Synthesis and biological evaluation of novel tamoxifen analogues

Article abstract of DOI:10.1016/j.bmc.2013.05.012

A collection of compounds, structurally related to the anticancer drug tamoxifen, used in breast cancer therapy, were designed and synthesized as potential anticancer agents. McMurry coupling reaction was used as the key synthetic step in the preparation of these analogues and the structural assignment of E, Z isomers was determined on the basis of 2D-NOESY experiments. The compounds were evaluated for their antiproliferative activity on breast cancer (MCF-7), cervix adenocarcinoma (HeLa) and biphasic mesothelioma (MSTO-211H) human tumor cell lines. The estrogen like properties of the novel compounds were compared with those of the untreated controls using an estrogen responsive element-based (ERE) luciferase reporter assay and compared to 17β-estradiol (E2). Finally, with the aim to correlate the antiproliferative activity with an intracellular target(s), the effect on relaxation activity of DNA topoisomerases I and II was assayed.

Full text of DOI:10.1016/j.bmc.2013.05.012

Bioorganic & Medicinal Chemistry 21 (2013) 4120–4131
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel tamoxifen analogues
d
Michael S. Christodoulou a,b, Nikolas Fokialakis c, Daniele Passarella b, , Aída Nelly García-Argáez ,
⇑
a,
Ornella Maria Gia d, Ingemar Pongratz e, Lisa Dalla Via d, , Serkos A. Haroutounian
⇑
⇑
a Chemistry Laboratory, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece
b Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy
c Division of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, Greece
d Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Via F. Marzolo 5, 35131 Padova, Italy
e Fenix Scientific
a r t i c l e i n f o
a b s t r a c t
Article history:
A collection of compounds, structurally related to the anticancer drug tamoxifen, used in breast cancer
therapy, were designed and synthesized as potential anticancer agents. McMurry coupling reaction
was used as the key synthetic step in the preparation of these analogues and the structural assignment
of E, Z isomers was determined on the basis of 2D-NOESY experiments. The compounds were evaluated
for their antiproliferative activity on breast cancer (MCF-7), cervix adenocarcinoma (HeLa) and biphasic
mesothelioma (MSTO-211H) human tumor cell lines. The estrogen like properties of the novel com-
pounds were compared with those of the untreated controls using an estrogen responsive element-based
(ERE) luciferase reporter assay and compared to 17b-estradiol (E2). Finally, with the aim to correlate the
antiproliferative activity with an intracellular target(s), the effect on relaxation activity of DNA topoi-
somerases I and II was assayed.
Received 19 March 2013
Revised 2 May 2013
Accepted 8 May 2013
Available online 15 May 2013
Keywords:
Tamoxifen derivatives
Breast cancer (MCF-7)
Cervix adenocarcinoma (HeLa)
Biphasic mesothelioma (MSTO-211H)
Topoisomerase
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
pling.15 The novel derivatives (Fig. 1), maintain the triarylethylene
skeleton of tamoxifen, bearing OH groups in position 4 of the phe-
Tamoxifen (1, Fig. 1) is a triarylethylene compound that has
been widely used in the treatment of breast cancer.1,2 In addition
to acute cancer therapy, tamoxifen is used as a preventative treat-
ment3 for high risk women as well as in long-term adjuvant ther-
apy.4 However, the use of tamoxifen has also been associated with
increased risk of endometrial5,6 and uterine7 cancers as well as in-
creased possibility of suffering a pulmonary embolism, stroke, or
deep vein thrombosis.8 Tamoxifen is converted to the active
metabolite 4-hydroxytamoxifen (2, Fig. 1) which acts primarily
through inhibition of the estrogen receptor transcriptional activ-
ity.9 Furthermore, other mechanisms have been documented
which may or may not be estrogen receptor dependent10 and in-
clude induction of apoptosis,11 interference with the insulin-like
growth factor I receptor,12 and suppression of telomerase activity
by inhibition of protein kinase C.13
nyl moieties of tamoxifen and substitute the side chain of tamox-
ifen with an amide side chain. The in vitro antiproliferative
activity of these compounds was evaluated against three human
tumor cell lines, MCF-7 (breast cancer), HeLa (cervix adenocarci-
noma) and MSTO-211H (biphasic mesothelioma). Furthermore,
we evaluated the estrogen like properties of the novel compounds
using an ERE luciferase assay in HC11 cells (mouse mammary epi-
thelial cells). The ERE luciferase reporter construct which is stably
integrated into these cells harbors a transgene composed of 3 con-
sensus ERE driving the expression of firefly luciferase protein,
which allows the detection of estrogen activity by light produced
by the luciferase enzyme. Finally, the effect on topoisomerases I
and II was assayed to determine the potential intracellular
target(s).
As a follow-up to our interest in the synthesis of anticancer
compounds,14 we describe in the present study, the synthesis of
novel tamoxifen derivatives (Fig. 1) via McMurry reaction cou-
2. Results and discussion
2.1. Chemistry
⇑
Corresponding authors. Tel.: +39 02 50314081; fax: +39 02 50314078 (D.P.);
tel.: +39 0498275712; fax: +39 0498275366 (L.D.V.); tel.: +30 210 529 4247; fax:
+30 210 529 4265 (S.A.H.).
The synthesis of the crucial keto-amides 6 is outlined in
Scheme 1. Specifically, various N-(4-bromophenyl)amides (4),
were reacted with 4-(benzyloxy)benzaldehyde (3) to produce the
hydroxy-amides (5) from moderate to good yields (25–55%).
E-mail addresses: daniele.passarella@unimi.it (D. Passarella), lisa.dallavia@
unipd.t (L. Dalla Via), sehar@aua.gr (S.A. Haroutounian).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.012

M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
4121
O
using 2D-NOESY data was employed for the stereochemical assign-
ment of compounds 7 and 8.
N
O
HN
R
2.2. Antiproliferative activity
The solubility of compounds 9 and 10 has been evaluated theo-
retically by clogP16 and have been compared to tamoxifen and 4-
hydroxytamoxifen (Table 1). The theoretical calculations for the
new compounds, showed values that are generally lower than that
of tamoxifen, which indicates that compounds 9 and 10 present a
reduced lipophilicity.
OH
R
HO
The novel compounds were evaluated by an in vitro assay per-
formed on three human cancer cell lines comprised of breast can-
cer (MCF-7), which is known to overexpress the estrogen
receptor,17 cervix adenocarcinoma (HeLa) and biphasic mesotheli-
oma (MSTO-211H), using tamoxifen and 4-hydroxytamoxifen as
reference compounds. The results, expressed as GI50 values, are
shown in Table 1.
As regard the estrogen sensitive MCF-7 cells, the obtained re-
sults highlighted for the compounds carrying in R a branched
group (b, d and e series) or the piperidine (9g and 10g) an antipro-
liferative activity comparable or even higher with respect to that of
tamoxifen (Scheme 2). In particular, the most active compound is
10b that shows an antiproliferative activity about 4 times higher
with respect to the reference drug. Nevertheless, the comparison
with 4-hydroxytamoxifen underlined for all new derivatives a sig-
nificantly lower cytotoxicity. In this connection, it seems that the
addition of a further 4-hydroxyl group in the triarylethylene moi-
ety could play a crucial role in the occurrence of cytotoxic effect. As
regard the compounds characterized in R by a linear substituent (a
and c series) or by a pyrrolidine (9f and 10f), they are practically
ineffective. Moreover, taking into consideration the stereochemis-
try, Z isomers seem generally slightly more active toward MCF-7
cells, showing GI50 values from 1.8 (compounds d) to 3.6 times
(compounds g) lower with respect to E isomers.
tamoxifen derivatives
1 R = H
2
R = OH
Figure 1. Structures of tamoxifen (1), 4-hydroxytamoxifen (2) and novel tamoxifen
derivatives.
Oxidation of hydroxy-amides 5, in the presence of Jones reagent,
furnished the corresponding keto-amides 6, in almost quantitative
yields (90–95%).
Subsequently, keto-amides 6, were treated under McMurry
olefination conditions with 1-(40-(benzyloxy)phenyl)propan-1-
one (Scheme 2), to provide a mixture of E/Z isomers (7 and 8) in
1:1 ratio, that were separated by flash column chromatography
in good yields (50–65%). Finally, deprotection of compounds 7
and 8 with BBr3 at ꢀ78 °C (Scheme 2), afforded the target tamoxi-
fen derivatives 9 and 10 in quantitative yields (90–95%).
The stereochemical assignments of the E, Z isomers were deter-
mined on the basis of 2D-NOESY experiments. For example the ste-
reochemistry of compound 7d (Scheme 2) was determined on its
2D-NOESY spectrum (Supplementary data), obtained at 400 MHz.
The CH2CH3 protons at 2.50 ppm demonstrate NOE cross peaks
with the proton at 7.07 ppm (H-20000) and the CH2CH3 protons at
0.96 ppm with the proton at 7.17 ppm (H-2000). These NOE data
establish the stereochemical assignment of compound 7d to be
in the E configuration. Similarly, the stereochemistry of compound
8d was determined to be in the Z configuration based on its 2D-
NOESY spectrum (Supplementary data), obtained at 400 MHz.
The CH2CH3 protons at 0.94 ppm demonstrate NOE cross peaks
with the proton at 7.17 ppm (H-30) and the CH2CH3 protons at
2.48 ppm with the proton at 7.06 ppm (H-20000). A similar strategy
The investigation of the antiproliferative effect on two estrogen-
independent human tumor cell lines, HeLa and MSTO-211H,
shows, as expected, a significant decrease of the sensitivity toward
tamoxifen. In contrast, for the novel derivatives the GI50 values ob-
tained for HeLa and MSTO-211H remain practically unchanged
with respect to those obtained for MCF-7, with the exception of
10b. Indeed, interestingly, for this latter compound a decrease in
O
OH
HN
R
O
a
O
H
+
BnO
N
H
R
BnO
Br
5
3
4
4a, 5a, 6a R = CH2CH3
4b, 5b, 6b R = CH(CH3)2
b
4c 5c 6c
,
,
R = (CH2)4CH3
R = Ν(CH3)2
O
4d 5d 6d
,
,
O
4e, 5e, 6e R = Ν(CH2CH3)2
BnO
N
H
R
N
4f, 5f, 6f R =
4g, 5g, 6g R =
6
N
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, ꢀ50 °C, 2 h; (b) Jones reagent, acetone, 0 °C, 30 min.

4122
M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
6
a
O
O
HN
R
HN
R
2'
3'
2'
3'
OR1
3''''
2'''
2''''
2''''
3''''
R1O
3'''
2'''
R1O
3'''
OR1
7: R1 = Bn
9: R1 = H
8: R1 = Bn
10: R1 = H
b
b
7a 8a 9a 10a
7e, 8e, 9e, 10e R = Ν(CH2CH3)2
,
,
,
R = CH2CH3
7b 8b 9b 10b
7f, 8f, 9f, 10f R =
,
,
,
R = CH(CH3)2
N
7g 8g 9g 10g
7c, 8c, 9c, 10c R = (CH2)4CH3
7d, 8d, 9d, 10d R = Ν(CH3)2
,
,
,
R = N
Scheme 2. Reagents and conditions: (a) Zn, TiCl4, 1-(40-(benzyloxy)phenyl)propan-1-one, THF, ꢀ10 °C to reflux, 5 h; (b) BBr3, DCM, ꢀ78 °C, 1 h.
Table 1
mic estrogen receptor response to agonists or antagonists. To
Cell growth inhibition in the presence of examined compounds
determine if the novel compounds were estrogenic, HC11 cells
were treated with E2 (10 nM, = 100%) in addition to 1 M concen-
clogPb
a
Compound
Cell lines GI50
HeLa
(lM)
l
tration of the tested compounds (Fig. 2). Overall, compounds 9a,
9b, 10e, and 9f displayed agonist activity (108%, 96%, 80% and
73% respectively), same as E2, with compound 9a to be the most
potent. Compound 9g showed reduced agonistic activity (51% acti-
vation compared to estradiol), while compounds 10b, 9d, 9e, 10f,
and 10g (18%, 25%, 27%, 20% and 15% respectively) were not able
to activate reporter gene expression.
MCF-7
MSTO-211H
9a
10a
9b
10b
9c
10c
9d
10d
9e
10e
9f
10f
9g
10g
>20
>20
6.7 1.5
2.9 0.4
>20
14.1 1.6
17.1 1.1
6.7 1.6
7.4 1.4
29.9 1.6
27.2 1.2
15.2 3.2
8.0 0.8
9.1 1.8
10.7 2.5
26.7 0.9
22.4 3.9
15.4 3.1
8.2 1.5
32.6 2.1
nd
12.3 0.3
15.5 1.5
10.9 1.3
8.4 1.3
18.1 3.4
16.6 1.4
17.8 3.2
8.7 0.8
8.3 1.1
8.3 0.3
>30
18.5 0.7
9.1 1.3
2.3 1.2
23.3 2.5
nd
5.076
5.076
5.320
5.320
6.646
6.646
4.382
4.382
5.524
5.524
5.175
5.175
5.680
5.680
6.064
5.585
>20
17.9 1.1
9.9 3.4
9.1 1.3
11.2 1.4
>20
2.4. Effect on topoisomerase relaxation activity
In a previous paper it was demonstrated the ability of tamoxi-
fen to inhibit the relaxation activity of topoisomerases I and II.20
In this connection, and with the aim to establish the possible
molecular target(s) accountable for the cytotoxicity of the new
derivatives, we investigate their effect on the catalytic activity of
both topoisomerase I and II. Figure 3A shows the effect of some
representative compounds (9b, 10b, 10d, 9e, 10g and 9a) on the
relaxation of plasmid pBR322 DNA, mediated by topoisomerase
II. The supercoiled DNA (DNA) was converted into relaxed forms
P20
16.5 1.2
4.5 1.1
12.0 1.6
0.107 0.081c
Tamoxifen
4-OH Tamoxifen
a
b
c
Values are the mean SD of at least three independent experiments.
clogP = calculated octanol–water partition coefficient.
Taken from Ref. 18.
by the enzyme (topo II). At 50 lM concentration both 9b and
cytotoxic effect of 2.5 and 2.8 times is observed in HeLa and MSTO-
211H, respectively.
10b exert a partial inhibitory effect, as demonstrated by the
appearance of supercoiled DNA and by the concurrent decrease
in relaxed products, while the other compounds do not affect the
enzymatic activity. Nevertheless, by increasing the concentration
2.3. Estrogen like properties of the compounds
up to 100 lM (Fig. 3B), a complete inhibition is observed for all
To assess the estrogenic activities of selected compounds, we
used HC11 (mouse mammary epithelial cell line) with an inte-
grated 3ꢁERE-Luciferase construct in their genome.19 Luciferase
activation, measured as amount of emitted light in the luciferase
assay, is proportional to the activation of gene transcription by
ERs and is, therefore, considered a measurement of classical geno-
the derivatives, apart from 9a. Indeed in this latter experimental
condition, 9a induces a significant, but only partial effect on topo-
isomerase II relaxation. Taking into account that 9a exerts the low-
er antiproliferative capacity, these data could suggest the
contribution of the inhibition on topoisomerase II to the cytotoxic-
ity. Otherwise, experiments performed with topoisomerase

M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
4123
Figure 2. Activity of selective compounds in HC11 cell line. HC11 cells stably transfected with 3ꢁERE–Luc construct were seeded in 24-well plates and after the cells reached
50–60% confluence, were treated with 1 M (final) tested compounds, 10 nM E2 or ethanol (control). After 24 h the percentage of estrogen activity was determined (see
l
experimental part). Data are presented as % to one of the arbitrary selected compounds of three independent experiments, performed in duplicates.
A
supercoiled DNA
B
supercoiled DNA
Figure 3. Effect on topoisomerase II-mediated DNA relaxation. Supercoiled pBR322 DNA (DNA) was incubated with topoisomerase II in the absence (topo II) and presence of
indicated compounds at 50 lM (A) or 100 lM (B) concentration.
I highlighted the inability of all derivatives to affect the enzymatic
marily through inhibition of the estrogen receptor transcriptional
activity. Indeed, the study on the molecular mechanism responsi-
ble for cytotoxicity highlighted the ability of compounds to inhibit
relaxation activity mediated by topoisomerase II, thus suggesting
the nuclear enzyme as a potential intracellular target.
activity, even at the higher tested concentration (100
shown).
lM, data not
3. Conclusions
Among the novel derivatives, 10b appears as the most interest-
ing. Indeed, this compound induces the highest antiproliferative ef-
fect on MCF-7 cells, about 4 times higher with respect to the
reference drug. In addition, both HeLa and MSTO-211H cell lines
show a significant less sensitivity toward 10b. On the whole, these
properties render this new triarylethylene-based compound a lead
structure that deserves to be further studied to obtain more spe-
cific drugs for the treatment of breast cancer.
The synthesis through McMurry coupling reaction of some
derivatives structurally related to the well known anti-estrogen
anticancer drug tamoxifen, clinically used to treat breast cancer,
is described. The investigation of the antiproliferative effect on hu-
man breast cancer cell line MCF-7 showed for some derivatives an
interesting cytotoxic activity, in some cases significantly higher
with respect to that obtained for tamoxifen. Nevertheless, in most
cases a comparable effect is obtained also on two estrogen inde-
pendent human tumor cell lines, HeLa (cervix adenocarcinoma)
and MSTO-211H (biphasic mesothelioma), thus suggesting the
ability to exert the antiproliferative effect through a mechanism
of action independent from the estrogen receptor. These results
could be also in agreement with the difference in cytotoxicity be-
tween the new derivatives and 4-hydroxytamoxifen that acts pri-
4. Experimental section
4.1. General experimental conditions
All reactions were carried out in oven-dried glassware under
argon atmosphere. All starting materials were purchased from

4124
M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
Aldrich, while the solvents used were purified by distillation prior
to use. Solvent mixtures employed in chromatography were re-
ported as volume to volume ratios. Thin layer chromatography
(TLC) was performed on Merck precoated aluminium sheets of Sil-
ica Gel 60 F254, visualization of products being accomplished by UV
absorbance at 254 nm and by an alcohol solution of anisaldehyde
with heating. Flash column chromatography was performed on a
4.2.1.3. Synthesis of N-(4-((40-(benzyloxy)phenyl)(hydroxy)-
methyl)phenyl) hexanamide (5c). According to the general
procedure, hydroxy-amide 5c was obtained from amide 4c after
purification by flash column chromatography (Hex/EtOAc 1:1)
and evaporation of the solvent as oil (30% yield). Rf = 0.22 (Hex/
EtOAc 1:1); IR (film)
m ;
: 1655, 1021 cmꢀ1 1H NMR (acetone-d6,
400 MHz): d = 9.08 (1H, s, NH), 7.60 (2H, d, J 8.4 Hz, H-2), 7.47
(2H, d, J 7.2 Hz, H-20), 7.39 (1H, t, J 7.2 Hz, PhH-para), 7.37–7.30
(6H, m, PhH-ortho, PhH-meta and H-3), 6.96 (2H, d, J 7.2 Hz, H-
30), 5.74 (1H, d, J 4.0 Hz, CHOH), 5.10 (2H, s, CH2O), 4.70 (1H, d, J
4.0 Hz, OH), 2.34 (2H, t, J 7.2 Hz, CH2CH2CH2CH2CH3), 1.67 (2H,
m, CH2CH2CH2CH2CH3), 1.33 (4H, m, CH2CH2CH2CH2CH3), 0.90
SDS silica gel (35–70 lm). All tested compounds possessed a purity
of >98% confirmed via elemental analyses (CHN). Melting points
were determined on a Stuart apparatus (SMP3) and are uncor-
rected. IR spectra were recorded on a Thermo electron corporation
Nicolet 6700 FT-IR spectrometer using CH2Cl2 in ZnSe round win-
dows. 1H NMR and 13C NMR spectra were recorded at 600, 400,
and 100, 50 MHz respectively on Bruker DRX-600, DRX-400 and
DRX-200 spectrometers in the indicated solvents. Chemical shifts
(d) for proton and carbon resonances are quoted in parts per mil-
lion (ppm) relative to tetramethylsilane (TMS), which was used
as an internal standard. MS spectra were recorded using electro-
spray ionization (ESI) technique on a Thermo Accela LC TSQ Quan-
tum Access MS–MS spectometer.
(3H, t,
J
7.2 Hz, CH2CH2CH2CH2CH3); 13C NMR (acetone-d6,
50 MHz): d = 170.2 (NHCO), 157.8 (C-40), 138.4 (C-4), 138.2 (C-1),
137.6 (PhCCH2O), 128.4 (C-20), 127.6 (PhC-meta and PhC-para),
127.4 (PhC-ortho), 126.6 (C-3), 120.8 (C-10), 118.8 (C-2), 114.3 (C-
30), 74.5 (CHOH), 69.5 (Ph-CH2O), 36.8 (CH2CH2CH2CH2CH3), 31.3
(CH2CH2CH2CH2CH3),
25.1
(CH2CH2CH2CH2CH3),
22.2
(CH2CH2CH2CH2CH3), 13.34 (CH2CH2CH2CH2CH3); MS: 404.57
(M+H+), 426.68 (M+Na+).
4.2.1.4. Synthesis of 3-(40-((400-(benzyloxy)phenyl)(hydroxy)-
4.2. Synthesis
methyl)phenyl)-1,1-dimethylurea (5d).
According to the
general procedure, hydroxy-amide 5d was obtained from amide
4d after purification by flash column chromatography (Hex/EtOAc
1:9) and evaporation of the solvent as foam (55% yield). Rf = 0.19
4.2.1. General procedure for the synthesis of analogues 5
4.2.1.1. Synthesis of N-(4-((40(benzyloxy)phenyl)(hydroxy)-
methyl)phenyl) propionamide (5a).
To a cold at ꢀ50 °C
(Hex/EtOAc 1:9); IR (film) m ;
: 1645, 1019 cmꢀ1 1H NMR (acetone-
solution of amide 4a (1.2 g, 5.2 mmol) in THF (60 mL), n-BuLi
(9.7 mL, 1.6 M in hexane, 16 mmol) was added dropwise and the
new solution was stirred for 10 min at the same temperature.
d6, 400 MHz): d = 7.72 (1H, s, NH), 7.47 (6H, m, H-20, PhH-ortho
and PhH-meta), 7.39 (1H, t, J 7.2 Hz, PhH-para), 7.31 (2H, d, J
8.0 Hz, H-30), 7.24 (2H, d, J 8.4 Hz, H-200), 6.95 (2H, d, J 8.4 Hz, H-
300), 5.72 (1H, s, CHOH), 5.10 (2H, s, CH2O), 4.71 (1H, s, OH), 2.97
Then,
a
solution of 4-(benzyloxy)benzaldehyde (3) (1.0 g,
4.7 mmol) in THF (10 mL) was added and the resulted solution
was stirred for another 2 h at ꢀ50 °C. After the completion of the
reaction, the solvent was evaporated in vacuum, EtOAc was added
and the organic layer was washed with 5% NH4Cl, water and brine,
dried with Na2SO4, filtered and evaporated. The residue was puri-
fied by flash column chromatography (Hex/EtOAc 1:1) to provide
hydroxy-amide 5a as oil (40% yield). Rf = 0.26 (Hex/EtOAc 1:1); IR
13
(6H, s, N(CH3)2); C NMR (acetone-d6, 50 MHz): d = 157.8 (C-400),
155.8 (NHCO), 139.5 (C-10), 139.2 (PhCCH2O), 138.3 (C-40), 137.6
(C-100), 128.4 (C-200), 127.6 (PhC-meta and PhC-para), 127.5 (PhC-
ortho), 126.4 (C-30), 119.2 (C-20), 114.3 (C-300), 74.6 (CHOH), 69.5
(Ph-CH2O), 35.6 (N(CH3)2); MS: 377.39 (M+H+), 399.40 (M+Na+).
4.2.1.5. Synthesis of 3-(40-((400-(benzyloxy)phenyl)(hydroxy)-
(film)
m: ;
1665, 1013 cmꢀ1 1H NMR (acetone-d6, 400 MHz):
methyl)phenyl)-1,1-diethylurea (5e).
According to the gen-
d = 9.06 (1H, s, NH), 7.60 (2H, d, J 8.4 Hz, H-2), 7.47 (2H, d, J
7.2 Hz, H-20), 7.39 (1H, t, J 8.0 Hz, PhH-para), 7.37–7.30 (6H, m,
PhH-ortho, PhH-meta and H-3), 6.96 (2H, d, J 7.2 Hz, H-30), 5.75
(1H, d, J 3.6 Hz, CHOH), 5.01 (2H, s, CH2O), 4.71 (1H, s, OH), 2.35
(2H, q, J 7.6 Hz, CH2CH3), 1.12 (3H, t, J 7.6 Hz, CH2CH3); 13C NMR
(acetone-d6, 50 MHz): d = 171.6 (NHCO), 157.9 (C-40), 140.5 (C-4),
138.4 (C-1), 138.2 (PhCCH2O), 137.6 (C-10), 128.4 (C-20), 127.6
(PhC-meta and PhC-para), 127.4 (PhC-ortho), 126.7 (C-3), 118.8
(C-2), 114.3 (C-30), 74.5 (CHOH), 69.5 (Ph-CH2O), 28.5 (CH2CH3),
9.0 (CH2CH3); MS: 362.47 (M+H+), 384.48 (M+Na+).
eral procedure, hydroxy-amide 5e was obtained from amide 4e
after purification by flash column chromatography (Hex/EtOAc
4:6) and evaporation of the solvent as oil (35% yield). Rf = 0.21
(Hex/EtOAc 4:6); IR (film) m ;
: 1642, 1012 cmꢀ1 1H NMR (acetone-
d6, 400 MHz): d = 7.62 (1H, s, NH), 7.49–7.47 (4H, m, H-20 and
PhH-meta), 7.39 (1H, t, J 7.6 Hz, PhH-para), 7.37–7.30 (4H, m, H-
30 and PhH-ortho), 7.24 (2H, d, J 7.6 Hz, H-200), 6.95 (2H, d, J
7.6 Hz, H-300), 5.72 (1H, s, CHOH), 5.10 (2H, s, CH2O), 4.70 (1H, s,
OH), 3.41 (4H, q, J 6.8 Hz, N(CH2CH3)2), 1.15 (6H, t, J 6.8 Hz,
13
N(CH2CH3)2); C NMR (acetone-d6, 50 MHz): d = 157.8 (C-400),
154.5 (NHCO), 139.6 (C-10), 139.2 (PhCCH2O), 138.3 (C-40), 137.6
(C-100), 128.4 (C-200), 127.6 (PhC-meta and PhC-para), 127.5 (PhC-
ortho), 126.4 (C-30), 119.4 (C-20), 114.3 (C-300), 74.6 (CHOH), 69.5
(Ph-CH2O), 40.9 (N(CH2CH3)2), 13.4 (N(CH2CH3)2); MS: 405.24
(M+H+), 427.24 (M+Na+).
4.2.1.2. Synthesis of N-(4-((40-(benzyloxy)phenyl)(hydroxy)-
methyl)phenyl) isobutyramide (5b).
According to the gen-
eral procedure, hydroxy-amide 5b was obtained from amide 4b
after purification by flash column chromatography (Hex/EtOAc
6:4) and evaporation of the solvent as oil (25% yield). Rf = 0.19
(Hex/EtOAc 6:4); IR (film) m ;
: 1663, 1019 cmꢀ1 1H NMR (acetone-
4.2.1.6. Synthesis of N-(40-((400-(benzyloxy)phenyl)(hydroxy)-
d6, 400 MHz): d = 9.03 (1H, s, NH), 7.61 (2H, d, J 8.4 Hz, H-2), 7.47
(2H, d, J 7.2 Hz, H-20), 7.39 (1H, t, J 7.2 Hz, PhH-para), 7.37–7.30
(6H, m, PhH-ortho, PhH-meta and H-3), 6.96 (2H, d, J 7.2 Hz, H-
30), 5.74 (1H, s, CHOH), 5.10 (2H, s, CH2O), 4.71 (1H, s, OH), 2.60
(1H, m, CH(CH3)2), 1.15 (6H, d, J 6.8 Hz, CH(CH3)2); 13C NMR (ace-
tone-d6, 50 MHz): d = 174.9 (NHCO), 157.8 (C-40), 138.4 (C-4),
138.2 (C-1), 137.6 (PhCCH2O), 128.4 (C-20), 127.6 (PhC-meta and
PhC-para), 127.5 (PhC-ortho), 126.6 (C-3), 120.9 (C-10), 118.9 (C-
2), 114.3 (C-30), 74.5 (CHOH), 69.5 (Ph-CH2O), 35.7 (CH(CH3)2),
19.0 (CH(CH3)2); MS: 376.37 (M+H+), 398.37 (M+Na+).
methyl)phenyl) pyrrolidine-1-carboxamide (5f).
According
to the general procedure, hydroxy-amide 5f was obtained from
amide 4f after purification by flash column chromatography
(Hex/EtOAc 4:6) and evaporation of the solvent as white solid
(35% yield). Rf = 0.13 (Hex/EtOAc 4:6); mp 164–165 °C; IR (film)
m
: 1646, 1024 cmꢀ1 1H NMR (acetone-d6, 400 MHz): d = 7.52–
;
7.49 (4H, m, H-20 and PhH-meta), 7.39 (1H, t, J 7.6 Hz, PhH-para),
7.34–7.30 (4H, m, H-30 and PhH-ortho), 7.23 (2H, d, J 7.6 Hz, H-
200), 6.95 (2H, d, J 7.6 Hz, H-300), 5.71 (1H, d, J 4.0 Hz, CHOH), 5.10
(2H, s, CH2O), 4.62 (1H, d, J 4.0 Hz, OH), 3.43 (4H, t, J 6.4 Hz,

M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
4125
N(CH2)2(CH2)2), 1.92 (4H, t, J 6.4 Hz, N(CH2)2(CH2)2); 13C NMR (ace-
tone-d6, 50 MHz): d = 157.9 (C-400), 153.9 (NHCO), 139.6 (C-10),
139.1 (PhCCH2O), 138.3 (C-40), 137.8 (C-100), 128.4 (C-200), 127.6
(PhC-ortho, PhC-meta, and PhC-para), 126.4 (C-30), 118.8 (C-20),
114.3 (C-300), 74.6 (CHOH), 69.5 (Ph-CH2O), 45.5 (N(CH2)2(CH2)2),
25.2 (N(CH2)2(CH2)2); MS: 403.50 (M+H+), 425.49 (M+Na+).
obtained from hydroxy-amide 5c as white solid (95% yield).
Rf = 0.24 (Hex/EtOAc 7:3); mp 168–169 °C; IR (film) : 1638,
1596 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d = 10.3 (1H, s, NH),
m
;
7.79–7.68 (6H, m, H-2, H-3 and H-20), 7.50 (2H, d, J 7.2 Hz, PhH-
ortho), 7.43 (2H, t, J 7.2 Hz, PhH-meta), 7.36 (1H, t, J 7.2 Hz, PhH-
para), 7.17 (2H, d, J 7.2 Hz, H-30), 5.23 (2H, s, CH2O), 2.34 (2H, t, J
7.2 Hz, CH2CH2CH2CH2CH3), 1.62 (2H, m, CH2CH2CH2CH2CH3),
4.2.1.7. Synthesis of N-(40-((400-(benzyloxy)phenyl)(hydroxy)-
1.32 (4H, m, CH2CH2CH2CH2CH3), 0.89 (3H, t,
J
7.2 Hz,
methyl)phenyl) piperidine-1-carboxamide (5g).
According
CH2CH2CH2CH2CH3); 13C NMR (DMSO-d6, 50 MHz): d = 193.7
(CO), 172.4 (NHCO), 162.2 (C-40), 143.5 (C-1), 137.0 (PhCCH2O),
132.4 (C-3), 132.2 (C-4), 131.2 (C-20), 130.5 (C-10), 129.0 (PhC-
ortho), 128.5 (PhC-para), 128.3 (PhC-meta), 118.6 (C-2), 115.0 (C-
to the general procedure, hydroxy-amide 5g was obtained from
amide 4g after purification by flash column chromatography
(Hex/EtOAc 3:7) and evaporation of the solvent as white solid
(35% yield). Rf = 0.30 (Hex/EtOAc 3:7); mp 166–167 °C; IR (film)
30),
(CH2CH2CH2CH2CH3),
70.0
(Ph-CH2O),
37.0
25.2
(CH2CH2CH2CH2CH3),
(CH2CH2CH2CH2CH3),
31.3
22.4
m
: 1643, 1027 cmꢀ1 1H NMR (acetone-d6, 400 MHz): d = 7.85
;
(1H, s, NH), 7.48–7.45 (4H, m, H-20 and PhH-meta), 7.39 (1H, t, J
7.6 Hz, PhH-para), 7.34–7.30 (4H, m, H-30 and PhH-ortho), 7.24
(2H, d, J 8.8 Hz, H-200), 6.95 (2H, d, J 8.8 Hz, H-300), 5.71 (1H, d, J
4.0 Hz, CHOH), 5.10 (2H, s, CH2O), 4.62 (1H, d, J 4.0 Hz, OH), 3.47
(CH2CH2CH2CH2CH3), 14.3 (CH2CH2CH2CH2CH3); MS: 402.57
(M+H+), 424.59 (M+Na+).
4.2.2.4. 3-(40-(400-(Benzyloxy)benzoyl)phenyl)-1,1-dimethylurea
(4H,
t,
J
6.4 Hz,
N(CH2)2(CH2)2CH2),
1.62
(4H,
m,
N(CH2)2(CH2)2CH2), 1.53 (2H, m, N(CH2)2(CH2)2CH2); 13C NMR
(acetone-d6, 50 MHz): d = 157.6 (C-400), 153.7 (NHCO), 139.3 (C-
10), 139.2 (PhCCH2O), 138.1 (C-40), 137.9 (C-100), 128.3 (C-200),
127.6 (PhC-meta and PhC-para), 127.5 (PhC-ortho), 126.4 (C-30),
119.0 (C-20), 114.3 (C-300), 74.6 (CHOH), 69.5 (Ph-CH2O), 44.9
(6d).
was obtained from hydroxy-amide 5d as white solid (90% yield).
Rf = 0.13 (Hex/EtOAc 1:1); mp 159–160 °C; IR (film) : 1643,
1599 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d = 8.75 (1H, s, NH),
According to the general procedure, keto-amide 6d
m
;
7.76–7.68 (6H, m, H-20, H-30 and H-200), 7.50 (2H, d, J 7.2 Hz, PhH-
ortho), 7.45 (2H, t, J 7.2 Hz, PhH-meta), 7.40 (1H, t, J 7.2 Hz, PhH-
para), 7.19 (2H, d, J 7.2 Hz, H-300), 5.25 (2H, s, CH2O), 2.99 (6H, s,
N(CH3)2); 13C NMR (DMSO-d6, 50 MHz): d = 193.7 (CO), 162.0 (C-
400), 155.7 (NHCO), 145.4 (C-10), 137.0 (PhCCH2O), 132.2 (C-30),
131.0 (C-200), 130.8 (C-40), 130.7 (C-100), 129.0 (PhC-ortho), 128.5
(PhC-para), 128.3 (PhC-meta), 118.7 (C-20), 115.0 (C-300), 70.0 (Ph-
CH2O), 36.7 (N(CH3)2); MS: 375.35 (M+H+), 397.34 (M+Na+).
(N(CH2)2(CH2)2CH2),
25.7
(N(CH2)2(CH2)2CH2),
24.4
(N(CH2)2(CH2)2CH2); MS: 417.46 (M+H+), 439.47 (M+Na+).
4.2.2. General procedure for the synthesis of analogues 6
4.2.2.1. Synthesis of N-(4-(40-(benzyloxy)benzoyl)phenyl)propi-
onamide (6a).
To a cold at 0 °C solution of hydroxy-amide 5a
(0.20 g, 0.55 mmol) in acetone (22 mL), Jones reagent (4.3 mL) was
added and the new solution was stirred for 30 min at the same
temperature. After the completion of the reaction, 2-propanol
was added and the solvent was evaporated in vacuum. Then, EtOAc
was added and the organic layer was washed with water and brine,
dried with Na2SO4, filtered and evaporated in vacuum till dryness
to provide keto-amide 6a as white solid (90% yield). Rf = 0.36
4.2.2.5.
(6e).
3-(40-(400-(Benzyloxy)benzoyl)phenyl)-1,1-diethylurea
According to the general procedure, keto-amide 6e was
obtained from hydroxy-amide 5e as white solid (95% yield).
Rf = 0.27 (Hex/EtOAc 1:1); mp 162–163 °C; IR (film) : 1644,
1597 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d = 8.60 (1H, s, NH),
m
(Hex/EtOAc 1:1); mp 162–163 °C; IR (film) m ;
: 1657, 1640 cmꢀ1
;
7.75–7.65 (6H, m, H-20, H-30 and H-200), 7.51 (2H, d, J 7.2 Hz, PhH-
ortho), 7.44 (2H, t, J 7.2 Hz, PhH-meta), 7.38 (1H, t, J 7.2 Hz, PhH-
para), 7.19 (2H, d, J 7.6 Hz, H-300), 5.24 (2H, s, CH2O), 3.41 (4H, q,
J 6.4 Hz, N(CH2CH3)2), 1.13 (6H, t, J 6.4 Hz, N(CH2CH3)2); 13C NMR
(DMSO-d6, 50 MHz): d = 193.7 (CO), 162.0 (C-400), 154.4 (NHCO),
145.5 (C-10), 137.0 (PhCCH2O), 132.2 (C-30), 131.0 (C-200), 130.8
(C-40), 130.7 (C-100), 129.0 (PhC-ortho), 128.5 (PhC-para), 128.3
(PhC-meta), 118.8 (C-20), 115.0 (C-300), 70.0 (Ph-CH2O), 41.0
(N(CH2CH3)2), 13.3 (N(CH2CH3)2); MS: 403.26 (M+H+), 425.34
(M+Na+).
1H NMR (DMSO-d6, 400 MHz): d = 10.2 (1H, s, NH), 7.78–7.69
(6H, m, H-2, H-3 and H-20), 7.49 (2H, d, J 7.2 Hz, PhH-ortho), 7.42
(2H, t, J 7.2 Hz, PhH-meta), 7.36 (1H, t, J 7.2 Hz, PhH-para), 7.17
(2H, d, J 7.2 Hz, H-30), 5.22 (2H, s, CH2O), 2.39 (2H, q, J 7.2 Hz,
CH2CH3), 1.11 (3H, t, J 7.6 Hz, CH2CH3); 13C NMR (DMSO-d6,
50 MHz): d = 193.7 (CO), 173.0 (NHCO), 162.2 (C-40), 143.5 (C-1),
137.0 (PhCCH2O), 132.4 (C-3), 132.2 (C-4), 131.2 (C-20), 130.5 (C-
10), 129.0 (PhC-ortho), 128.5 (PhC-para), 128.3 (PhC-meta), 118.6
(C-2), 115.0 (C-30), 67.0 (Ph-CH2O), 30.1 (CH2CH3), 10.0 (CH2CH3);
MS: 360.47 (M+H+), 382.48 (M+Na+).
4.2.2.2. Synthesis of N-(4-(40-(benzyloxy)benzoyl)phenyl)isobu-
4.2.2.6. N-(40-(400-(Benzyloxy) benzoyl)phenyl)pyrrolidine-1-car-
tyramide (6b).
According to the general procedure, keto-
boxamide (6f).
According to the general procedure, keto-
amide 6b was obtained from hydroxy-amide 5b as white solid
amide 6f was obtained from hydroxy-amide 5f as white solid
(95% yield). Rf = 0.41 (Hex/EtOAc 1:1); mp 165–166 °C; IR (film)
: 1698, 1635 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d = 10.2 (1H,
;
(90% yield). Rf = 0.47 (Hex/EtOAc 3:7); mp 178–179 °C; IR (film)
m
m
: 1645, 1598 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d = 8.54 (1H,
;
s, NH), 7.80–7.68 (6H, m, H-2, H-3 and H-20), 7.49 (2H, d, J
7.2 Hz, PhH-ortho), 7.42 (2H, t, J 7.2 Hz, PhH-meta), 7.36 (1H, t, J
7.2 Hz, PhH-para), 7.16 (2H, d, J 7.2 Hz, H-30), 5.22 (2H, s, CH2O),
2.65 (1H, m, CH(CH3)2), 1.13 (6H, d, J 6.8 Hz, CH(CH3)2); 13C NMR
(DMSO-d6, 50 MHz): d = 193.7 (CO), 176.3 (NHCO), 162.2 (C-40),
143.6 (C-1), 137.0 (PhCCH2O), 132.4 (C-3), 132.2 (C-4), 131.2 (C-
20), 130.5 (C-10), 129.0 (PhC-ortho), 128.5 (PhC-para), 128.3 (PhC-
meta), 118.7 (C-2), 115.0 (C-30), 70.0 (Ph-CH2O), 35.5 (CH(CH3)2),
19.9 (CH(CH3)2); MS: 374.47 (M+H+), 396.48 (M+Na+).
s, NH), 7.74–7.71 (6H, m, H-20, H-30 and H-200), 7.48 (2H, d, J
7.2 Hz, PhH-ortho), 7.42 (2H, t, J 7.2 Hz, PhH-meta), 7.36 (1H, t, J
7.2 Hz, PhH-para), 7.17 (2H, d, J 7.6 Hz, H-300), 5.22 (2H, s, CH2O),
3.80 (2H, t,
J 7.2 Hz, N(CH2)2(CH2)2), 3.40 (2H, t, J 7.2 Hz,
N(CH2)2(CH2)2), 2.67 (2H, t, J 7.2 Hz, N(CH2)2(CH2)2), 2.00 (2H, t, J
7.2 Hz, N(CH2)2(CH2)2); 13C NMR (DMSO-d6, 50 MHz): d = 193.7
(CO), 162.0 (C-400), 154.6 (NHCO), 145.5 (C-10), 137.0 (PhCCH2O),
132.2 (C-30), 131.0 (C-200), 130.8 (C-40), 130.7 (C-100), 129.0 (PhC-
ortho), 128.5 (PhC-para), 128.3 (PhC-meta), 118.6 (C-20), 115.0
(C-300),
(N(CH2)2(CH2)2).; MS: 401.39 (M+H+), 423.39 (M+Na+).
70.0
(Ph-CH2O),
45.5
(N(CH2)2(CH2)2),
25.3
4.2.2.3.
(6c).
N-(4-(40-(benzyloxy)benzoyl)phenyl)hexanamide
According to the general procedure, keto-amide 6c was

4126
M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
4.2.2.7. N-(40-(400-(Benzyloxy)benzoyl)phenyl)piperidine-1-car-
boxamide (6g). According to the general procedure, keto-
200), 130.6 (C-2000), 130.1 (C-3), 129.9 (C-1), 128.5 (PhC0-ortho and
PhC-ortho), 127.9 (PhC0-para and PhC-para), 127.5 (PhC0-meta
and PhC-meta), 119.4 (C-2), 114.3 (C-3000), 113.8 (C-300), 69.9
(C4000-OCH2), 69.9 (C400-OCH2), 30.8 (COCH2CH3), 28.9 (CH2CH3),
13.7 (CH2CH3), 9.7 (COCH2CH3); MS: 568.45.44 (M+H+), 590.44
(M+Na+).
amide 6g was obtained from hydroxy-amide 5g as white solid
(90% yield). Rf = 0.30 (Hex/EtOAc 1:1); mp 205–206 °C; IR (film)
m
: 1644, 1599 cmꢀ1 1H NMR (DMSO-d6, 400 MHz): d = 8.89 (1H,
;
s, NH), 7.78 (2H, d, J 7.6 Hz, H-20), 7.74–7.66 (6H, m, H-30 and H-
200), 7.51 (2H, d, J 7.2 Hz, PhH-ortho), 7.44 (2H, t, J 7.2 Hz, PhH-
meta), 7.38 (1H, t, J 7.2 Hz, PhH-para), 7.18 (2H, d, J 7.6 Hz, H-300),
5.24 (2H, s, CH2O), 3.47 (4H, t, J 6.4 Hz, N(CH2)2(CH2)2CH2), 1.61
(2H, m, N(CH2)2(CH2)2CH2), 1.53 (4H, m, N(CH2)2(CH2)2CH2 and
N(CH2)2(CH2)2CH2); 13C NMR (DMSO-d6, 50 MHz): d = 193.7 (CO),
162.0 (C-400), 154.8 (NHCO), 145.6 (C-10), 137.0 (PhCCH2O), 132.2
(C-30), 131.1 (C-200), 130.8 (C-40), 130.6 (C-100), 129.0 (PhC-ortho),
128.5 (PhC-para), 128.3 (PhC-meta), 118.5 (C-20), 115.0 (C-300),
4.2.3.2.
Synthesis
of
(E)-N-(4-(10,20-bis(400,4000-(benzyl-
oxy)phenyl)but-10-enyl)phenyl)isobutyramide (7b) and (Z)-N-
(4-(10,20-bis(400,4000-(benzyloxy) phenyl) but-10-enyl)phenyl)iso-
butyramide (8b).
According to the general procedure, ana-
logues 7b and 8b were obtained from keto-amide 6b after
purification by flash column chromatography (Hex/EtOAc 6:4)
and evaporation of the solvent as white solid and oil respectively
(65% total yield). Compound 7b: Rf = 0.69 (Hex/EtOAc 6:4); mp
70.0
(Ph-CH2O),
45.2
(N(CH2)2(CH2)2CH2),
26.0
(N(CH2)2(CH2)2CH2), 24.5 (N(CH2)2(CH2)2CH2); MS: 415.44
156–157 °C; IR (film) m ;
: 1664 cmꢀ1 1H NMR (CDCl3, 600 MHz):
(M+H+), 437.49 (M+Na+).
d = 7.47–7.35 (10H, m, PhH and PhH0), 7.20 (2H, d, J 8.4 Hz, H-2),
7.14 (2H, d, J 8.4 Hz, H-200), 7.03 (2H, d, J 8.4 Hz, H-2000), 7.00 (1H,
s, NH), 6.95 (2H, d, J 8.4 Hz, H-300), 6.83 (2H, d, J 8.4 Hz, H-3), 6.79
(2H, d, J 8.4 Hz, H-3000), 5.07 (2H, s, C400-OCH2), 5.00 (2H, s, C4000-
OCH2), 2.49 (2H, q, J 7.8 Hz, CH2CH3), 2.46–2.41 (1H, m, CH(CH3)2),
4.2.3. General procedure for the synthesis of analogues 7 and 8
4.2.3.1.
Synthesis
of
(E)-N-(4-(10,20-bis(400,4000-(benzyl-
oxy)phenyl)but-10-enyl)phenyl)propionamide (7a) and (Z)-N-
(4-(10,20-bis(400,4000-(benzyloxy) phenyl)but-10-enyl)phenyl) pro-
1.21 (6H, d, J 6.6 Hz, CH(CH3)2), 0.945 (3H, t, J 7.8 Hz, CH2CH3); 13
C
NMR (CDCl3, 50 MHz): d = 175.0 (CO), 157.6 (C-400), 157.2 (C-4000),
141.2 (C-20), 140.0 (C-4), 137.3 (C-1), 137.2 (PhCCH2O and
PhC0CH2O), 136.5 (C-10), 136.0 (C-100), 135.6 (C-1000), 131.6 (C-3),
130.8 (C-2000), 130.7 (C-200), 128.6 (PhC0-ortho and PhC-ortho),
128.0 (PhC0-para and PhC-para), 127.6 (PhC0-meta and PhC-meta),
118.6 (C-2), 114.4 (C-300), 113.4 (C-3000), 70.1 (C400-OCH2), 70.0
(C4000-OCH2), 36.7 (CH(CH3)2), 28.7 (CH2CH3), 19.7 (CH(CH3)2),
13.7 (CH2CH3); MS: 582.54 (M+H+), 604.53 (M+Na+). Compound
pionamide (8a).
To a cold at ꢀ10 °C suspension of Zn (0.21 g,
3.2 mmol) in THF (3.1 mL), TiCl4 (0.14 mL, 1.3 mmol) was added
dropwise and the new mixture was stirred for another 10 min in
the same temperature and after was refluxed for 2 h. Then, a solu-
tion of keto-amide 6a (0.12 g, 0.33 mmol) and 1-(40-(benzyl-
oxy)phenyl)propan-1-one (0.086 g, 0.36 mmol) in THF (6.6 mL),
was added to the cooled suspension of the titanium reagent at
0 °C, and the reaction mixture was refluxed for 2.5 h. After the
completion of the reaction, the reaction mixture was cooled to rt
and poured into a 10% aq K2CO3 solution (10 mL). Vigorous stirring
was maintained for 5 min, and the dispersed insoluble material
was removed by vacuum filtration using celite. The organic
layer was separated and the aqueous layer was extracted 3 times
with EtOAc. The combined EtOAc extracts were washed with water
and brine, dried with Na2SO4, filtered and evaporated. The residue
was purified by flash column chromatography (Hex/EtOAc 6:4) to
provide analogue 7a as white solid and analogue 8a as oil (50% to-
tal yield). Compound 7a: Rf = 0.43 (Hex/EtOAc 6:4); mp 154–
8b: Rf = 0.62 (Hex/EtOAc 6:4); IR (film)
m ;
: 1664 cmꢀ1 1H NMR
(CDCl3, 600 MHz): d = 7.51 (2H, d, J 8.4 Hz, H-2), 7.46–7.29 (10H,
m, PhH and PhH0), 7.19 (2H, d, J 8.4 Hz, H-3), 7.16 (1H, s, NH),
7.04 (2H, d, J 8.4 Hz, H-2000), 6.80 (2H, d, J 8.4 Hz, H-3000), 6.78 (2H,
d, J 8.4 Hz, H-200), 6.64 (2H, d, J 8.4 Hz, H-300), 5.01 (2H, s, C4000-
OCH2), 4.93 (2H, s, C400-OCH2), 2.53–2.48 (1H, m, CH(CH3)2), 2.44
(2H, q, J 7.8 Hz, CH2CH3), 1.27 (6H, d, J 6.6 Hz, CH(CH3)2), 0.935
(3H, t, J 7.8 Hz, CH2CH3); 13C NMR (CDCl3, 50 MHz): d = 175.2
(CO), 157.1 (C-4000), 156.7 (C-400), 141.0 (C-20), 139.9 (C-4), 137.2
(PhCCH2O and PhC0CH2O), 136.4 (C-10), 136.0 (C-100), 134.9 (C-1000),
132.0 (C-200), 130.8 (C-1), 130.7 (C-2000), 130.1 (C-3), 128.5 (PhC0-
ortho and PhC-ortho), 127.9 (PhC0-para and PhC-para), 127.5
(PhC0-meta and PhC-meta), 119.4 (C-2), 114.3 (C-3000), 113.7 (C-
300), 69.9 (C4000-OCH2), 69.8 (C400-OCH2), 36.8 (CH(CH3)2), 29.7
(CH2CH3), 19.6 (CH(CH3)2), 13.7 (CH2CH3); MS: 582.50 (M+H+),
604.54 (M+Na+).
155 °C; IR (film)
m: ;
1688 cmꢀ1 1H NMR (CDCl3, 600 MHz):
d = 7.46–7.31 (10H, m, PhH and PhH0), 7.18 (2H, d, J 8.4 Hz, H-2),
7.13 (2H, d, J 8.4 Hz, H-200), 7.02 (2H, d, J 8.4 Hz, H-2000), 7.00 (1H,
s, NH), 6.95 (2H, d, J 8.4 Hz, H-300), 6.83 (2H, d, J 8.4 Hz, H-3), 6.78
(2H, d, J 8.4 Hz, H-3000), 5.07 (2H, s, C400-OCH2), 4.99 (2H, s, C4000-
OCH2), 2.47 (2H, q, J 7.2 Hz, CH2CH3), 2.32 (2H, q, J 7.2 Hz,
COCH2CH3), 1.20 (3H, t, J 7.2 Hz, COCH2CH3), 0.940 (3H, t, J
7.2 Hz, CH2CH3); 13C NMR (CDCl3, 50 MHz): d = 171.7 (CO), 157.6
(C-400), 157.2 (C-4000), 141.3 (C-20), 139.6 (C-4), 137.3 (C-1), 137.2
(PhCCH2O and PhC0CH2O), 136.4 (C-10), 136.0 (C-100), 135.4 (C-1000),
131.5 (C-3), 130.8 (C-2000), 130.7 (C-200), 128.6 (PhC0-ortho), 128.5
(PhC-ortho), 128.0 (PhC0-para), 127.9 (PhC-para), 127.6 (PhC0-
meta), 127.5 (PhC-meta), 118.6 (C-2), 114.4 (C-300), 114.3 (C-3000),
70.1 (C400-OCH2), 70.0 (C4000-OCH2), 30.8 (COCH2CH3), 29.7
(CH2CH3), 13.7 (CH2CH3), 9.7 (COCH2CH3); MS: 568.45 (M+H+),
590.44 (M+Na+). Compound 8a: Rf = 0.35 (Hex/EtOAc 6:4); IR (film)
4.2.3.3.
Synthesis
of
(E)-N-(4-(10,20-bis(400,4000-
(benzyloxy)phenyl)but-10-enyl)phenyl)hexanamide (7c) and
(Z)-N-(4-(10,20-bis(400,4000-(benzyloxy)phenyl) but-10-enyl)phenyl)
hexanamide (8c).
According to the general procedure, ana-
logues 7c and 8c were obtained from keto-amide 6c after purifica-
tion by flash column chromatography (Hex/EtOAc 8:2) and
evaporation of the solvent as white solid and oil respectively
(50% total yield). Compound 7c: Rf = 0.33 (Hex/EtOAc 8:2); mp
162–163 °C; IR (film) m ;
: 1657 cmꢀ1 1H NMR (CDCl3, 400 MHz):
m
: 1688 cmꢀ1 1H NMR (CDCl3, 600 MHz): d = 7.52 (2H, d, J 8.4 Hz,
;
d = 7.48–7.35 (10H, m, PhH and PhH0), 7.22 (2H, d, J 8.4 Hz, H-2),
7.16 (2H, d, J 8.4 Hz, H-200), 7.05 (2H, d, J 8.4 Hz, H-2000), 7.04 (1H,
s, NH), 6.98 (2H, d, J 8.4 Hz, H-300), 6.85 (2H, d, J 8.4 Hz, H-3), 6.81
(2H, d, J 8.4 Hz, H-3000), 5.09 (2H, s, C400-OCH2), 5.02 (2H, s, C4000-
OCH2), 2.45 (2H, q, J 7.8 Hz, CH2CH3), 2.30 (2H, t, J 7.2 Hz,
CH2CH2CH2CH2CH3), 1.72–1.69 (2H, m, CH2CH2CH2CH2CH3),
1.36–1.32 (4H, m, CH2CH2CH2CH2CH3), 0.960–0.900 (6H, m,
H-2), 7.43–7.29 (10H, m, PhH and PhH0), 7.18 (3H, m, H-3 and NH),
7.04 (2H, d, J 8.4 Hz, H-2000), 6.80 (2H, d, J 8.4 Hz, H-3000), 6.78 (2H, d,
J 8.4 Hz, H-200), 6.64 (2H, d, J 8.4 Hz, H-300), 5.01 (2H, s, C4000-OCH2),
4.93 (2H, s, C400-OCH2), 2.45 (2H, q, J 7.2 Hz, CH2CH3), 2.40 (2H, q, J
7.2 Hz, COCH2CH3), 1.26 (3H, t, J 7.2 Hz, COCH2CH3), 0.928 (3H, t, J
7.2 Hz, CH2CH3); 13C NMR (CDCl3, 50 MHz): d = 171.9 (CO), 157.1
(C-400), 156.8 (C-4000), 141.0 (C-20), 140.0 (C-4), 137.2 (PhCCH2O
and PhC0CH2O), 136.4 (C-10), 136.0 (C-100), 134.9 (C-1000), 132.0 (C-
CH2CH3 ja
I
CH2CH2CH2CH2CH3); 13C NMR (CDCl3, 50 MHz):

M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
4127
d = 171.3 (CO), 157.6 (C-400), 157.2 (C-4000), 141.0 (C-20), 139.6 (C-4),
137.3 (C-1), 137.2 (PhCCH2O and PhC0CH2O), 136.4 (C-10), 136.0 (C-
100), 135.4 (C-1000), 131.5 (C-3), 130.8 (C-2000), 130.7 (C-200), 128.6
(PhC0-ortho and PhC-ortho), 128.0 (PhC0-para and PhC-para),
127.6 (PhC0-meta and PhC-meta), 118.6 (C-2), 114.4 (C-300),
114.3 (C-3000), 70.0 (C400-OCH2), 69.9 (C4000-OCH2), 37.9
(CH2CH2CH2CH2CH3), 31.5 (CH2CH2CH2CH2CH3), 28.9 (CH2CH3),
25.4 (CH2CH2CH2CH2CH3), 22.5 (CH2CH2CH2CH2CH3), 14.0
(CH2CH2CH2CH2CH3) 13.7 (CH2CH3); MS: 610.84 (M+H+), 632.89
4.2.3.5.
Synthesis
of
(E)-3-(40-(100,200-bis(4000,40000-(benzyl-
oxy)phenyl)but-100-enyl)phenyl)-1,1-diethylurea (7e) and (Z)-3-
(40-(100,200-bis(4000,40000-(benzyloxy)phenyl)but-100-enyl)phenyl)-
1,1-diethylurea (8e).
According to the general procedure,
analogues 7e and 8e were obtained from keto-amide 6e after puri-
fication by flash column chromatography (Hex/EtOAc 6:4) and
evaporation of the solvent as white solid and oil respectively
(65% total yield). Compound 7e: Rf = 0.38 (Hex/EtOAc 6:4); mp
177–178 °C; IR (film) m ;
: 1645 cmꢀ1 1H NMR (CDCl3, 600 MHz):
(M+Na+). Compound 8c: Rf = 0.31 (Hex/EtOAc 8:2); IR (film)
1657 cmꢀ1
m:
d = 7.47–7.31 (10H, m, PhH and PhH0), 7.15 (2H, d, J 8.8 Hz, H-
2000), 7.07 (2H, d, J 8.8 Hz, H-20000), 7.06 (2H, d, J 8.8 Hz, H-20), 6.95
(2H, d, J 8.8 Hz, H-3000), 6.80 (2H, d, J 8.8 Hz, H-30), 6.79 (2H, d, J
8.8 Hz, H-30000), 6.12 (1H, s, NH), 5.07 (2H, s, C4000-OCH2), 5.00 (2H,
s, C40000-OCH2), 3.32 (4H, q, J 7.2 Hz, N(CH2CH3)2), 2.48 (2H, q, J
7.2 Hz, CH2CH3), 1.18 (6H, t, J 7.2 Hz, N(CH2CH3)2), 0.943 (3H, t, J
7.2 Hz, CH2CH3); 13C NMR (CDCl3, 50 MHz): d = 157.6 (C-4000),
157.2 (C-40000), 154.5 (CO), 140.8 (C-200), 138.1 (C-40), 137.5 (C-100),
137.2 (C-10 and C-1000), 136.7 (PhCCH2O and PhC0CH2O), 135.0 (C-
10000), 131.4 (C-30), 130.8 (C-20000), 130.7 (C-2000), 128.5 (PhC0-ortho
and PhC-ortho), 128.0 (PhC0-para and PhC-para), 127.6 (PhC0-meta
and PhC-meta), 118.6 (C-20), 114.4 (C-3000), 114.2 (C-30000), 70.1 (C4000-
OCH2), 69.9 (C40000-OCH2), 41.6 (N(CH2CH3)2), 28.9 (CH2CH3), 13.9
(N(CH2CH3)2), 13.7 (CH2CH3); MS: 611.57 (M+H+), 633.55
;
1H NMR (CDCl3, 400 MHz): d = 7.52 (2H, d, J 8.4 Hz,
H-2), 7.46–7.32 (10H, m, PhH and PhH0), 7.20 (2H, d, J 8.4 Hz, H-
3), 7.19 (1H, s, NH), 7.07 (2H, d, J 8.4 Hz, H-2000), 6.84 (2H, d, J
8.4 Hz, H-3000), 6.80 (2H, d, J 8.4 Hz, H-200), 6.67 (2H, d, J 8.4 Hz, H-
300), 5.03 (2H, s, C4000-OCH2), 4.96 (2H, s, C400-OCH2), 2.47 (2H, q, J
7.8 Hz, CH2CH3), 2.38 (2H, t,
J 7.2 Hz, CH2CH2CH2CH2CH3),
1.79–1.75 (2H, m, CH2CH2CH2CH2CH3), 1.42–1.38 (4H, m,
CH2CH2CH2CH2CH3), 0.980–0.920 (6H, m, CH2CH3 and
CH2CH2CH2CH2CH3); 13C NMR (CDCl3, 50 MHz): d = 171.4 (CO),
157.1 (C-4000), 156.8 (C-400), 141.3 (C-20), 140.0 (C-4), 137.2
(PhCCH2O and PhC0CH2O), 136.4 (C-10), 136.0 (C-100), 134.9 (C-1000),
132.0 (C-200), 130.8 (C-1), 130.7 (C-2000), 130.1 (C-3), 128.5 (PhC0-
ortho and PhC-ortho), 127.9 (PhC0-para and PhC-para), 127.5
(PhC0-meta and PhC-meta), 119.4 (C-2), 114.3 (C-3000), 113.7 (C-
300), 69.9 (C4000-OCH2), 69.8 (C400-OCH2), 37.9 (CH2CH2CH2CH2CH3),
31.5 (CH2CH2CH2CH2CH3), 28.9 (CH2CH3), 25.4 (CH2CH2CH2
CH2CH3), 22.5 (CH2CH2CH2CH2CH3), 14.0 (CH2CH2CH2CH2CH3)
13.7 (CH2CH3); MS: 610.85 (M+H+), 632.88 (M+Na+).
(M+Na+). Compound 8e: Rf = 0.33 (Hex/EtOAc 6:4); IR (film)
m:
1644 cmꢀ1 1H NMR (CDCl3, 600 MHz): d = 7.49–7.30 (12H, m,
;
PhH, PhH0 and H-20), 7.15 (2H, d, J 8.8 Hz, H-30), 7.04 (2H, d, J
8.8 Hz, H-20000), 6.80 (2H, d, J 8.8 Hz, H-30000), 6.79 (2H, d, J 8.8 Hz,
H-2000), 6.64 (2H, d, J 8.8 Hz, H-3000), 6.33 (1H, s, NH), 5.01 (2H, s,
C40000-OCH2), 4.93 (2H, s, C4000-OCH2), 3.39 (4H
q J 7.2 Hz,
4.2.3.4.
Synthesis
of
(E)-3-(40-(100,200-bis(4000,40000-(benzyl-
N(CH2CH3)2), 2.46 (2H, q, J 7.2 Hz, CH2CH3), 1.24 (6H, t, J 7.2 Hz,
N(CH2CH3)2), 0.920 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR (CDCl3,
50 MHz): d = 157.1 (C-4000), 156.7 (C-40000), 154.1 (CO), 140.8 (C-200),
139.0 (C-10), 137.3 (C-100 jaI C-40), 137.1 (PhCCH2O and PhC0CH2O),
136.1 (C-1000), 135.0 (C-10000), 132.0 (C-2000), 130.8 (C-20000), 130.1 (C-
30), 128.5 (PhC0-ortho and PhC-ortho), 127.9 (PhC0-para and PhC-
para), 127.6 (PhC0-meta and PhC-meta), 119.5 (C-20), 114.3 (C-
30000), 113.7 (C-3000), 69.9 (C4000-OCH2), 69.8 (C40000-OCH2), 41.7
(N(CH2CH3)2), 29.7 (CH2CH3), 14.0 (N(CH2CH3)2) 13.7 (CH2CH3);
MS: 611.58 (M+H+), 633.55 (M+Na+).
oxy)phenyl)but-100-enyl)phenyl)-1,1-dimethylurea (7d) and (Z)-
3-(40-(100,200-bis(4000,40000-(benzyloxy)phenyl)but-100-enyl)phenyl)-
1,1-dimethylurea (8d).
According to the general procedure,
analogues 7d and 8d were obtained from keto-amide 6d after puri-
fication by flash column chromatography (Hex/EtOAc 1:1) and
evaporation of the solvent as white solid and oil respectively
(50% total yield). Compound 7d: Rf = 0.23 (Hex/EtOAc 1:1); mp
174–175 °C; IR (film) m ;
: 1646 cmꢀ1 1H NMR (CDCl3, 400 MHz):
d = 7.49–7.33 (10H, m, PhH and PhH0), 7.17 (2H, d, J 8.8 Hz, H-
2000), 7.07 (2H, d, J 8.8 Hz, H-20000), 7.05 (2H, d, J 8.8 Hz, H-20), 6.98
(2H, d, J 8.8 Hz, H-3000), 6.82 (2H, d, J 8.8 Hz, H-30), 6.81 (2H, d, J
8.8 Hz, H-30000), 6.20 (1H, s, NH), 5.09 (2H, s, C4000-OCH2), 5.01 (2H,
s, C40000-OCH2), 2.98 (6H, s, N(CH3)2), 2.50 (2H, q, J 7.2 Hz, CH2CH3),
0.960 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR (CDCl3, 50 MHz): d = 157.5
(C-4000), 157.1 (C-40000), 155.6 (CO), 140.9 (C-200), 138.3 (C-40), 137.4
(C-100), 137.2 (PhCCH2O and PhC0CH2O), 136.4 (C-10 and C-1000),
135.0 (C-10000), 131.4 (C-30), 130.8 (C-20000), 130.7 (C-2000), 128.6
(PhC0-ortho and PhC-ortho), 128.0 (PhC0-para and PhC-para),
127.6 (PhC0-meta and PhC-meta), 118.6 (C-20), 114.4 (C-3000),
114.2 (C-30000), 70.0 (C4000-OCH2), 69.9 (C40000-OCH2), 36.5 (N(CH3)2),
29.0 (CH2CH3), 13.7 (CH2CH3); MS: 583.64 (M+H+), 605.64
4.2.3.6.
oxy)phenyl)but-100-enyl)phenyl)pyrrolidine-1-carboxamide (7f)
and
(Z)-N-(40-(100,200-bis(4000,40000-(benzyloxy)phenyl)but-100-
enyl)phenyl)pyrrolidine-1-carboxamide (8f). According to
the general procedure, analogues 7f and 8f were obtained from
keto-amide 6f after purification by flash column chromatography
(Hex/EtOAc 1:1) and evaporation of the solvent as white solid
and oil respectively (50% total yield). Compound 7f: Rf = 0.32
Synthesis
of
(E)-N-(40-(100,200-bis(4000,40000-(benzyl-
(Hex/EtOAc 1:1); mp 185–186 °C; IR (film)
m ;
: 1641 cmꢀ1 1H
NMR (CDCl3, 600 MHz): d = 7.46–7.31 (10H, m, PhH and PhH0),
7.16 (2H, d, J 8.8 Hz, H-2000), 7.07 (2H, d, J 8.8 Hz, H-20000), 7.03 (2H,
d, J 8.8 Hz, H-20), 6.95 (2H, d, J 8.8 Hz, H-3000), 6.79 (2H, d, J 8.8 Hz,
H-30), 6.78 (2H, d, J 8.8 Hz, H-30000), 6.09 (1H, s, NH), 5.07 (2H, s,
C4000-OCH2), 5.00 (2H, s, C40000-OCH2), 3.43 (4H, m, N(CH2)2(CH2)2),
2.42 (2H, q, J 7.2 Hz, CH2CH3), 1.94 (4H, m, N(CH2)2(CH2)2), 0.913
(3H, t, J 7.2 Hz, CH2CH3); 13C NMR (CDCl3, 50 MHz): d = 157.5 (C-
4000), 157.0 (C-40000), 153.9 (CO), 140.7 (C-200), 138.1 (C-40), 137.5
(C-100), 137.1 (C-10 jaI C-1000),136.7 (PhCCH2O and PhC0CH2O),
135.4 (C-10000), 131.4 (C-30), 130.8 (C-20000), 130.7 (C-2000), 128.5
(PhC0-ortho and PhC-ortho), 127.9 (PhC0-para and PhC-para),
127.6 (PhC0-meta and PhC-meta), 118.3 (C-20), 114.3 (C-3000),
114.2 (C-30000), 70.0 (C4000-OCH2), 69.9 (C40000-OCH2), 45.8
(N(CH2)2(CH2)2), 28.9 (CH2CH3), 25.6 (N(CH2)2(CH2)2), 13.7
(CH2CH3); MS: 609.65 (M+H+), 631.63 (M+Na+). Compound 8f:
(M+Na+). Compound 8d: Rf = 0.13 (Hex/EtOAc 1:1); IR (film)
1643 cmꢀ1 1H NMR (CDCl3, 400 MHz): d = 7.46–7.32 (12H, m,
m:
;
PhH, PhH0 and H-20), 7.17 (2H, d, J 8.8 Hz, H-30), 7.06 (2H, d, J
8.8 Hz, H-20000), 6.82 (2H, d, J 8.8 Hz, H-30000), 6.81 (2H, d, J 8.8 Hz,
H-2000), 6.66 (2H, d, J 8.8 Hz, H-3000), 6.34 (1H, s, NH), 5.03 (2H, s,
C40000-OCH2), 4.95 (2H, s, C4000-OCH2), 3.06 (6H, s, N(CH3)2), 2.48
(2H, q, J 7.2 Hz, CH2CH3), 0.940 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR
(CDCl3, 50 MHz): d = 157.0 (C-4000), 156.7 (C-40000), 155.7 (CO),
140.7 (C-200), 138.8 (C-10), 137.5 (C-100), 137.3 (C-40), 137.1
(PhCCH2O and PhC0CH2O), 136.1 (C-1000), 135.0 (C-10000), 132.0 (C-
2000), 130.8 (C-20000), 130.0 (C-30), 128.5 (PhC0-ortho and PhC-ortho),
127.9 (PhC0-para and PhC-para), 127.5 (PhC0-meta and PhC-meta),
119.5 (C-20), 114.2 (C-30000), 113.7 (C-3000), 69.9 (C4000-OCH2), 69.8
(C40000-OCH2), 36.5 (N(CH3)2), 29.7 (CH2CH3), 13.7 (CH2CH3); MS:
583.65 (M+H+), 605.64 (M+Na+).
Rf = 0.24 (Hex/EtOAc 1:1); IR (film)
m ;
: 1642 cmꢀ1 1H NMR (CDCl3,
600 MHz): d = 7.49–7.30 (12H, m, PhH, PhH0 and H-20), 7.14 (2H, d,

4128
M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
J 8.8 Hz, H-30), 7.03 (2H, d, J 8.8 Hz, H-20000), 6.79 (2H, d, J 8.8 Hz, H-
30000), 6.78 (2H, d, J 8.8 Hz, H-2000), 6.64 (2H, d, J 8.8 Hz, H-3000), 6.17
(1H, s, NH), 5.00 (2H, s, C40000-OCH2), 4.93 (2H, s, C4000-OCH2), 3.48
(4H, m, N(CH2)2(CH2)2), 2.46 (2H, q, J 7.2 Hz, CH2CH3), 1.98 (4H,
m, N(CH2)2(CH2)2), 0.922 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR (CDCl3,
50 MHz): d = 157.6 (C-4000), 156.9 (C-40000), 154.1 (CO), 140.7 (C-200),
138.6 (C-10), 137.5 (C-100), 137.4 (C-40), 137.1 (PhCCH2O and
PhC0CH2O), 136.2 (C-1000), 135.1 (C-10000), 132.0 (C-2000), 130.8
(C-20000), 130.0 (C-30), 128.5 (PhC0-ortho and PhC-ortho), 127.9
(PhC0-para and PhC-para), 127.6 (PhC0-meta and PhC-meta), 119.2
tracted twice with EtOAc. The combined organic extracts were
washed with water and brine, dried with Na2SO4, filtered and
evaporated. The residue was purified by flash column chromatog-
raphy (Hex/EtOAc 1:1) to provide analogue 9a as oil (90% yield).
Rf = 0.20 (Hex/EtOAc 1:1); IR (film)
m: ;
1689 cmꢀ1 1H NMR
(CD3OD-d4, 400 MHz): d = 7.20 (2H, d, J 8.6 Hz, H-2), 7.02 (2H, d, J
8.6 Hz, H-200), 6.94 (2H, d, J 8.6 Hz, H-2000), 6.81 (2H, d, J 8.6 Hz, H-
300), 6.78 (2H, d, J 8.6 Hz, H-3), 6.60 (2H, d, J 8.6 Hz, H-3000), 2.47
(2H, q, J 7.5 Hz, CH2CH3), 2.33 (2H, q, J 7.6 Hz, COCH2CH3), 1.19
(3H, t, J 7.8 Hz, COCH2CH3), 0.936 (3H, t, J 7.8 Hz, CH2CH3); 13C
NMR (CD3OD-d4, 100 MHz): d = 173.8 (CO), 155.8 (C-400), 155.4
(C-4000), 141.2 (C-20), 139.8 (C-4), 137.5 (C-1), 135.9 (C-1000), 135.0
(C-100), 133.4 (C-10), 130.9 (C-3), 130.5 (C-2000), 130.2 (C-200), 118.7
(C-2), 114.5 (C-300), 114.3 (C-3000), 29.6 (COCH2CH3), 28.4 (CH2CH3),
12.6 (CH2CH3), 8.8 (COCH2CH3); MS: 388.35 (M+H+), 410.33
(M+Na+). Anal. Calcd for C25H25NO3: C, 77.49; H, 6.50; N, 3.61.
Found: C, 81.40; H, 6.22; N, 3.38.
(C-20), 114.3 (C-30000), 113.7 (C-3000), 69.9 (C4000-OCH2), 69.8 (C40000
-
OCH2), 45.8 (N(CH2)2(CH2)2), 29.7 (CH2CH3), 25.6 (N(CH2)2(CH2)2)
13.7 (CH2CH3); MS: 609.67 (M+H+), 631.67 (M+Na+).
4.2.3.7.
Synthesis
of
(E)-N-(40-(100,200-bis(4000,4000-(benzyl-
oxy)phenyl)but-100-enyl)phenyl)piperidine-1-carboxamide (7g)
and (Z)-N-(40-(100,200-bis(4000,4000-(benzyloxy)phenyl)but-100-enyl)-
phenyl)piperidine-1-carboxamide (8g).
According to the
general procedure, analogues 7g and 8g were obtained from
keto-amide 6g after purification by flash column chromatography
(Hex/EtOAc 7:3) and evaporation of the solvent as white solid
and oil respectively (50% total yield). Compound 7g: Rf = 0.28
4.2.4.2.
Synthesis
of
(Z)-N-(4-(10,20-bis(400,4000-hydroxy-
According
phenyl)but-10-enyl) phenyl)propionamide (10a).
to the general procedure, analogue 10a was obtained from ana-
logue 8a after purification by flash column chromatography
(Hex/EtOAc 1:1) and evaporation of the solvent as oil (90% yield).
(Hex/EtOAc 7:3); mp 210–211 °C; IR (film)
m ;
: 1644 cmꢀ1 1H
NMR (CDCl3, 400 MHz): d = 7.49–7.34 (10H, m, PhH and PhH0),
7.16 (2H, d, J 8.8 Hz, H-2000), 7.06 (2H, d, J 8.8 Hz, H-20000), 7.04 (2H,
d, J 8.8 Hz, H-20), 6.97 (2H, d, J 8.8 Hz, H-3000), 6.80 (4H, d, J 8.8 Hz,
H-30 and H-30000), 6.23 (1H, s, NH), 5.09 (2H, s, C4000-OCH2), 5.01
(2H, s, C40000-OCH2), 3.40 (4H, t, J 6.4 Hz, N(CH2)2(CH2)2CH2), 2.49
(2H, q, J 7.2 Hz, CH2CH3), 1.60 (6H, m, N(CH2)2(CH2)2CH2 and
N(CH2)2(CH2)2CH2), 0.957 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR
(CDCl3, 50 MHz): d = 157.5 (C-4000), 157.1 (C-40000), 154.9 (CO),
140.8 (C-200), 138.2 (C-40), 137.4 (C-100), 137.1 (PhCCH2O and
PhC0CH2O), 136.7 (C-10), 136.6 (C-1000), 135.0 (C-10000), 131.4 (C-30),
130.8 (C-20000), 130.7 (C-2000), 128.5 (PhC0-ortho and PhC-ortho),
127.9 (PhC0-para and PhC-para), 127.6 (PhC0-meta and PhC-meta),
118.6 (C-20), 114.3 (C-3000), 114.2 (C-30000), 70.0 (C4000-OCH2), 69.9
(C40000-OCH2), 45.2 (N(CH2)2(CH2)2CH2), 29.7 (CH2CH3), 25.7
(N(CH2)2(CH2)2CH2), 24.4 (N(CH2)2(CH2)2CH2), 13.7 (CH2CH3);
MS: 623.87 (M+H+), 645.81 (M+Na+). Compound 8g: Rf = 0.22
Rf = 0.18 (Hex/EtOAc 1:1); IR (film) m: ;
1690 cmꢀ1 1H NMR
(CD3OD-d4, 400 MHz): d = 7.53 (2H, d, J 8.5 Hz, H-2), 7.15 (2H, d, J
8.5 Hz, H-3), 6.94 (2H, d, J 8.6 Hz, H-2000), 6.69 (2H, d, J 8.6 Hz, H-
200), 6.61 (2H, d, J 8.6 Hz, H-3000), 6.46 (2H, d, J 8.6 Hz, H-300), 2.48–
2.40 (4H, m, CH2CH3 and COCH2CH3), 1.23 (3H, t, J 7.6 Hz,
COCH2CH3), 0.932 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR (CD3OD-d4,
100 MHz): d = 174.0 (CO), 155.3 (C-4000), 154.9 (C-400), 140.6 (C-20),
140.1 (C-4), 137.4 (C-10), 136.9 (C-1), 134.8 (C-100), 133.5 (C-1000),
131.7 (C-200), 130.6 (C-2000), 129.5 (C-3), 119.6 (C-2), 114.3 (C-3000),
113.8 (C-300), 29.7 (COCH2CH3), 28.3 (CH2CH3), 12.6 (CH2CH3), 8.9
(COCH2CH3); MS: 388.36 (M+H+), 410.35 (M+Na+). Anal. Calcd for
C25H25NO3: C, 77.49; H, 6.50; N, 3.61. Found: C, 81.42; H, 6.24;
N, 3.36.
4.2.4.3. Synthesis of (E)-N-(4-(10,20-bis(400,4000-hydroxyphenyl)-
but-10-enyl) phenyl)isobutyramide (9b).
According to the
(Hex/EtOAc 7:3); IR (film)
m
:
1642 cmꢀ1
;
1H NMR (CDCl3,
general procedure, analogue 9b was obtained from analogue 7b
after purification by flash column chromatography (Hex/EtOAc
1:1) and evaporation of the solvent as oil (95% yield). Rf = 0.30
400 MHz): d = 7.46–7.32 (12H, m, PhH, PhH0 and H-20), 7.16 (2H,
d, J 8.8 Hz, H-30), 7.05 (2H, d, J 8.8 Hz, H-20000), 6.82 (2H, d, J
8.8 Hz, H-30000), 6.81 (2H, d, J 8.8 Hz, H-2000), 6.66 (2H, d, J 8.8 Hz,
H-3000), 6.38 (1H, s, NH), 5.03 (2H, s, C40000-OCH2), 4.95 (2H, s, C4000-
OCH2), 3.48 (4H, t, J 6.4 Hz, N(CH2)2(CH2)2CH2), 2.48 (2H, q, J
7.2 Hz, CH2CH3), 1.66 –1.61 (6H, m, N(CH2)2(CH2)2CH2 and
N(CH2)2(CH2)2CH2), 0.940 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR
(CDCl3, 50 MHz): d = 157.0 (C-4000), 156.7 (C-40000), 155.7 (CO),
140.7 (C-200), 138.7 (C-40), 137.6 (C-10), 137.4 (C-100), 137.1
(PhCCH2O and PhC0CH2O), 136.1 (C-1000), 135.1 (C-10000), 132.0 (C-
2000), 130.8 (C-20000), 130.0 (C-30), 128.5 (PhC0-ortho and PhC-ortho),
127.9 (PhC0-para and PhC-para), 127.6 (PhC0-meta and PhC-meta),
119.4 (C-20), 114.2 (C-30000), 113.7 (C-3000), 69.9 (C4000-OCH2), 69.8
(C40000-OCH2), 45.3 (N(CH2)2(CH2)2CH2), 29.7 (CH2CH3), 25.7
(N(CH2)2(CH2)2CH2), 24.4 (N(CH2)2(CH2)2CH2), 13.7 (CH2CH3);
MS: 623.84 (M+H+), 645.81 (M+Na+).
(Hex/EtOAc 1:1); IR (film)
m ;
: 1665 cmꢀ1 1H NMR (acetone-d6,
600 MHz): d = 7.35 (2H, d, J 8.4 Hz, H-2), 7.05 (2H, d, J 8.4 Hz, H-
200), 6.97 (2H, d, J 8.4 Hz, H-2000), 6.83 (2H, d, J 8.4 Hz, H-300), 6.80
(2H, d, J 8.4 Hz, H-3), 6.65 (2H, d, J 8.4 Hz, H-3000), 2.54 (1H, m,
CH(CH3)2), 2.45 (2H, q, J 7.8 Hz, CH2CH3), 1.10 (6H, d, J 6.6 Hz,
CH(CH3)2), 0.915 (3H, t, J 7.8 Hz, CH2CH3); 13C NMR (acetone-d6,
50 MHz): d = 174.8 (CO), 156.1 (C-400), 155.7 (C-4000), 140.8 (C-20),
138.9 (C-4), 137.6 (C-10), 137.2 (C-1), 135.1 (C-100), 133.4 (C-1000),
130.9 (C-3), 130.7 (C-2000), 130.4 (C-200), 118.1 (C-2), 114.9 (C-300),
114.8 (C-3000), 35.7 (CH(CH3)2), 28.8 (CH2CH3 from HSQC), 19.6
(CH(CH3)2), 13.1 (CH2CH3); MS: 402.38 (M+H+), 424.36 (M+Na+).
Anal. Calcd for C26H27NO3: C, 77.78; H, 6.78; N, 3.49. Found: C,
81.82; H, 6.43; N, 3.12.
4.2.4.4. Synthesis of (Z)-N-(4-(10,20-bis(400,4000-hydroxyphenyl)-
4.2.4. General procedure for the deptotection of analogues 7
and 8
but-10-enyl) phenyl)isobutyramide (10b).
According to the
general procedure, analogue 10b was obtained from analogue 8b
after purification by flash column chromatography (Hex/EtOAc
1:1) and evaporation of the solvent as oil (95% yield). Rf = 0.29
4.2.4.1. Synthesis of (E)-N-(4-(10,20-bis(400,4000-hydroxyphenyl)-
but-10-enyl) phenyl)propionamide (9a).
To a cold at ꢀ78 °C
solution of analogue 7a (0.018 g, 0.032 mmol) in CH2Cl2 (2.2 mL),
BBr3 (0.07 mmol, 1.0 M in CH2Cl2, 5 equiv per bond, 3.6 mL) was
added dropwise and the reaction was kept for another 1 h at the
same temperature. Then, the solution was cooled at 0 °C, neutral-
ized with MeOH and HCl 1 N was added. The organic phase was ex-
(Hex/EtOAc 1:1); IR (film) m ;
: 1665 cmꢀ1 1H NMR (acetone-d6,
600 MHz): d = 7.67 (2H, d, J 8.4 Hz, H-2), 7.13 (2H, d, J 8.4 Hz, H-
3), 6.97 (2H, d, J 8.4 Hz, H-2000), 6.71 (2H, d, J 8.4 Hz, H-200), 6.66
(2H, d, J 8.4 Hz, H-3000), 6.50 (2H, d, J 8.4 Hz, H-300), 2.63 (1H, m,
CH(CH3)2), 2.43 (2H, q, J 7.8 Hz, CH2CH3), 1.17 (6H, d, J 6.6 Hz,

M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
4129
CH(CH3)2), 0.920 (3H, t, J 7.8 Hz, CH2CH3); 13C NMR (acetone-d6):
d = 171.8 (CO), 155.6 (C-4000), 155.3 (C-400), 140.4 (C-20), 139.2 (C-
4), 138.2 (C-10), 137.6 (C-1), 134.8 (C-100), 133.4 (C-1000), 131.8 (C-
200), 130.7 (C-2000), 129.6 (C-3), 118.9 (C-2), 114.7 (C-3000), 114.2 (C-
300), 36.0 (CH(CH3)2), 28.9 (CH2CH3 from HSQC), 19.0 (CH(CH3)2),
13.1 (CH2CH3); MS: 402.37 (M+H+), 424.37 (M+Na+). Anal. Calcd
for C26H27NO3: C, 77.78; H, 6.78; N, 3.49. Found: C, 81.84; H,
6.42; N, 3.11.
20), 114.4 (C-3000), 114.3 (C-30000), 35.3 (N(CH3)2), 28.4 (CH2CH3),
12.6 (CH2CH3). MS: 403.51 (M+H+), 425.52 (M+Na+). Anal. Calcd
for C25H26N2O3: C, 74.60; H, 6.51; N, 6.96. Found: C, 79.73; H,
6.18; N, 6.55.
4.2.4.8. Synthesis of (Z)-3-(40-(100,200-bis(4000,4000-hydroxyphenyl)-
but-100-enyl) phenyl)-1,1-dimethylurea (10d).
According to
the general procedure, analogue 10d was obtained from analogue
8d after purification by flash column chromatography (EtOAc)
and evaporation of the solvent as oil (95% yield). Rf = 0.41 (EtOAc);
4.2.4.5. Synthesis of (E)-N-(4-(10,20-bis(400,4000-hydroxyphenyl)-
but-10-enyl)phenyl) hexanamide (9c).
According to the gen-
IR (film) m ;
: 1644 cmꢀ1 1H NMR (CD3OD-d4, 400 MHz): d = 7.35
eral procedure, analogue 9c was obtained from analogue 7c after
purification by flash column chromatography (Hex/EtOAc 1:1)
and evaporation of the solvent as oil (90% yield). Rf = 0.31 (Hex/
(2H, d, J 8.4 Hz, H-20), 7.11 (2H, d, J 8.4 Hz, H-30), 6.94 (2H, d, J
8.4 Hz, H-20000), 6.68 (2H, d, J 8.4 Hz, H-2000), 6.61 (2H, d, J 8.4 Hz,
H-30000), 6.44 (2H, d, J 8.4 Hz, H-3000), 3.05 (6H, s, N(CH3)2), 2.46
(2H, q, J 7.2 Hz, CH2CH3), 0.930 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR
(CD3OD-d4, 50 MHz): d = 157.7 (CO), 155.7 (C-4000), 155.2 (C-40000),
140.4 (C-200), 137.2 (C-10), 136.8 (C-100), 135.2 (C-40), 133.5 (C-1000),
131.4 (C-10000), 130.7 (C-2000), 130.6 (C-20000), 130.3 (C-30), 119.7 (C-
20), 114.4 (C-30000), 114.3 (C-3000), 35.3 (N(CH3)2), 28.4 (CH2CH3),
12.6 (CH2CH3); MS: 403.50 (M+H+), 425.50 (M+Na+). Anal. Calcd
for C25H26N2O3: C, 74.60; H, 6.51; N, 6.96. Found: C, 79.77; H,
6.20; N, 6.58.
EtOAc 1:1); IR (film)
m: ;
1658 cmꢀ1 1H NMR (CD3OD-d4,
400 MHz): d = 7.20 (2H, d, J 8.8 Hz, H-2), 7.02 (2H, d, J 8.8 Hz, H-
200), 6.93 (2H, d, J 8.4 Hz, H-2000), 6.81 (2H, d, J 8.8 Hz, H-3), 6.77
(2H, d, J 8.8 Hz, H-300), 6.60 (2H, d, J 8.4 Hz, H-3000), 2.47 (2H, q, J
7.4 Hz, CH2CH3), 2.31 (2H, t, J 7.4 Hz, CH2CH2CH2CH2CH3), 1.68–
1.63 (2H, m, CH2CH2CH2CH2CH3), 1.38–1.33 (4H, m,
CH2CH2CH2CH2CH3), 0.933 (6H, t,
J
7.4 Hz, CH2CH3 and
CH2CH2CH2CH2CH3); 13C NMR (CD3OD-d4, 100 MHz): d = 173.8
(CO), 156.5 (C-400), 156.0 (C-4000), 141.8 (C-20), 140.5 (C-4), 138.1
(C-10), 136.5 (C-1), 135.7 (C-100), 134.1 (C-1000), 131.5 (C-3), 131.2
(C-2000), 130.9 (C-200), 119.4 (C-2), 115.1 (C-300), 115.0 (C-3000), 37.2
(CH2CH2CH2CH2CH3), 31.8 (CH2CH2CH2CH2CH3), 29.1 (CH2CH3),
25.9 (CH2CH2CH2CH2CH3), 22.7 (CH2CH2CH2CH2CH3), 13.2
(CH2CH2CH2CH2CH3 and CH2CH3); MS: 430.57 (M+H+), 461.57
(M+Na+). Anal. Calcd for C28H31NO3: C, 78.29; H, 7.27; N, 3.26.
Found: C, 83.13; H, 6.98; N, 2.88.
4.2.4.9. Synthesis of (E)-3-(40-(100,200-bis(4000,4000-hydroxyphenyl)-
but-100-enyl) phenyl)-1,1-diethylurea (9e).
According to the
general procedure, analogue 9e was obtained from analogue 7e after
purification by flash column chromatography (Hex/EtOAc 1:1) and
evaporation of the solvent as foam (95% yield). Rf = 0.15 (Hex/EtOAc
1:1);IR(film)m
:1646 cmꢀ1; 1HNMR(acetone-d6, 600 MHz):d = 7.41
(1H, s, NH), 7.21 (2H, d, J 8.4 Hz, H-20), 7.05 (2H, d, J 8.4 Hz, H-2000),
6.96 (2H, d, J 8.4 Hz, H-20000), 6.83 (2H, d, J 8.4 Hz, H-3000), 6.73 (2H, d,
J 8.4 Hz, H-30), 6.65 (2H, d, J 8.4 Hz, H-30000), 3.36 (4H, q, J 7.2 Hz,
N(CH2CH3)2), 2.45 (2H, q, J 7.2 Hz, CH2CH3), 1.11 (6H, t, J 7.2 Hz,
N(CH2CH3)2), 0.911 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR (acetone-d6,
50 MHz): d = 156.1 (CO), 155.6 (C-4000), 154.3 (C-40000), 140.3 (C-200),
138.3 (C-40), 137.8 (C-100), 137.4 (C-10), 135.3 (C-1000), 133.6 (C-10000),
130.7 (C-30), 130.6 (C-20000), 130.4 (C-2000), 118.4 (C-20), 114.7 (C-3000
and C-30000), 40.9 (N(CH2CH3)2), 28.9 (CH2CH3 from HSQC), 13.4
(N(CH2CH3)2), 13.1 (CH2CH3); MS: 431.39 (M+H+), 453.40 (M+Na+).
Anal. Calcd for C27H30N2O3: C, 75.32; H, 7.02; N, 6.51. Found: C,
80.87; H, 6.69; N, 6.04.
4.2.4.6. Synthesis of (Z)-N-(4-(10,20-bis(400,4000-hydroxyphenyl)-
but-10-enyl)phenyl) hexanamide (10c).
According to the
general procedure, analogue 10c was obtained from analogue 8c
after purification by flash column chromatography (Hex/EtOAc
1:1) and evaporation of the solvent as oil (90% yield). Rf = 0.29
(Hex/EtOAc 1:1); IR (film)
m ;
: 1658 cmꢀ1 1H NMR (CD3OD-d4,
400 MHz): d = 7.53 (2H, d, J 8.8 Hz, H-2), 7.15 (2H, d, J 8.8 Hz, H-
3), 6.94 (2H, d, J 8.8 Hz, H-2000), 6.68 (2H, d, J 8.8 Hz, H-200), 6.61
(2H, d, J 8.8 Hz, H-3000), 6.45 (2H, d, J 8.8 Hz, H-300), 2.47 (2H, q, J
7.4 Hz, CH2CH3), 2.37 (2H, t, J 7.7 Hz, CH2CH2CH2CH2CH3), 1.75–
170 (2H, m, CH2CH2CH2CH2CH3), 1.41–1.38 (4H, m,
CH2CH2CH2CH2CH3), 0.933 (6H, t,
J
7.4 Hz, CH2CH3 and
4.2.4.10. Synthesis of (Z)-3-(40-(100,200-bis(4000,4000-hydroxyphenyl)-
CH2CH2CH2CH2CH3); 13C NMR (CD3OD-d4, 100 MHz): d = 173.8
(CO), 157.0 (C-4000), 155.5 (C-400), 141.3 (C-20), 140.8 (C-4), 137.5
(C-10), 135.7 (C-1), 135.4 (C-100), 134.1 (C-1000), 132.3 (C-200), 131.2
(C-2000), 130.1 (C-3), 120.2 (C-2), 115.0 (C-3000), 114.4 (C-300), 37.2
(CH2CH2CH2CH2CH3), 32.1 (CH2CH2CH2CH2CH3), 29.0 (CH2CH3),
25.9 (CH2CH2CH2CH2CH3), 22.7 (CH2CH2CH2CH2CH3), 13.5
(CH2CH2CH2CH2CH3 and CH2CH3); MS: 430.58 (M+H+), 461.56
(M+Na+). Anal. Calcd for C28H31NO3: C, 78.29; H, 7.27; N, 3.26.
Found: C, 83.12; H, 6.99; N, 2.87.
but-100-enyl) phenyl)-1,1-diethylurea (10e).
According to
the general procedure, analogue 10e was obtained from analogue
8e after purification by flash column chromatography (Hex/EtOAc
1:1) and evaporation of the solvent as oil (95% yield). Rf = 0.14
(Hex/EtOAc 1:1); IR (film)
m ;
: 1643 cmꢀ1 1H NMR (acetone-d6,
600 MHz): d = 7.62 (1H, s, NH), 7.54 (2H, d, J 8.4 Hz, H-20), 7.07
(2H, d, J 8.4 Hz, H-30), 6.97 (2H, d, J 8.4 Hz, H-20000), 6.71 (2H, d, J
8.4 Hz, H-2000), 6.65 (2H, d, J 8.4 Hz, H-30000), 6.50 (2H, d, J 8.4 Hz,
H-3000), 3.43 (4H, q, J 7.2 Hz, N(CH2CH3)2), 2.44 (2H, q, J 7.2 Hz,
CH2CH3), 1.17 (6H, t, J 7.2 Hz, N(CH2CH3)2), 0.911 (3H, t, J 7.2 Hz,
CH2CH3); 13C NMR (acetone-d6, 50 MHz): d = 157.1 (CO), 155.6
(C-4000), 154.4 (C-40000), 140.1 (C-200), 137.8 (C-10), 137.2 (C-100),
135.0 (C-40), 133.6 (C-1000), 131.8 (C-10000), 130.7 (C-2000), 130.4 (C-
20000), 129.2 (C-30), 119.2 (C-20), 114.7 (C-30000), 114.1 (C-3000), 40.9
(N(CH2CH3)2), 29.0 (CH2CH3 from HSQC), 13.4 (N(CH2CH3)2), 13.1
(CH2CH3); MS: 431.38 (M+H+), 453.40 (M+Na+). Anal. Calcd for
C27H30N2O3: C, 75.32; H, 7.02; N, 6.51. Found: C, 80.86; H, 6.66;
N, 6.08.
4.2.4.7. Synthesis of (E)-3-(40-(100,200-bis(4000,4000-hydroxyphenyl)-
but-100-enyl) phenyl)-1,1-dimethylurea (9d).
According to
the general procedure, analogue 9d was obtained from analogue
7d after purification by flash column chromatography (EtOAc)
and evaporation of the solvent as oil (95% yield). Rf = 0.44 (EtOAc);
IR (film)
m ;
: 1647 cmꢀ1 1H NMR (CD3OD-d4, 400 MHz): d = 7.03
(2H, d, J 8.4 Hz, H-20), 7.02 (2H, d, J 8.4 Hz, H-2000), 6.94 (2H, d, J
8.4 Hz, H-20000), 6.78 (2H, d, J 8.4 Hz, H-3000), 6.76 (2H, d, J 8.4 Hz,
H-30), 6.58 (2H, d, J 8.4 Hz, H-30000), 2.98 (6H, s, N(CH3)2), 2.46 (2H,
q, J 7.2 Hz, CH2CH3), 0.930 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR
(CD3OD-d4, 50 MHz): d = 157.5 (CO), 155.3 (C-4000), 154.8 (C-40000),
140.8 (C-200), 138.8 (C-40), 137.6 (C-100), 136.9 (C-10), 135.2 (C-1000),
133.5 (C-10000), 130.7 (C-30), 130.5 (C-20000), 130.2 (C-2000), 119.8 (C-
4.2.4.11. Synthesis of (E)-N-(40-(100,200-bis(4000,40000-hydroxy-
phenyl)but-100-enyl)phenyl)pyrrolidine-1-carboxamide
(9f).
According to the general procedure, analogue 9f was
obtained from analogue 7f after purification by flash column

4130
M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
chromatography (Hex/EtOAc 4:6) and evaporation of the solvent as
(2H, d, J 8.4 Hz, H-3000), 3.53 (4H, t, J 6.4 Hz, N(CH2)2(CH2)2CH2),
2.46 (2H, q, J 7.2 Hz, CH2CH3), 1.70 (4H, m, N(CH2)2(CH2)2CH2),
1.64 (2H, m, N(CH2)2(CH2)2CH2), 0.931 (3H, t, J 7.2 Hz, CH2CH3);
13C NMR (CD3OD-d4, 50 MHz): d = 156.9 (C-4000), 155.7 (C-40000),
152.7 (CO), 140.8 (C-200), 139.0 (C-10), 137.6 (C-100), 137.0 (C-40),
135.2 (C-1000), 133.5 (C-10000), 131.6 (C-2000), 130.2 (C-20000), 129.3 (C-
30), 119.6 (C-20), 114.3 (C-30000), 113.7 (C-3000), 44.9
(N(CH2)2(CH2)2CH2), 29.3 (CH2CH3), 25.6 (N(CH2)2(CH2)2CH2 and
N(CH2)2(CH2)2CH2), 12.6 (CH2CH3); MS: 443.58 (M+H+), 465.52
(M+Na+). Anal. Calcd for C28H30N2O3: C, 75.99; H, 6.83; N, 6.33.
Found: C, 80.15; H, 6.47; N, 6.01.
oil (90% yield). Rf = 0.11 (Hex/EtOAc 4:6); IR (film) m ;
: 1640 cmꢀ1
1H NMR (acetone-d6, 600 MHz): d = 7.23 (2H, d, J 8.4 Hz, H-20),
7.05 (2H, d, J 8.4 Hz, H-2000), 6.96 (2H, d, J 8.4 Hz, H-20000), 6.83 (2H,
d, J 8.4 Hz, H-3000), 6.73 (2H, d, J 8.4 Hz, H-30), 6.64 (2H, d, J 8.4 Hz,
H-30000), 3.38 (4H, t, J 7.2 Hz, N(CH2)2(CH2)2), 2.44 (2H, q, J 7.2 Hz,
CH2CH3), 1.88 (4H, t, J 7.2 Hz, N(CH2)2(CH2)2), 0.909 (3H, t, J
13
7.2 Hz, CH2CH3); C NMR (acetone-d6, 50 MHz): d = 156.1 (C-4000),
155.4 (C-40000), 154.8 (CO), 140.1 (C-200), 138.3 (C-40), 137.9 (C-100),
137.1 (C-10), 135.5 (C-1000), 133.2 (C-10000), 130.5 (C-30), 130.2 (C-
20000), 130.1 (C-2000), 118.4 (C-20), 114.9 (C-3000), 114.8 (C-30000), 45.4
(N(CH2)2(CH2)2), 29.0 (CH2CH3 from HSQC), 25.2 (N(CH2)2(CH2)2),
13.3 (CH2CH3); MS: 429.40 (M+H+), 451.39 (M+Na+). Anal. Calcd
for C27H28N2O3: C, 75.68; H, 6.59; N, 6.54. Found: C, 80.12; H,
6.22; N, 6.18.
4.3. Biological assays
4.3.1. Inhibition growth assay
MCF-7 (human mammary gland adenocarcinoma cells), MSTO-
211H (human biphasic mesothelioma cells) and HeLa (human
cervix adenocarcinoma cells) were grown in Dulbecco’s Modified
Eagle’s Medium (Sigma Chemical Co.), in RPMI 1640 (Sigma Chem-
ical Co.) supplemented with 2.38 g/L Hepes, 0.11 g/L pyruvate so-
dium and in Nutrient Mixture F-12 [HAM] (Sigma Chemical Co.),
respectively. 10% Heat-inactivated fetal calf serum (Invitrogen),
4.2.4.12. Synthesis of (Z)-N-(40-(100,200-bis(4000,40000-hydroxy-
phenyl)but-100-enyl)phenyl)pyrrolidine-1-carboxamide
(10f).
According to the general procedure, analogue 10f was
obtained from analogue 8f after purification by flash column chro-
matography (Hex/EtOAc 4:6) and evaporation of the solvent as oil
(90% yield). Rf = 0.10 (Hex/EtOAc 4:6); IR (film) m ;
: 1642 cmꢀ1 1H
NMR (acetone-d6, 600 MHz): d = 7.55 (2H, d, J 8.4 Hz, H-20), 7.52
(1H, s, NH), 7.07 (2H, d, J 8.4 Hz, H-30), 6.96 (2H, d, J 8.4 Hz, H-
20000), 6.71 (2H, d, J 8.4 Hz, H-2000), 6.65 (2H, d, J 8.4 Hz, H-30000), 6.50
(2H, d, J 8.4 Hz, H-3000), 3.45 (4H, t, J 7.2 Hz, N(CH2)2(CH2)2), 2.44
(2H, q, J 7.2 Hz, CH2CH3), 1.93 (4H, t, J 7.2 Hz, N(CH2)2(CH2)2),
0.910 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR (acetone-d6, 50 MHz):
d = 155.6 (C-4000), 155.3 (C-40000), 153.9 (CO), 140.0 (C-200), 137.8 (C-
10), 137.6 (C-100), 135.9 (C-40), 135.0 (C-1000), 131.8 (C-10000), 130.7
(C-2000), 129.3 (C-20000 and C-30), 118.7 (C-20), 114.7 (C-30000), 114.1
(C-3000), 45.5 (N(CH2)2(CH2)2), 29.0 (CH2CH3 from HSQC), 25.3
(N(CH2)2(CH2)2), 13.2 (CH2CH3); MS: 429.43 (M+H+), 451.41
(M+Na+). Anal. Calcd for C27H28N2O3: C, 75.68; H, 6.59; N, 6.54.
Found: C, 80.10; H, 6.21; N, 6.17.
100 U/mL penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL
amphotericin B (Sigma Chemical Co.) were added to the media.
The cells were cultured at 37 °C in a moist atmosphere of 5% carbon
dioxide in air. MCF-7 and MSTO-211H (3–4 ꢁ 104) were seeded into
each well of a 24-well cell culture plate. After incubation for 24 h,
various concentrations of the test agents were added to the com-
plete medium and cells were incubated for a further 72 h. HeLa
(3–4 ꢁ 104) were seeded into each well of a 24-well cell culture
plate. After incubation for 24 h, the medium was replaced with an
equal volume of fresh medium, and various concentrations of the
test agents were added. The cells were then incubated in standard
conditions for a further 72 h. A trypan blue assay was performed
to determine cell viability. Cytotoxicity data were expressed as
GI50 values, that is, the concentration of the test agent inducing
50% reduction in cell growth compared with control cultures.
4.2.4.13. Synthesis of (E)-N-(40-(100,200-bis(4000,40000-hydroxy-
phenyl)but-100-enyl)
phenyl)piperidine-1-carboxamide
According to the general procedure, analogue 9g was ob-
(9g).
4.3.2. Estrogenic activity: luciferase assay (HC11 cells)
tained from analogue 7g after purification by flash column chro-
HC11 cells (mouse mammary breast epithelial cells) stably
transfected with 3ꢁERE–Luc construct were seeded in 24-well
plates and after the cells reached 50–60% confluence, were treated
matography (Hex/EtOAc 1:1) and evaporation of the solvent as
oil (90% yield). Rf = 0.18 (Hex/EtOAc 1:1); IR (film) m ;
: 1645 cmꢀ1
1H NMR (CD3OD-d4, 400 MHz): d = 7.01 (2H, d, J 8.4 Hz, H-20),
7.00 (2H, d, J 8.4 Hz, H-2000), 6.94 (2H, d, J 8.4 Hz, H-20000), 6.77 (2H,
d, J 8.4 Hz, H-3000), 6.76 (2H, d, J 8.4 Hz, H-30), 6.59 (2H, d, J 8.4 Hz,
H-30000), 3.45 (4H, t, J 6.4 Hz, N(CH2)2(CH2)2CH2), 2.46 (2H, q, J
7.2 Hz, CH2CH3), 1.66 (4H, m, N(CH2)2(CH2)2CH2), 1.58 (2H, m,
N(CH2)2(CH2)2CH2), 0.929 (3H, t, J 7.2 Hz, CH2CH3); 13C NMR
(CD3OD-d4, 50 MHz): d = 155.9 (C-4000), 155.2 (C-40000), 154.9 (CO),
142.0 (C-200), 138.7 (C-40), 138.0 (C-100), 137.8 (C-10), 135.1 (C-1000),
133.6 (C-10000), 131.6 (C-30), 130.5 (C-20000), 130.2 (C-2000), 120.5 (C-
20), 114.4 (C-3000), 114.3 (C-30000), 44.8 (N(CH2)2(CH2)2CH2), 29.3
(CH2CH3), 25.5 (N(CH2)2(CH2)2CH2 and N(CH2)2(CH2)2CH2), 12.8
(CH2CH3); MS: 443.59 (M+H+), 465.54 (M+Na+). Anal. Calcd for
with 1 lM (final) tested compounds, 1 lM 4-OH tamoxifen and
10 nM E2 or ethanol (control). After 24 h cells were collected in
1ꢁ Passive Lysis Buffer (Promega) according to manufacturer’s
instructions, and luciferase activity was measured using BioThema
Luciferase Assay. Protein concentration of samples was determined
using Bradford assay. The luciferase values were correlated to the
protein concentration of each sample. Data are indicated as % of
E2 activity above bars of three independent experiments, per-
formed in duplicates.
4.3.3. DNA topoisomerase relaxation assay
Supercoiled pBR322 plasmid DNA (0.25 lg, Fermentas Life Sci-
C28H30N2O3: C, 75.99; H, 6.83; N, 6.33. Found: C, 80.17; H, 6.49;
ences) was incubated with 1U topoisomerase II (USB Corporation)
N, 5.99.
or 2U topoisomerase I (Topogen) and the test compounds as indi-
4.2.4.14. Synthesis of (Z)-N-(40-(100,200-bis(4000,40000-hydroxy-
cated, for 60 min at 37 °C in 20
Reactions were stopped by adding 4
0.125% bromophenol blue and 25% glycerol), 50
l
L reaction buffer.
L stop buffer (5% SDS,
g/mL proteinase
phenyl)but-100-enyl)
phenyl)piperidine-1-carboxamide
According to the general procedure, analogue 10g
l
l
(10g).
K (Sigma) and incubating for a further 30 min at 37 °C. The samples
was obtained from analogue 8g after purification by flash column
were separated by electrophoresis on a 1% agarose gel at room
chromatography (Hex/EtOAc 1:1) and evaporation of the solvent
temperature. The gels were stained with ethidium bromide 1 lg/
as oil (90% yield). Rf = 0.17 (Hex/EtOAc 1:1); IR (film)
m:
1643 cmꢀ1
;
1H NMR (CD3OD-d4, 400 MHz): d = 7.32 (2H, d, J
mL in TAE buffer, transilluminated by UV light, and fluorescence
emission was visualized by a CCD camera coupled to a Bio-Rad
Gel Doc XR apparatus.
8.4 Hz, H-20), 7.11 (2H, d, J 8.4 Hz, H-30), 6.94 (2H, d, J 8.4 Hz, H-
20000), 6.68 (2H, d, J 8.4 Hz, H-2000), 6.61 (2H, d, J 8.4 Hz, H-30000), 6.44

M. S. Christodoulou et al. / Bioorg. Med. Chem. 21 (2013) 4120–4131
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This research has been developed under the umbrella of
CM0602 COST Action ‘Inhibitors of Angiogenesis: design, synthesis
and biological exploitation’ and the ongoing CM1106 COST Action
‘Chemical Approaches for Targeting Drug Resistance in Cancer
Stem Cells’.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.012.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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