 to M.F.). The authors thank Dr. Noam
Tal (School of Chemistry, Tel Aviv University) for his professional
assistance in performing HR–ULC–ESIMS measurements. The au-
thors also thank Dr. Liora Lindenboim and Dr. Lior Faigenbloom
(Department of Neurobiology, Tel Aviv University) for advice and
assistance in performing the in vitro cell cytotoxicity experiments.
Keywords: antitumor agents
intercalating drugs · drug metabolism · mitoxantrone
· cytotoxic agents · DNA
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Published online on July 31, 2015
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