SAR-guided development and characterization of a potent antitumor compound toward b-cell neoplasms with no detectable cytotoxicity toward healthy cells

Article abstract of DOI:10.1021/jm501848m

Acute hematological diseases (leukemias and aggressive lymphomas) can be cured in approximately half of the patients, while the other patients die from their disease. Chronic leukemias and indolent lymphomas can be well controlled for years in most cases. However, the cure rate of these patients is low and the course of the disease is characterized by frequent recurrence. Therefore, novel agents for monotherapies or combination therapies still need to be explored. The presented study describes the identification of the chalcone derivative 15 on different types of human malignant cells of the lymphoid and myeloid lineage. Further experiments performed with compound 15 on peripheral blood mononuclear cells (PBMCs) of chronic lymphocytic leukemia (CLL) patients clearly stated a higher cytotoxicity in PBMC from CLL patients compared to healthy donors (HD). The newly identified chalcone derivative 15 showed a higher therapeutic potential than fludarabine, a drug already in use in lymphoma treatment.

Full text of DOI:10.1021/jm501848m

Article
pubs.acs.org/jmc
SAR-Guided Development and Characterization of a Potent
Antitumor Compound toward B‑Cell Neoplasms with No Detectable
Cytotoxicity toward Healthy Cells
Gerda Brunhofer-Bolzer,†,‡ Trang Le,‡,§ Nils Dyckmanns,‡,§ Hanna A. Knaus,‡,§ Clemens Pausz,‡,§
Patricia Freund,‡,§ Ulrich Jager,‡,§ Thomas Erker,*,† and Katrina Vanura‡,§
̈
†Department of Medicinal Chemistry, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria
‡Department of Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, 1090 Vienna, Austria
§Comprehensive Cancer Center, Medical University of Vienna, 1090 Vienna, Austria
ABSTRACT: Acute hematological diseases (leukemias and
aggressive lymphomas) can be cured in approximately half of
the patients, while the other patients die from their disease.
Chronic leukemias and indolent lymphomas can be well
controlled for years in most cases. However, the cure rate of
these patients is low and the course of the disease is
characterized by frequent recurrence. Therefore, novel agents
for monotherapies or combination therapies still need to be explored. The presented study describes the identification of the
chalcone derivative 15 on different types of human malignant cells of the lymphoid and myeloid lineage. Further experiments
performed with compound 15 on peripheral blood mononuclear cells (PBMCs) of chronic lymphocytic leukemia (CLL) patients
clearly stated a higher cytotoxicity in PBMC from CLL patients compared to healthy donors (HD). The newly identified
chalcone derivative 15 showed a higher therapeutic potential than fludarabine, a drug already in use in lymphoma treatment.
One of the approaches for the development of novel
anticancer drugs is the assessment of naturally occurring
compounds for cancer therapy. In this study, we were focusing
on the chalcone scaffold as a template for the structure−activity
guided development of selective cytotoxic compounds toward
B-cell neoplasms. Chemically, chalcones are biosynthetic
precursors of flavonoids, and both natural as well as synthetic
derivatives have shown biologic activity in cancer cells.6−10
Recent studies have shown that chalcone derivatives exhibit
cytotoxic activity in different tumor cell lines including
hematological malignancies. (E)-α-Benzylthiochalcones were
able to significantly inhibit proliferation of K562, a BCR-ABL
positive chronic myeloid leukemia cell line.11 Nevertheless little
is known about the mode of action of chalcone-based
compounds. Their apoptosis inducing properties seemed to
be mediated by interference in microtubule formation,12−20
inhibition of nuclear factor κB (NF-κB),21 and/or depletion of
mitochondrial glutathione.8
In this contribution, a promising cytotoxic agent with
specificity to neoplastic cells, in particular to hematological
malignancies, was developed by using a lead structure
optimization procedure in the B-cell lines MEC1, U-2940,
and OCI-LY7. Considering the different biological character-
istics and therapeutic responses of lymphoma, we continued to
study the effect of the most interesting compound on six
additional types of human lymphoma and leukemia cells of
INTRODUCTION
■
Hematologic malignancies are a highly diverse group of cancers,
which affect bone marrow, blood, and the lymph nodes. They
rank just outside the top 10 list of cancers worldwide1 and are
expected to constitute 9% of cancers and to account for 9.4% of
deaths from cancers in the U.S. in 2013.2 Roughly 75% of these
neoplasms are of lymphoid, and the rest is of myeloid origin,
incidence rates being ∼20 for lymphoid and ∼7 for myeloid
entities, respectively, per 100 000 persons per year in Western
countries.2−5
Among lymphoid malignancies, mature B-cell neoplasms not
only constitute the largest proportion of diseases (52%)3 but
also show the highest incidence rates in Europe, 19.14 per
100 000, with similar numbers reported from other Western
countries,4 and are predominately diagnosed in elderly
persons.3 Because of the diversity of these malignancies,
biology and pathological characteristics are highly heteroge-
neous as are clinical presentation and behavior and,
subsequently, therapeutic requirements. Conventional thera-
peutic regimens include chemotherapy, chemoimmunotherapy,
and hematopoietic stem cell transplantation; experimental
treatments comprise small and targeting molecules. Still, 30−
50% of patients are resistant to treatment, progress rapidly, and
die of their disease. Novel first line therapies are often very
effective with response rates from 70% to 90%; however, many
patients relapse or develop resistant disease. For these
refractory patients and for patients characterized by particularly
adverse factors, novel treatment opportunities need to be
provided and novel, targeted therapies have to be explored.
Received: September 24, 2014
© XXXX American Chemical Society
A
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both the lymphoid and myeloid lineage (HL60, K562, CCRF,
Jurkat, SU-DHL6, SU-DHL9). Studies of the effect of the
identified hit on normal cells position this compound as a
potent and novel candidate in the hunt for new antitumor
agents.
RESULTS AND DISCUSSION
■
In an initial screening of an in-house library mainly based on
naturally occurring scaffolds, e.g., curcumins, imidazoles,
benzanilides, as well as chalcones, we have identified 3-(2-
naphthyl)-1-(2-ethoxyphenyl)-2-propen-1-one (1) as a highly
potent cytotoxic agent with IC50 values in the nanomolar range.
As initial screening panel, we used the three cell lines MEC1
(chronic lymphocytic leukemia), U-2940 (diffuse large B-cell
lymphoma), and OCI-LY7 (non-Hodgkin lymphoma) repre-
senting B-cell neoplasms.
Structurally seen, the small molecule compound 1 represents
a typical chalcone scaffold. It is characterized by an
orthoethoxy-substituted phenyl part as ring A and a 2-naphthyl
moiety symbolizing ring B. The focus of the present study was
to guide the systematic identification of a growth inhibitor with
specificity to neoplastic cells, as cytotoxicity toward healthy
cells is mostly associated with adverse effects, which can be
related to morbidity and mortality of the patient.22 To
discriminate in vitro between structural elements necessary
for potent cytotoxicity toward tumor cells, which at the same
time clearly show less activity on normal cells, the compounds
were additionally tested on the murine bone marrow fibroblast
cell line M2-10B4. This cell line is regularly used as feeder layer
when cultured with patient cells, providing additional support
and survival signals in particular in drug screens.23
Beginning with compound 1 as lead, we first started with
homologous variation of the 2-alkoxy substituent of ring A. In
the second step we used a bioisosteric replacement strategy to
optimize the ring B site of compound 1 (Figure 1). In the last
set of compounds we focused on the substitution pattern of
ring A with respect to the ethoxy group. The ortho, meta, and
para congeners of the three most active compounds were
generated and biologically investigated (Table 2). All the
compounds were obtained by base-catalyzed reaction of the
corresponding acetophenone with an appropriate benzaldehyde
derivative (Claisen−Schmidt condensation) (Scheme 1).
Initially, compounds were biologically tested in six
concentration steps (0.1, 1, 5, 10, 50, and 100 μM) using the
CellTiter-Blue cell viability assay (Promega, Madison, WI,
USA) and an incubation time of 48 h. In the case of a highly
active compound (IC50 < 1 μM) the tested concentration range
was shifted to 0.005, 0.01, 0.05, and 0.1 μM to define the IC50
more precisely. IC50 values were calculated from at least two
independent experiments with three replicates each. In order to
evaluate the biologic activity of the new compounds,
cytotoxicity was compared to that of fludarabine, an already
approved drug for the treatment of various B-cell malignancies.
The cytotoxic potential (IC50) of fludarabine was 42 μM in
MEC1, 2.0 μM in U-2940, and 106 μM in OCI-LY7 cells,
respectively. Differences in IC50 values for the different cell
types can be explained by the biological and genetic
particularities of each subtype of B-cell non-Hodgkin
lymphoma.24−28
Figure 1. SAR studies based on the lead compounds 1 were obtained
by structural modifications of ring A and ring B and resulted in
compound 15 as new hit compound. Cytotoxic impact of compounds
1 and 15 on three malignant hematological cell lines (MEC1, U-2940,
and OCI-LY7) as well as the mouse fibroblast cell line (M2−10B4)
was used in the lead structure optimization process.
a
Scheme 1. Synthetic Route of Compounds 1−21
a
Reagents and conditions: 50% NaOH, EtOH, room temp, 1−24 h.
vary the substituents of ring A in position 2. Hence, the first
derivatives synthesized represented the homologous series of
the alkoxy substituent and started by methoxy, followed by
propoxy and butoxy. The biological data (Table 1) clearly
showed the significance of this structural element: the methoxy
derivative 2 demonstrated an almost 10-fold loss of activity on
MEC1 and OCI-LY7 cells (0.43 and 0.46 μM) and half of the
activity toward U-2940 (4.1 μM), although the unwanted
cytotoxic potential on M2-10B4 (IC50 = 42 μM) was reduced.
Elongation of the ethoxy group of compound 1 by one more
methylene group to obtain the propoxy derivative 3
represented only half of the cytotoxic potency on the malignant
kind of cell lines (0.13, 5.3, and 0.10 μM vs. 0.06, 2.4, and 0.06
μM). The compound did not show any activity on the mouse
fibroblast cell line up to 100 μM. The inhibition of cell growth
demonstrated clear SAR depending on the length of the alkoxy
chain, as butoxy (compound 4) showed reduced activity
compared to the propoxy compound 3. Compound 5 with an
isopropoxy residue presented even weaker activity than
First Optimization Step. We used compound 1 as lead
structure to study the influence of the ortho ethoxy group with
respect to the biological activity. We kept the 3-(2-naphthyl)-1-
phenyl-2-propen-1-one backbone and started to systematically
B
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Table 1. Cytotoxic Activity of Compounds 1−15 on Three Malignant Hematological Cell Lines (MEC1, U-2940, and OCI-LY7)
b
as Well as the Mouse Fibroblast Cell Line (M2-10B4)
a
b
nt: not tested. For comparison, IC50 values (in μM) upon incubation with fludarabine, an approved drug for hematologic malignancies, were 42
(MEC1), 2.0 (U-2940), 105 (OCI-LY7), and >100 (M2- 10B4).
compounds 2 and 3. Moreover, by use of methyl instead of an
alkoxy substituent as in the case in compound 6, the inhibitory
activity on all three malignant cell lines decreased further to the
lower micromolar range (6.4, 10, and 4.9 μM) and also a
cytotoxic potential toward M2-10B4 was observed (72 μM).
With regard to the biological activity, already small changes of
the 2-substituent of ring A considerably influenced the
inhibitory activity with ethoxy being the most favorable
structural feature.
Second Optimization Step. As outlined above and
demonstrated in Figure 1, the second optimization step focused
on a bioisosteric replacement of ring B. The presence of a 6-
methoxy substituent on the naphthyl part (compound 7)
resulted in a cell line dependent loss of activity with no
cytotoxic potential on M2-10B4 cells. Further modifications of
the naphthyl moiety led to compound 8 with a “dissected”
aromatic ring. The vinylphenyl derivative was almost
equipotent in killing U-2940 cells (2.5 μM) but less active on
MEC1 and OCI-LY7 (0.20 and 0.50 μM) compared to
compound 1 but did not show any activity on mouse
fibroblasts. Further variations of ring B included a benzodioxole
moiety (compound 9) as well as the 1-naphthyl congener of
derivative 1, compound 10. Both modifications exhibited lower
activity on MEC1 (1.1 and 1.9 μM), U-2940 (9.5 and 7.4 μM),
and OCI-LY7 (0.13 and 1.5 μM). Compound 10 showed an
IC50 value of 17 μM on M2-10B4, which is about half as much
as for compound 1 (7.8 μM). The insertion of a third phenyl
ring led in the case of the anthracene derivative 11 to a
complete loss of activity (up to 100 μM) toward all four cell
lines, whereas with a phenanthrene moiety (compound 12) the
cytotoxic activity on the malignant cell lines was retained even
though it was a little bit lower compared to compound 1
(MEC1 1.6 μM, U-2940 40 μM, and OCI-LY7 0.68 μM).
Introduction of 5- and 3-thionaphthene (compound 13 and
14), respectively, resulted in a 10-fold loss of cytotoxic activity
in MEC1 and OCI-LY7 cells, whereas regarding U-2940 the 3-
C
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thionaphthene (0.86 μM) proved to be more potent than its
congener 13 (9.1 μM). This fact applies also for the healthy
fibroblasts: compound 13 (21 μM) shows lower and
compound 14 (8.2 μM) similar activity than the lead
compound 1 (7.8 μM). In compound 15 the 2-naphthyl was
replaced by a 2-indole ring system, which presented an
improvement of the biological activity on the malignant cell
lines (MEC 0.022 μM, U-2940 2.9 μM, and OCI-LY7 0.052
μM). Interestingly, the compound showed less cytotoxic
potential on healthy cells (19 μM) in comparison to compound
1 (7.8 μM). Together, from steps 1 and 2 compound 15
resulted as a hit with respect to hematological B-cell
malignancies.
Comparison of the Ortho, Meta, and Para Derivatives.
We systematically varied the position of the ethoxy group,
which proved as biologically essential in the first optimization
step. We have chosen compounds 1 (as original lead), 14, and
15 (the two most active compounds arising from the
optimization process) and synthesized and tested the
corresponding congeners. Table 2 gives the obtained biological
results. In all three sets the meta and para isomers were 10- to
100-fold less active than the ortho isomer. It can be clearly seen
that position 4 is being less preferred than position 3, which is
very obvious in the case of the sets of compounds 1 and 15.
Therefore, the position of the ethoxy substituent plays an
important role for the compounds’ cytotoxic activity and ortho
is proved to be the most favored pattern.
Together, compound 15 developed by two lead optimization
processes turned out to have the optimal structural require-
ments for potent inhibition of malignant hematological cell
lines with less biological activity on normal mouse fibroblasts.
Especially in conjunction with chalcone derivatives, the 3,4,5-
trimethoxyphenyl moiety is described as an essential structural
feature of potent cytotoxic compounds.29−32 However, also in
the case of other small molecules, for example, 1,5-diary-
limidazole derivatives, this substitution pattern of the aromatic
site proved to be the most favored.33 In the hunt for specified
cytotoxic compounds, we identified the 2-ethoxyphenyl part as
a further highly active structural principle in the context of
cytotoxic chalcone derivatives. Biological investigation of the
homologous series of this structural feature proved the ethoxy
substituent as most preferred. A recent study conducted by
Maioral et al. investigating apoptosis induction of 1-
naphthylchalcones in human acute leukemia cell lines described
the derivative with a 2,5-dimethoxy substituent on the site of
ring A as the most potent cytotoxic representative of the series
tested with an IC50 value of 40 μM on K562 and 21 μM on
Jurkat cells after 24 h of incubation.34 Moreover, the compound
was shown to be nontoxic to peripheral blood lymphocytes
with a cell viability of 99.89 10.69% at 50 μM compound
concentration after 24 h. These data can be seen in accordance
with the results obtained by the herein presented study: the 1-
naphthylchalcone with a 2-methoxy substituent in ring A
proved to be the most potent one. However, through
optimization of both ring A and ring B we were able to obtain
an even more potent cytotoxic compound on hematological cell
lines.
Table 2. Comparison of the Biological Activity of the Meta
(Position 3) and Para (Position 4) (MEC1, U-2940, and
a
OCI-LY7)
Effect of Compound 15 on Other Hematological Cell
Lines. As described above, hematologic malignancies are highly
heterogeneous in their biology and behavior. This and the fact
that some patient groups are resistant to therapy underline the
a
nd: no significant cytotoxic activity was detected up to 100 μM.
Figure 2. Cytotoxic effect of compound 15 on a variety of hematologic cell lines. CCRF, Jurkat: T-cell acute lymphoblastic leukemia. HL60, K562:
myeloid leukemia. SU-DHL6, SUDHL9: B-cell lymphoma. Viability was determined as described in the methods section and normalized to cells
incubated with vehicle only. Mean values plus standard deviation are shown.
D
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a
Table 3. IC50 Values in μM Determined for Compound 15 in Different Cell Types
CCRF
Jurkat
HL60
K562
SUDHL6
SUDHL9
CLL suspension
HD suspension
CLL coculture
15
0.032
nd
0.038
nd
0.016
nd
n.d.
nd
0.056
nd
0.031
nd
1.2
1.9
14
14
0.68
10
fludarabine
a
For comparison, the standard therapeutic drug fludarabine was tested in parallel in experiments using primary cells. CCRF, Jurkat: T-cell acute
leukemia. HL60, K562: myeloid leukemia. SU-DHL6, SU-DHL9: B-cell lymphoma. CLL suspension: primary CLL cells in suspension culture. HD
suspension: healthy donor cells in suspension culture. CLL coculture: CLL cells in coculture with M2-10B4 fibroblast cells. nd: not determined.
Figure 3. Comparing cytotoxic efficacies of compound 15 and fludarabine in three cell lines of the B-cell lineage: (A) MEC1; (B) U-2940; (C) OCI-
LY7. Results were normalized to incubations with vehicle only. Mean values plus standard deviation are shown. IC50 values (μM) were 0.022 and 42
(MEC1), 2.9 and 2.0 (U-2940), 0.052 and 106 (OCI-LY7) for compound 15 and fludarabine, respectively.
urgent need for new therapeutic drugs. Thus, after having
developed compound 15 in our screening panel, we sought to
test this potent compound on other cell lines representing a
variety of hematologic neoplasms: two T-cell acute leukemia
E
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Figure 4. Comparison of cytotoxic efficacy of compound 15 and fludarabine in primary cells: (A) peripheral blood mononuclear cells (PBMCs) of
chronic lymphocytic leukemia (CLL) patients (N = 15) tested in suspension culture; (B) PBMC of healthy individuals (N = 4) in suspension
culture; (C) PBMC of CLL patients (N = 9) cultured over a layer of mouse fibroblast cells (M2-10B4). Results were normalized to incubations with
vehicle only. Mean values plus standard deviation are shown. IC50 values are listed in Table 3.
cell lines (CCRF, Jurkat), two myeloid leukemia cell lines
(HL60, K562), and two additional B-cell lines (SU-DHL6, SU-
DHL9). Viability was assessed as described in the methods
section; data are shown in Figure 2. Compound 15
considerably reduced the viability of cell lines to less than
30% of control already at concentrations of <1 μM. The
exception was the chronic myeloid leukemia line K562.
Although viability was reduced to 60% already at low
concentrations, toxicity did not increase with increasing
concentrations of compound 15 (Figure 2). However, this
cell line is derived from a patient in blast crisis and is
characterized by a number of genetic aberrations associated
with aggressive disease and resistance to treatment.35 In all
other cell lines, the biologic activity of compound 15 reached
levels comparable to standard drugs used in therapy.36−42 IC50
values for compound 15 are listed in Table 3.
Comparison of Compound 15 with Fludarabine on
MEC1, OCI-LY7, and U-2940. To put the efficacy of
compound 15 into perspective, we screened the cell lines of
our screening panel also with fludarabine. [(2R,3R,4S,5R)-5-(6-
F
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and J values are reported in hertz. Mass spectra were obtained by a
Hewlett-Packard (GC, 5890; MS, 5970) spectrometer. The purity of
the synthesized compounds was established by combustion analysis
with a PerkinElmer 2400 CHN elemental analyzer and was within
0.4%. All biologically evaluated compounds were demonstrated to
exist in >95% purity by CHN analysis. Solutions in organic solvents
were dried over anhydrous sodium sulfate.
General Synthetic Procedure for Compounds 1−21. A
solution of 2 mmol of the appropriate acetophenone derivative and
3 mL of 50% NaOH in 5 mL of ethanol was stirred at room
temperature for 30 min. Then an amount of 2 mmol of the
corresponding aldehyde derivative, dissolved in 3 mL of ethanol, was
added and stirred at room temperature. After complete conversion of
the starting materials (monitored by TLC), the reaction mixture was
poured into ice−water or extracted with ethyl acetate. The organic
layer was dried over anhydrous Na2SO4 and evaporated. The so-
obtained crude product was purified by column chromatography or by
recrystallization in ethanol.
3-(2-Naphthyl)-1-(2-ethoxyphenyl)-2-propen-1-one (1).
Yield: 0.16 g (27%) yellow solid. Mp: 96−97 °C. 1H NMR
(CDCl3) δ 8.14−7.39 (m, 11H), 7.21−6.85 (m, 2H), 4.14 (q, J =
6.9 Hz, 2H), 1.43 (t, J = 6.9 Hz, 3H). 13C NMR (CDCl3) δ 192.7,
157.7, 142.6, 134.2, 133.4, 133.0, 132.8, 130.6, 130.3, 129.3, 128.6,
128.5, 127.7, 127.4, 127.1, 126.6, 123.6, 120.7, 112.6, 64.2, 14.8. MS
m/z 302 (13%, M+), 287 (2%), 273 (3%), 151 (77%), 68 (100%).
Anal. Calcd for C21H18O2·0.2H2O: C 82.44; H 6.06. Found: C 82.07;
H 5.62.
1-(2-Methoxyphenyl)-3-(2-naphthyl)-2-propen-1-one (2).
Yield: 0.35 g (60%) yellow solid. Mp: 81−83 °C. 1H NMR
(CDCl3) δ 7.96 (s, 1H), 7.91−7.70 (m, 5H), 7.65 (dd, J = 7.6 Hz, J
= 1.6 Hz, 1H), 7.68−7.38 (m, 4H), 7.12−6.96 (m, 2H), 3.92 (s, 3H).
13C NMR (CDCl3: δ 193.0, 158.1, 143.4, 134.2, 133.3, 132.8, 132.6,
130.4, 130.3, 129.3, 128.6, 128.5, 127.8, 127.2, 127.2, 126.6, 123.7,
120.7, 111.6, 55.8. MS m/z 288 (61%, M+), 287 (42%), 229 (39%),
152 (100%), 135 (47%). Anal. Calcd for C20H16O2: C 83.31; H 5.59.
Found: C 83.07; H 5.64.
Amino-2-fluoropurin-9-yl)-3,4-dihydroxyoxolan-2-yl]-
methoxyphosphonic acid is a purine analogue that inhibits
DNA synthesis through block of function of a number of
enzymes required for DNA replication. It is used in
combinations with rituximab, cyclophosphamide, dexametha-
sone, and mitoxantrone in various treatment regimens for
indolent non-Hodgkin lymphomas. It also is part of the
standard FCR therapy regimen for chronic lymphocytic
leukemia (CLL) (fludarabine, cyclophosphamide, plus ritux-
imab) as defined by the German CLL study group.43 Although
the function of compound 15 most likely is completely different
compared to fludarabine, we decided to test the chalcone
against a drug that right now may be considered as one of the
standard chemotherapeutic agents in the treatment of
hematologic malignancies.
As can be seen in Figure 3, compound 15 displayed much
higher toxicity compared to fludarabine in MEC1 and OCI-
LY7; furthermore it had comparable efficacy in U-2940 cells.
This was also reflected in IC50 values, which were 1−3 log
smaller for compound 15 compared to fludarabine (listed in
Figure 3). Of note, while fludarabine had variable impact on
viability in these three cell lines, the effect of compound 15 was
more consistent. This not only reflects the biological and
genetic differences involved but also suggests a high therapeutic
potential of compound 15 in future antitumor regimens.24−26,28
Investigating the Effect of Compound 15 in Primary
Cells. As a last step, we wanted to evaluate cytotoxicity of
compound 15 in primary cells and compare its activity to the
impact of fludarabine. For this, peripheral blood mononuclear
cells (PBMCs) of CLL patients were used in two experimental
settings. They were cultured in conventional suspension culture
being exposed to increasing concentrations of the tested
compound. However, CLL cells need close contact with the
tumor microenvironment depending on and receiving survival
signals from stromal cells, which has impact on response to
therapy. Therefore, as a second approach, CLL cells were
cultured over a layer of mouse fibroblasts (M2-10B4) to
include this biological aspect in drug evaluation. Such an
approach is often used in preclinical assessment and testing of
drugs in CLL cells.23 Cells from healthy donors (HDs) were
tested as control.
3-(2-Naphthyl)-1-(2-propoxyphenyl)-2-propen-1-one (3).
1
Yield: 0.42 g (66%) yellow oil. H NMR (CDCl3) δ 7.96 (s, 1H),
7.89−7.65 (m, 6H), 7.65−7.39 (m, 4H), 7.09−6.93 (m, 2H), 4.02 (t, J
= 6.4 Hz, 2H), 1.92−1.70 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13C NMR
(CDCl3: δ 192.7, 157.8, 142.5, 134.2, 133.4, 133.0, 132.7, 130.6, 130.3,
129.4, 128.6, 128.5, 127.7, 127.5, 127.1, 126.6, 123.7, 120.6, 112.4,
70.1, 22.6, 10.7. MS m/z 316 (1%, M+), 141 (9%), 85 (27%), 43
(100%), 41 (20%). Anal. Calcd for C22H20O2: C 83.51; H 6.37.
Found: C 83.11; H 6.09.
Figure 4 shows the reduction of viability in the tested
settings, Table 3 lists the IC50 values for both compound 15
and fludarabine in CLL and HD primary cells. We observed
higher cytotoxicity of compound 15 in PBMC from CLL
compared to HD in suspension culture (Figure 4A and Figure
4B). Also, in comparison, fludarabine had less effect on tumor
cells and displayed a greater reduction of viability in healthy
cells. This indicates a more favorable therapeutic ratio for
compound 15. In coculture a protective effect for CLL cells
usually is observed, as in our experiments with fludarabine
(Figure 4C). In contrast, when incubated with compound 15,
CLL cells showed a reduction in viability comparable to that
observed in suspension culture. Again, this indicates a high
therapeutic potential of this novel compound.
1-(2-Butoxyphenyl)-3-(2-naphthyl)-2-propen-1-one (4).
Yield: 0.23 g (48%) yellow oil. H NMR (CDCl3) δ 7.98 (s, 1H),
1
7.90−7.67 (m, 6H), 7.63 (s, 1H), 7.57−7.41 (m, 3H), 7.11−6.95 (m,
2H), 4.09 (t, J = 6.3 Hz, 2H), 1.88−1.70 (m, 2H), 1.59−1.38 (m, 2H),
0.87 (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3) δ 192.7, 157.9, 142.5,
134.2, 133.4, 133.0, 132.8, 130.6, 130.3, 129.3, 128.6, 128.5, 127.8,
127.5, 127.1, 126.6, 123.7, 120.6, 112.4, 68.3, 31.3, 19.4, 13.7. MS m/z
330 (12%, M+), 273 (38%), 189 (45%), 141 (100%), 121 (41%). Anal.
Calcd for C23H22O2: C 83.60; H 6.71. Found: C 83.17; H 6.49.
3-(2-Naphthyl)-1-[2-(2-propanyloxy)phenyl]-2-propen-1-
1
one (5). Yield: 0.48 g (76%) yellow solid. Mp: 82−85 °C. H NMR
(CDCl3) δ 7.98 (s, 1H), 7.91−7.40 (m, 10H), 7.09−6.96 (m, 2H),
4.66 (sept, J = 7.0 Hz, 1H), 1.37 (d, J = 7.0 Hz, 3H). 13C NMR
(CDCl3) δ 192.9, 156.5, 142.2, 134.0, 133.3, 132.7, 130.6, 130.3, 130.2,
128.5, 128.4, 127.6, 127.4, 127.0, 126.5, 123.4, 120.6, 114.2, 71.0, 22.0.
MS m/z 316 (25%, M+), 273 (82%), 154 (100%), 152 (95%), 43
(39%). Anal. Calcd for C22H20O2·0.3H2O: C 82.11; H 6.45. Found: C
82.05; H 6.33.
EXPERIMENTAL SECTION
■
1-(2-Methylphenyl)-3-(2-naphthyl)-2-propen-1-one (6).
Chemistry. Unless stated otherwise, all chemicals were obtained
from Sigma-Aldrich or Apollo Europe and were of analytical grade.
Melting points were determined on a Kofler hot stage apparatus and
1
Yield: 0.25 g (45%) yellow oil. H NMR (CDCl3) δ 7.93 (s, 1H),
7.90−7.78 (m, 3H), 7.76−7.27 (m, 8H), 7.20 (s, 1H), 2.47 (s, 3H).
13C NMR (CDCl3) δ 197.0, 146.6, 139.6, 137.4, 134.8, 133.7, 132.6,
131.8, 131.1, 130.9, 129.2, 129.1, 128.5, 128.3, 127.9, 127.3, 127.2,
125.9, 124.0, 20.7. MS m/z 272 (30%, M+), 141 (61%), 119 (100%),
1
are uncorrected. The H and 13C NMR spectra were recorded on a
BrukerAvance DPx200 (200 and 50 MHz). Chemical shifts are
reported in δ units (ppm) relative to Me4Si line as internal standard,
G
DOI: 10.1021/jm501848m
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Journal of Medicinal Chemistry
Article
91 (55%), 43 (37%). Anal. Calcd for C20H16O: C 88.20; H 5.92.
Found: C 87.88; H 5.66.
(m, 3H), 7.80−7.56 (m, 3H), 7.50−7.33 (m, 3H), 7.10−6.90 (m, 2H),
4.12 (q, J = 7.0 Hz, 2H), 1.41 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3)
δ 192.3, 157.7, 140.5, 137.3, 134.1, 133.1, 132.5, 130.7, 129.2, 128.3,
127.4, 125.0, 124.8, 122.9, 122.2, 120.7, 112.6, 64.2, 14.8. MS m/z 308
(43%, M+), 147 (100%), 121 (98%), 43 (89%), 41 (30%). Anal. Calcd
for C19H16O2S: C 74.00; H 5.23. Found: C 73.71; H 4.92.
1-(2-Ethoxyphenyl)-3-(1H-indole-2-yl)-2-propen-1-one (15).
Yield: 0.22 g (38%) brown solid. Mp: 124−126 °C. 1H NMR (CDCl3)
δ 9.05 (sbr, 1H), 7.72−7.54 (m, 3H), 7.49−6.89 (m, 7H), 6.87−6.80
(m, 1H), 4.08 (q, J = 7.0 Hz, 2H), 1.37 (t, J = 7.0 Hz, 3H). 13C NMR
(CDCl3) δ 192.9, 157.4, 138.0, 134.3, 133.3, 132.8, 130.3, 129.2, 128.5,
124.9, 124.5, 121.4, 120.6, 120.4, 112.6, 111.3, 108.8, 64.3, 14.7. MS
m/z 291 (63%, M+), 262 (42%), 130 (61%), 119 (100%), 91 (49%).
Anal. Calcd for C19H17NO2: C 78.33; H 5.88; N 4.81. Found: C 78.24;
H 5.67; N 4.79.
3-(2-Naphthyl)-1-(3-ethoxyphenyl)-2-propen-1-one (16).
Yield: 0.21 g (34%) yellow solid. Mp: 97−100 °C. 1H NMR
(CDCl3) δ 8.03−7.76 (m, 6H), 7.66−6.37 (m, 6H), 7.15−7.05 (m,
1H), 4.12 (q, J = 7.0 Hz, 2H), 1.45 (t, J = 7.0 Hz, 3H). 13C NMR
(CDCl3) δ 190.4, 159.4, 145.0, 139.8, 134.6, 133.5, 132.6, 130.8, 129.8,
128.9, 128.8, 128.0, 127.5, 126.9, 123.9, 122.4, 121.1, 119.8, 113.8,
63.9, 15.0. MS m/z 302 (100%, M+), 181 (36%), 152 (84%), 128
(18%), 65 (15%). Anal. Calcd for C21H18O2: C 83.42; H 6.00. Found:
C 83.12; H 5.95.
3-(2-Naphthyl)-1-(4-ethoxyphenyl)-2-propen-1-one (17).
Yield: 0.44 g (73%) yellow solid. Mp: 159−161 °C. 1H NMR
(CDCl3) δ 8.09−7.48 (m, 11H), 6.97 (d, J = 8.8 Hz, 2H), 4.11 (q, J =
7.0 Hz, 2H), 1.45 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3) δ 188.8,
163.0, 144.1, 134.4, 133.5, 132.8, 131.1, 131.0, 130.6, 128.8, 128.8,
127.9, 127.4, 126.9, 123.9, 122.2, 114.5, 63.9, 14.9. MS m/z 302 (99%,
M+), 273 (36%), 152 (100%), 121 (59%), 65 (37%). Anal. Calcd for
C21H18O2: C 83.42; H 6.00. Found: C 83.07; H 5.87.
1-(2-Ethoxyphenyl)-3-(6-methoxy-2-naphthyl)-2-propen-1-
1
one (7). Yield: 0.46 g (68%) yellow solid. Mp: 97−99 °C. H NMR
(CDCl3) δ 7.90 (s, 1H), 7.84−7.64 (m, 5H), 7.60−7.39 (m, 2H),
7.20−7.10 (m, 2H), 7.08−6.94 (m, 2H), 4.13 (q, J = 7.0 Hz, 2H), 3.92
(s, 3H), 1.43 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3) δ 192.9, 158.8,
157.6, 143.1, 135.7, 132.9, 130.7, 130.5, 130.2, 130.1, 129.5, 128.8,
127.5, 126.5, 124.4, 120.7, 119.4, 112.6, 106.0, 64.3, 55.4, 14.9. MS m/
z 332 (39%, M+), 171 (100%), 139 (65%), 121 (61%), 65 (35%).
Anal. Calcd for C22H20O3: C 79.50; H 6.06. Found: C 79.45; H 5.86
3-(3-Ethenylphenyl)-1-(2-ethoxyphenyl)-2-propen-1-one
1
(8). Yield: 0.31 g (56%) yellow oil. H NMR (CDCl3) δ 7.72−7.24
(m, 8H), 7.08−6.91 (m, 2H), 6.74 (dd, J = 17.6 Hz, J = 10.8 Hz, 1H),
5.78 (dd, J = 17.6 Hz, J = 0.7 Hz, 1H), 5.29 (dd, J = 10.8 Hz, J = 0.7
Hz, 1 H), 4.12 (q, J = 7.0 Hz, 2H), 1.42 (t, J = 7.0 Hz, 3H). 13C NMR
(CDCl3) δ 192.5, 157.6, 142.2, 138.1, 136.1, 135.4, 133.0, 130.5, 129.1,
129.0, 127.7, 127.4, 126.1, 120.6, 114.6, 112.5, 64.1, 14.8. MS m/z 278
(7%, M+), 161 (100%), 128 (36%), 121 (74%), 65 (23%). HRMS for
C19H19O2: 279.1385. Found: 279.1389
3-(1,3-Benzodioxol-5-yl)-1-(2-ethoxyphenyl)-2-propen-1-
1
one (9). Yield: 0.29 g (49%) brown solid. Mp: 65−70 °C. H NMR
(CDCl3) δ 7.69−7.27 (m, 4H), 7.13−6.92 (m, 4H), 6.86−6.77 (m,
1H), 6.00 (s, 2H), 4.13 (q, J = 7.0 Hz, 2H), 1.43 (t, J = 7.0 Hz, 3H).
13C NMR (CDCl3) δ 192.6, 157.6, 149.5, 148.3, 142.5, 132.8, 130.5,
129.7, 129.5, 125.4, 124.8, 120.7, 112.6, 108.6, 106.5, 101.5, 64.2, 14.9.
MS m/z 296 (4%, M+), 149 (19%), 135 (29%), 121 (25%), 43
(100%). Anal. Calcd for C18H16O4·0.5H2O: C 70.81; H 5.60. Found:
C 70.82; H 5.35.
1-(2-Ethoxyphenyl)-3-(1-naphthyl)-2-propen-1-one (10).
Yield: 0.47 g (78%) yellow solid. Mp: 109−110 °C. 1H NMR
(CDCl3) δ 8.57−8.42 (m, 1H), 8.30−8.28 (m, 1H), 7.95−7.78 (m,
3H), 7.77−7.68 (m, 1H), 7.64−7.38 (m, 5H), 7.11−6.92 (m, 2H),
4.13 (q, J = 7.0 Hz, 2H), 1.42 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3)
δ 192.6, 157.7, 139.3, 133.7, 133.1, 132.6, 131.7, 130.7, 130.3, 129.7,
129.3, 128.7, 126.7, 126.2, 125.4, 124.9, 123.6, 120.7, 112.6, 64.2, 14.8.
MS m/z 302 (40%, M+), 152 (88%), 141 (54%), 121 (98%), 43
(100%). Anal. Calcd for C21H18O2·0.25toluene: C 83.97; H 6.20.
Found: C 84.00; H 5.82.
3-(9-Anthranyl)-1-(2-ethoxyphenyl)-2-propen-1-one (11).
Yield: 0.54 g (76%) yellow solid. Mp: 93−96 °C. 1H NMR
(CDCl3) δ 8.66−8.51 (m, 1H), 8.44−8.25 (m, 3H), 8.05−7.91 (m,
2H), 7.81−7.72 (m, 1H), 7.55−7.34 (m, 6H), 7.12−6.99 (m, 1H),
6.98−6.88 (m, 1H), 4.08 (q, J = 6.9 Hz, 2H), 1.34 (t, J = 6.9 Hz, 3H).
13C NMR (CDCl3) δ 192.5, 157.7, 139.7, 135.7, 133.2, 131.3, 130.7,
130.4, 129.5, 129.3, 128.8, 128.1, 126.1, 125.5, 125.3, 120.8, 112.4,
64.2, 14.8. MS m/z 352 (31%, M+), 202 (65%), 149 (61%), 121
(100%), 65 (21%). Anal. Calcd for C25H20O2·0.14H2O: C 84.60; H
5.76. Found: C 84.62; H 5.52.
3-(1-Benzothien-3-yl)-1-(3-ethoxyphenyl)-2-propen-1-one
1
(18). Yield: 0.21 g (34%) yellow solid. Mp: 103−106 °C. H NMR
(CDCl3) δ 8.15−8.07 (m, 2H), 7.92−7.88 (m, 2H), 7.65−7.38 (m,
6H), 7.16−7.11 (m, 1H), 4.12 (q, J = 7.0 Hz, 2H), 1.45 (t, J = 7.0 Hz,
3H). 13C NMR (CDCl3) δ 190.3, 159.5, 140.7, 139.7, 137.5, 136.5,
132.5, 129.8, 128.9, 125.3, 125.2, 123.3, 122.7, 122.4, 121.0, 119.9,
113.8, 63.9, 15.0. MS m/z 308 (60%, M+), 279 (26%), 187 (48%), 115
(100%), 69 (56%). Anal. Calcd for C19H16O2S: C 74.00; H 5.23.
Found: C 73.99; H 4.87.
3-(1-Benzothien-3-yl)-1-(3-ethoxyphenyl)-2-propen-1-one
1
(19). Yield: 0.45 g (74%) yellow solid. Mp: 129−130 °C. H NMR
(CDCl3) δ 8.14−8.04 (m, 4H), 7.92−7.88 (m, 2H), 7.65 (AB-system,
JAB = 15.6 Hz, 1H), 7.50−7.41 (m, 2H), 7.00−6.96 (m, 2H), 4.12 (q, J
= 7.0 Hz, 2H), 1.46 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3) δ 188.7,
163.1, 140.7, 137.6, 132.7, 131.1, 131.0, 128.3, 125.3, 125.2, 123.2,
122.5, 122.4, 114.5, 64.0, 14.9. MS m/z 308 (100%, M+), 279 (27%),
251 (25%), 115 (35%), 65 (10%). Anal. Calcd for C19H16O2S·
0.13H2O: C 73.44; H 5.27. Found: C 73.24; H 4.87.
1-(2-Ethoxyphenyl)-3-(9-phenanthrenyl)-2-propen-1-one
1
1-(3-Ethoxyphenyl)-3-(1H-indole-2-yl)-2-propen-1-one (20).
Yield: 0.18 g (31%) brown solid. Mp: 156−158 °C. 1H NMR (CDCl3)
δ 8.99 (s, 1H), 7.85 (d, J = 15.6 Hz, 1H), 7.65−7.07 (m, 9H), 6.91 (s,
1H), 4.03 (q, J = 7.0 Hz, 2H), 1.40 (t, J = 7.0 Hz, 3H). 13C NMR
(CDCl3) δ190.3, 159.4, 139.7, 135.0, 134.3, 129.8, 128.7, 125.1, 121.9,
121.0, 120.9, 119.9, 119.6, 113.7, 111.5, 110.3, 63.9, 14.9. MS m/z 291
(100%, M+), 262 (71%), 234 (49%), 170 (35%), 115 (32%). Anal.
Calcd for C19H17NO2: C 78.33; H 5.88. Found: C 78.20; H 5.56.
1-(4-Ethoxyphenyl)-3-(1H-indole-2-yl)-2-propen-1-one (21).
Yield: 0.09 g (15%) brown solid. Mp: 178−181 °C. 1H NMR (CDCl3)
δ 8.83 (sbr, 1H), 8.03−6.90 (m, 11H), 4.07 (q, J = 7.0 Hz, 2H), 1.43
(t, J = 7.0 Hz, 3H). 13C NMR (CDCl3) δ 188.5, 163.1, 138.1, 134.0,
131.0, 130.9, 128.8, 125.0, 121.8, 120.9, 119.5, 114.5, 111.4, 109.8,
64.0, 14.9. MS m/z 291 (100%, M+), 262 (43%), 234 (29%), 117
(44%), 65 (30%). Anal. Calcd for C19H17NO2: C 78.33; H 5.88, N
4.81. Found: C 78.18; H 5.40, N 4.77.
(12). Yield: 0.38 g (54%) yellow solid. Mp: 117−120 °C. H NMR
(CDCl3) δ 8.78−8.61 (m, 2H), 8.54−8.41 (m, 1H), 8.33−8.21 (m,
1H), 8.06 (s, 1H), 7.94−7.84 (m, 1H), 7.80−7.59 (m, 6H), 7.57−7.41
(m, 1H), 7.12−6.94 (m, 2H), 4.18 (q, J = 6.9 Hz, 2H), 1.48 (t, J = 6.9
Hz, 3H). 13C NMR (CDCl3) δ 192.5, 157.8, 140.0, 133.2, 131.8, 131.2,
131.0, 130.7, 130.4, 130.3, 129.3, 129.1, 127.5, 127.0, 126.9, 126.4,
124.5, 123.1, 122.6, 120.8, 112.6, 64.3, 14.9. MS m/z 352 (49%, M+),
202 (83%), 191 (46%), 121 (100%), 57 (53%). Anal. Calcd for
C25H20O2: C 85.20; H 5.72. Found: C 85.05; H 5.44.
3-(5-Benzo[b]thienyl)-1-(2-ethoxyphenyl)-2-propen-1-one
1
(13). Yield: 0.090 g (14%) yellow solid. Mp: 104−106 °C. H NMR
(CDCl3) δ 8.03−7.97 (m, 1H), 7.94−7.33 (m, 8H), 7.10−6.94 (m,
2H), 4.15 (q, J = 7.0 Hz, 2H), 1.44 (t, J = 7.0 Hz, 3H). 13C NMR
(CDCl3) δ 192.8, 157.7, 142.9, 140.1, 132.9, 131.7, 130.6, 129.5, 127.5,
126.9, 124.6, 124.1, 123.2, 122.9, 120.7, 112.6, 64.3, 14.9. MS m/z 308
(11%, M+), 279 (14%), 161 (38%), 147 (100%), 121 (37%), 65
(21%). Anal. Calcd for C19H16O2S·0.15H2O: C 73.36; H 5.28. Found:
C 73.44; H 4.95.
Cell Lines and Patients. Hematological cell lines were acquired
from the Leibniz Institute DSMZ-German Collection of Micro-
organisms and Cell Cultures (www.dsmz.de) and the American Type
Culture Collection (http://www.lgcstandards-atcc.org/). Sixteen
3-(1-Benzothien-3-yl)-1-(2-ethoxyphenyl)-2-propen-1-one
1
(14). Yield: 0.39 g (63%) yellow oil. H NMR (CDCl3) δ 8.13−7.81
H
DOI: 10.1021/jm501848m
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Journal of Medicinal Chemistry
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patients diagnosed with chronic lymphocytic leukemia at the Division
of Hematology and Hemostaseology at the Vienna General Hospital
and four healthy donors were included in the study. All participants
signed informed consent according to the Declaration of Helsinki
(Ethics Committee No. EK 1722/2012). Peripheral blood mono-
nuclear cells (PBMCs) were isolated using standardized Ficoll−
Hypaque gradient centrifugation (Seromed, Berlin, Germany) and
stored in liquid nitrogen until use.
Cell Culture. JURKAT and CCRF-CEM (T-cell acute lympho-
blastic leukemia), HL60 and K562 representing acute and chronic
myeloid leukemia, respectively, the chronic lymphocytic leukemia cell
line MEC1, and the diffuse large B-cell lymphoma cell line U-2940
were cultured in Gibco RPMI 1640 + GlutaMAX (Life Technologies,
Carlsbad, CA, USA) containing phenol red and supplemented with
10% fetal bovine serum gold (FBS gold; PAA Laboratories, Pasching,
Austria) and 1% PenStrep (100 U/mL penicillin and 100 μg/mL
streptomycin; PAA Laboratories) and maintained at 37 °C in
humidified atmosphere with 5% CO2. The diffuse large B-cell
lymphoma cell line OCI-LY7 was cultured in Gibco IMDM +
GlutaMAX and 25 mM HEPES (Life Technologies, Carlsbad, CA,
USA) containing phenol red and 20% FBS gold and 1% PenStrep.
Primary Cells. Primary cells were thawed in phenol red-free Gibco
RPMI 1640 + L-glutamine (Life Technologies, Carlsbad, CA, USA)
supplemented with 20% FBS gold and cultured overnight at standard
conditions before cells were incubated with the compounds.
Viability Tests. All experiments with hematological cell lines were
done using phenol red-free Gibco RPMI 1640 + L-glutamine
supplemented with 1% PenStrep and 10% FBS gold except
experiments with OCI-LY7 which were carried out in Gibco IMEM
+ GlutaMAX and 25 mM HEPES containing phenol red, 20% FBS
gold, and 1% PenStrep. Cells were incubated in triplicates with
increasing concentrations of the compounds for 48 h. Changes in
viability were determined using the CellTiter-Blue cell viability assay
(Promega, Madison, WI, USA) following instructions of the
manufacturer. IC50 values were calculated from two independent
experiments using GraphPad Prism5 software.
Primary cells were incubated at a density of 300 000 cells per well in
96-well plates in a total volume of 100 μL of phenol red-free Gibco
RPMI 1640 + L-glutamine supplemented with 20% FBS gold with
increasing concentrations of compounds for 48 h. For coculture
experiments, 150 000 M2-10B4 mouse fibroblast cells were seeded
into 12-well plates and allowed to form a confluent layer overnight.
Supernatant was replaced by 3 million primary cells in a total volume
of 1 mL of medium and incubated with increasing concentrations of
compounds for 48 h. Viability was measured as described above.
Experiments with primary cells were done in triplicate. IC50 values
were calculated from the mean values of all samples used in the various
experiments using GraphPad Prism5 software.
ABBREVIATIONS USED
■
CCR2, CC chemokine receptor 2; CCL2, CC chemokine
ligand 2; CCR5, CC chemokine receptor 5; TLC, thin layer
chromatography
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +43 (0)1 4277 55003. Fax: +43 (0)1 4277 95514. E-
mail: thomas.erker@univie.ac.at.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Joint Research Cluster of the
University of Vienna and Medical University of Vienna
“Platform for Accelerated Drug Optimization in Malignant
Disease (PADO)” http://forschungscluster.meduniwien.ac.at/
pado/?L=0.
I
DOI: 10.1021/jm501848m
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Journal of Medicinal Chemistry
Article
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