Synthesis of estradiol-cinnamide conjugates

Article abstract of DOI:10.3184/174751912X13419346932954

The synthesis of a number of structurally related estradiol-17α- ylmethyl hydroxycinnamides and of one novel estra- 1,3,5(10),6-tetraen-3-ol- 17β-yl hydroxycinnamide is described, using a microwave assisted amidation of steroidal amines with pentafluorophenol activated, non-protected hydroxycinnamic acids as a key step. A selection of the compounds and of other estra-1,3,5(10)-trien-3-ol 17β-yl hydroxycinnamides was screened against 60 human cancer cell lines, derived from nine neoplastic diseases. From the overall results of the screening, it could be inferred that dihydroxycinnamide derived estradiol conjugates exhibit a better cytotoxic profile when compared with hydroxymethoxycinnamide derived estradiol conjugates.

Full text of DOI:10.3184/174751912X13419346932954

JOURNAL OF CHEMICAL RESEARCH 2012
RESEARCH PAPER 549
SEPTEMBER, 549–554
Synthesis of estradiol-cinnamide conjugates
Goreti Ribeiro Moraisa and Thies Thiemannb,c,*
aInstituto SuperiorTécnico/InstitutoTecnológico e Nuclear, Estrada Nacional 10, Sacavem, Portugal
bInstitute of Materials Chemistry and Engineering, Kyushu University, Kasuga 2686-953, Fukuoka 816-8580, Japan
cPresent address: Faculty of Science, United Arab Emirates University, PO Box 17551, Al Ain, UAE
The synthesis of a number of structurally related estradiol-17α-ylmethyl hydroxycinnamides and of one novel estra-
1,3,5(10),6-tetraen-3-ol-17β-yl hydroxycinnamide is described, using a microwave assisted amidation of steroidal
amines with pentafluorophenol activated, non-protected hydroxycinnamic acids as a key step. A selection of the
compounds and of other estra-1,3,5(10)-trien-3-ol 17β-yl hydroxycinnamides was screened against 60 human cancer
cell lines, derived from nine neoplastic diseases. From the overall results of the screening, it could be inferred that
dihydroxycinnamide derived estradiol conjugates exhibit a better cytotoxic profile when compared with hydroxy-
methoxycinnamide derived estradiol conjugates.
Keywords: steroids, estradiols, cinnamides, antiproliferative effect, cytotoxicity
Simple phenolic and polyphenolic structures, many of which
are aromatic metabolites of plants, have attracted much atten-
tion because of their potential protective role against diseases
associated with oxidative damage. They have been studied
extensively, and their free radical scavenging and antioxidant
activities have been well documented.1,2 In addition to reports
describing the antioxidant properties of phenolic structures,
communications have revealed the anticancer activity of some
phenolic acids.3–8 The mechanism associated with the observed
antiproliferative and cytotoxic effects of those structures is still
not clear, and may depend on the dose and the model studied.
The pro-oxidant effect and consequent capacity for generating
free radicals has been one of the factors thought to be respon-
sible for the cytotoxicity associated with these molecular
structures.8
Phytoestrogens, naturally occurring chemicals derived from
plants, are known to retard chemical carcinogenesis in experi-
mental models.3,4 These natural compounds have attracted par-
ticular attention due to their structural similarity to steroidal
estrogens and their capacity to mimic biological responses of
estrogens. On the other hand, steroidal estrogens themselves
(e.g. estradiol) have been found to exhibit the biological prop-
erties of phytoestrogens, in particular, antioxidant activity.9
This realisation has driven researchers to introduce structural
modifications on the estradiol nucleus, with the purpose of
increasing the antioxidant properties of these compounds. It
has been shown that the introduction of a radicophilic moiety,
preferentially of a phenolic group, at the 17α-position of the
estradiol framework increases the antioxidant activity of the
derivatives.10,11
For this reason, we designed a small series of estrogen
hybridised with phenolic acids, in an effort to prepare mole-
cules that were biologically active towards neoplastic cells.
Three different hydroxycinnamic acid residues were selected
to be attached to steroidal derivatives of the estrane series
to give the structurally related steroidal-hydroxycinnamide
conjugates 1 and 2 (Fig. 1). As candidates, steroids were
chosen, in which hydroxycinnamic moieties were connected
via an amido function on the β face of the steroids (1). Also,
steroidal amides were to be prepared, in which the hydroxycin-
namate moiety was connected to the α face of the steroids (2,
Fig. 1). As hydroxycinnamic residues, hydroxyphenyl moieties
with different phenolic patterns were chosen, including α,β-
dihydroxy- (caffeic acid derived) and α-hydroxy-β-methoxy-
cinnamic residues (ferulic acid and isoferulic acid derived). In
the steroidal moiety, the structural differences were obtained
by replacing the 17β-hydroxyl group of the estradiol with
an amino function or by introducing an amino function in
the form of a methylamino group at the 17α-position, while
preserving the hydroxyl group at the 17β-position. As the
influence of a protection of the phenolic function of the estra-
diol residue in form of a methoxy group and the influence of
an unsaturation at the 6,7-position of the steroidal framework
on the biological activity of the conjugates were to be studied,
the novel steroid 1a was to be one of the main synthetic targets.
Furthermore, with the planned key step being a microwave
assisted amidation of non-protected hydroxycinnamic acids,
previously developed by the authors,12 we wished to examine
whether this reaction could be used in the neighbourhood of a
tertiary hydroxyl group in the amine component, as is present
in the steroidal amines leading to target molecules 2.
Fig. 1 Steroidal-hydroxycinnamide conjugates.
* Correspondent. E-mail: thies@uaeu.ac.ae

550 JOURNAL OF CHEMICAL RESEARCH 2012
Results and discussion
the respective steroidal amine in water, with no possibility to
extract it with several organic phases tried. Thus, before carry-
ing out the reduction, the hydroxyl groups in the cyanohydrin
6 were protected as tetrahydropyranyl ethers. The steroidal
amine 9 was then isolated without problem. Further, steroidal
amine 9 was used without purification, in order to avoid
hydrolysis of the acid sensitive protective groups.
The syntheses of the compounds 1b–e have been described
previously.12 Treatment of the 3-methoxy-estra-1,3,5(10),6-
tetraene-17-one 3c, prepared according to the literature,13 with
hydroxylamine hydrochloride gave the estra-1,3,5(10),6-
tetraene oxime 4c.14 The oxime function was reduced using
the aluminum amalgam method,15 in which the 17-amino
group was obtained predominantly in the β-configuration
(Scheme 1). This method has shown to be selective, with
no significant hydrogenation of the olefin bond at the 6,7-
position. 3-O-Methyl-17β-aminoestra-1,3,5(10)-triene-3-ol (5b)
and 17β-aminoestra-1,3,5(10)-triene-3-ol (5a) were prepared
by reducing the respective oximes 4a and 4b with sodium in
refluxing propanol.
In the other type of E-hydroxycinnamides 2, the amide link-
age was to be created on the α face of the steroidal derivatives.
Preparation of the steroidal derivative with an amine function
on the α side involves the stereoselective transformation of the
17-ketone into a cyanohydrin group and further reduction to
the methylamine. Thus, estrone 3a and 3-methoxyestrone 3b,
respectively, were reacted with trimethylsilyl cyanide in the
presence of a Lewis acid (ZnI2) followed by treatment with
acid (HCl).16 Under these conditions, the addition of the cyano
group is α-stereoselective. Reduction of the cyano group was
carried out with lithium aluminum hydride, in dry THF at room
temperature (Scheme 2).17
With the steroidal amines prepared, the next step consisted
of the amidation reaction of the steroids with the phenolic
acids, caffeic and ferulic acids. In our group, it was previously
established12 that a solventless mixture of the amine with
caffeic acid readily gave the corresponding caffeic amide
under microwave (MW) irradiation, when activated with dicy-
clohexylcarbodiimide (DCC) and pentafluorophenol (PFP).
This method was shown to be a general synthetic approach to
prepare amides from unprotected hydroxyphenylalkenoic
acids.12 Normally, the pentafluorophenyl alkenoates as the
activated acid components are prepared first and the isolated
activated alkenoates are then reacted in a second step under
MW irradiation. As reported previously by the authors, ferulic
and isoferulic acid gave the corresponding, easily isolable
pentafluorophenyl esters 12b and 12c in high yield, when
following a procedure by Zhao et al.,18 adapted by the authors
to the reaction of alkenoic acids. Thus, the phenolic acids were
reacted with DCC and PFP in anhydrous dioxane at room
temperature (Scheme 3). The formed dicyclohexylurea (DCU)
was easily removed by filtration and the product was obtained
by column chromatography on silica gel.
Attempts to isolate the product of the reduction of the
cyanohydrin 6 failed. The reason was an excessive solubility of
Scheme 1
Scheme 2

JOURNAL OF CHEMICAL RESEARCH 2012 551
Scheme 3
In the case of caffeic acid (11a), the reaction was found to
lead to a by-product along with the formation of the respective,
desired activated acid derivative 12a. However, the by-product
was shown not to be the typical ureide, described in the litera-
ture as a by-product in the reaction of 2-alkenoic acids with
nucleophiles, when using DCC as coupling reagent. Rather,
it was the 1,3-dicyclohexyl-1-(3′,4′-dihydroxycinnamoyl)-2-
pentafluorophenyl-isourea 13 (Scheme 3). This by-product,
which was formed in significant amounts, complicated the
work-up of 12a.
Nevertheless, the reaction of the steroidal amines 5c, 9, and
10 with a mixture of 12a and 13 gave the E-3,4-dihydroxy-
phenylpropenamides 1a, 14a, and 2b in good yield under MW
irradiation (Scheme 4). The steroidal amines 9 and 10, when
reacted with the pentafluorophenyl ester of ferulic acid 12b
gave the E-3-methoxy-4-hydroxyphenylpropenamides 14c
and 2d under the same conditions. A small amount of dioxane,
necessary to help to mix the reagents adequately, was used.
Before column chromatographic separation of the amidation
products, the steroidal amides 14a and 14c were treated with
Scheme 4

552 JOURNAL OF CHEMICAL RESEARCH 2012
Table 1 In vitro tumour 50% growth inibition (GI50) of 1c, 2b,
2d and 5-fluorouracil (FU)25
Conclusion
A number of novel steroidal-cinnamide conjugates 1a and 2
were prepared according to a straightforward synthetic strat-
egy in good overall yield. The microwave assisted amidation
of non-protected cinnamic acids was found to be possible in
the presence of a neighbouring hydroxyl group in the amine
component. The in vitro anti-tumor screening against a full
panel of 60 cancer cell lines revealed the anti-tumor activity of
most of the steroidal cinnamide conjugates. The dihydroxy-
phenyl moiety of the derivatives confers a higher antiprolifera-
tion capacity to the derivatives. The presence of the methoxy
group at the 3-position of the A ring in the steroid subunit is
necessary for a good antiproliferative activity within this series
of compounds. Compounds possessing an additional hydroxyl
group at 17β (2b and 2d) preserved the desired antiprolifera-
tive effect with a moderate decrease in the cytostatic effect and
a marked loss of the cytotoxicity.
Panel/cell line
1c
log10GI50
2b
log10GI50
2d
FU
log10GI50 log10GI50
Leukemia
CCRF-CEM
–6.14
–6.06
–6–30
–6.03
<–8.00
–4.41
–5.00
–5.10
–7.20
–4.70
–4.42
–5.80
–5.60
–5.00
Non-small cell lung cancer
NCI-H522
CNS cancer
SF-539
Ovarian cancer
SK-OV-3
Melanoma
SK-MEL-2
Renal cancer
UO-31
Prostate cancer
PC-3
–5.77
–4.94
–5.56
–5.90
–5.66
–5.26
–4.83
–5.25
–5.99
–5.72
–5.40
Breast cancer
BT-549
Experimental
Melting points were measured on a Yanaco hot-stage microscope.
IR spectra were measured with a JASCO IR-700 instrument. 1H
(270 MHz) and 13C (67.8 MHz) spectra were recorded with a JEOL
EX-270 spectrometer. The chemical shifts are relative to TMS. Mass
spectra were measured with a JMS-01-SG-2 spectrometer. Column
chromatography was carried out on Wakogel 300.
For the microwave irradiation experiments a domestic microwave
National NE-S12 (with power settings at 170 W/500 W and 750 W,
2450 MHz) was used. Pyrex glass beakers (20 mL) with a small open-
ing were used. The beakers were sealed with Saran Wrap®, which
allows for pressure equilibration. Temperature calibration showed that
a temperature plateau of 130 ºC (bulk temperature of the mixture) was
reached after 3 min. All chemical reagents were of reagent grade.
Estrone was obtained commercially from Wako. 3-Methoxyestrone
(3b) was prepared according to the literature (KOH, MeI, DMSO).22
3-O-Methyl-17β-aminoestra-1,3,5(10)-triene-3-ol (5b) and 17β-
aminoestra-1,3,5(10)-triene-3-ol (5a) were prepared from the corre-
sponding steroidal oximes23 by reduction with sodium in refluxing
propanol.24 The synthesis of steroids 1b–1e has been described
earlier.12
para-toluenesulfonic acid in order to hydrolyse the tetrahydro-
pyranyloxy groups, giving the non-protected hydroxysteroidal
amides 2a and 2c.
The starting protocol consists of an in vitro pre-screening
of the reported compounds 1a–e and 2a–d using the three
cell lines MCF 7 (breast cancer), NCI-H460 (lung cancer), and
SF-268 (Central Nervous System cancer-CMS), in accordance
with the protocol of the Drug Evaluation Branch, National
Cancer Institute, Bethesda.19–21 The minimum condition for
satisfying this test is the reduction of at least 32% of the cell
growth in any one of the three lines. Among all the evaluated
compounds, compounds 1d and 1e failed the pre-screening,
while compounds 1a–c and 2a–d exhibited positive results and
have been further evaluated in vitro against a full panel of 60
human cancer cell lines derived from nine neoplastic diseases.
The response parameters 50% growth inhibition (GI50), total
growth inhibition (TGI), and 50% lethal concentration (LC50)
of the compounds obtained for each cell line are given in
Table 1 and in the Electronic Supplementary Information
(ESI, Tables S1–S3). Compounds 1a–c and 2a–d exhibited a
non-selective broad spectrum of antitumor activity.
Compounds 1a, 1c, 2b, and 2d exhibited a better antiprolif-
erative profile than the parents 1b, 2a, and 2c. In the most
active compounds, the hydroxyl function at the 3-position of
the A-ring of the steroid was protected in the form of methoxy
group. Given that a good compromise between lipophilicity
versus hydrophilicity is required for the molecules to reach the
cell, it is believed that the presence of the methoxy group might
contribute to the better in vitro performance exhibited by the
more lipophilic 1a, 1c, 2b, and 2d. It should be noted that
the unsaturation in the B ring in 1a does not improve the
antiproliferative effect of the derivatives. In comparison with
5-fluorouracil (FU), 1c shows a better antiproliferative effect
against a number of cancer cell lines.
Compound 2d was found to exhibit a very high antiprolif-
erative effect against the CNS cancer cell line SF-539 (Table 1)
and the leukemic cancer cell line CCRF-CEM. In both cancer
cell lines, the growth inhibition effect of 1d surpassed the
observed effect for the FU. However, it was compound 2b that
exhibited a more linear tumour cell growth inhibitory effect
among the 60 cancer cell lines, while also exhibiting a better
antiproliferative effect than FU in several cancer cell lines
(Table 1; ESI, Table S1). The good antiproliferative effect of
2b, in addition to the low cytotoxicity (ESI, Table S3) suggests
this compound to be the most favourable candidate as a lead
compound for further development of new derivatives along
these lines.
3-O-Methyl-estra-1,3,5(10),6-tetraene-3-ol-17-one-17-oxime (4c):
3-O-Methyl-estra-1,3,5(10),6-tetraene-3-ol-17-one (3c, 1.3 g, 4.7 mmol)
was dissolved in EtOH (22 mL) and a solution of NH2OH.HCl (1.3 g,
18.8 mmol) in H2O (5 mL) was added at room temperature. The
reaction mixture was heated at 50 ºC for 11 h 30 min. The solvent
was then evaporated, and the crude mixture was submitted to column
chromatography on silica gel (EtOAc/CHCl3 1:3 → 1:2) to give 4c
(850 mg, 61%) as a colourless solid, m.p. 180–184 ºC. (Found: M+,
297.1730. C19H23O2N requires M+, 297.1729). KBr/cm−1 νmax 3262,
2924, 1682, 1630, 1603, 1568, 1493, 1452, 1257, 1046, 929; 1δH
(CDCl3) 0.96 (3H, s, CH3), 1.50–2.43 (9H, m), 2.58–2.63 (2H, m),
3.80 (3H, s, OCH3), 6.04 (1H, d, 3J = 9.4 Hz), 6.49 (1H, dd,
3
4
4J = 2.7 Hz J = 9.4 Hz), 6.66 (1H, d, J = 2.7 Hz), 6.75 (1H, dd,
4J = 2.7 Hz, 3J 8.3 Hz), 7.17 (1H, d, 3J = 8.3 Hz), 7.50 (1H, bs, OH);
δc (CDCl3) 16.9, 22.8, 24.0, 25.1, 33.5, 38.1, 42.1, 44.7, 51.2, 55.3,
111.8, 112.0, 124.2, 128.3, 131.1, 132.0, 135.3, 158.2, 170.7; MS
(EI+, 70 eV) m/z (%) 297 (100) [M+].
3-O-Methyl-17β-aminoestra-1,3,5(10),6-tetraene-3-ol (5c): Alu-
minium powder (297 mg, 11 mmol) was added to a solution of estrone
oxime 4c (320 mg, 1.07 mmol) in EtOH (10 mL). A solution of HgCl2
(240 mg, 0.88 mmol) in H2O (1 mL) and EtOH (2 mL) was added.
The reaction mixture was stirred at reflux temperature for 24 h. The
reaction mixture was then submitted to centrifugation. The superna-
tant was separated and was evaporated in vacuo. The centrifugate was
stirred with conc. HCl (5 mL) and resubmitted to centrifugation. This
procedure was repeated twice. An aqueous solution of HCl (50 mL)
was added to the acidic supernatant phases was added and poured onto
the crude of the first supernatant, followed by extraction with EtOAc
(2 x 50 mL). Aqueous 10% NaOH sol. was then added to the aqueous
phase until basic pH, and the mixture was extracted with EtOAc
(3 × 50 mL). The combined organic phases were washed with H2O
(100 mL), dried over anhydrous Na2SO4, and evaporated in vacuo
to provide 3-O-methyl-17β-aminoestra-1,3,5(10),6-tetraene-3-ol, 3c
(140 mg, 46%) as an oil. (Found: M+, 283.1933. C19H25ON requires

JOURNAL OF CHEMICAL RESEARCH 2012 553
M+, 283.1936). Neat/cm−1 νmax 3372, 2924, 1603, 1570, 1491, 1460,
1260; δH (CDCl3) 0.68 (3H, s, CH3), 1.20–2.47 (13H, m), 2.79 (1H,
requires MH+, 316.2277). KBr/cm−1 νmax 3448, 2932, 1614, 1507,
1448; δH (CDCl3) 0.93 (3H, s, CH3), 1.25–2.3 (14H, m), 2.59 (1H, d,
gJ = 12.5 Hz), 2.82–2.86 (2H, m), 3.01 (1H, d, gJ = 12.5 Hz), 3.77 (3H,
s, OCH3), 6.62 (1H, d, J = 2.7 Hz), 6.70 (1H, dd, J = 8.4 Hz J =
2.7 Hz), 7.19 (1H, d, 3J = 8.4 Hz); δC (CDCl3) 14.4, 23.2, 26.3, 27.5,
29.8, 32.6, 34.9, 39.4, 43.8, 45.8, 47.5, 50.0, 55.2, 81.7, 111.5, 113.8,
126.2, 132.6, 138.0, 157.5; MS (FAB+, 3-nitrobenzyl alcohol) m/z (%)
316 (44) [MH+].
3
3
m), 3.79 (3H, s, OCH3), 5.99 (1H, dd, J = 1.6 Hz J = 9.6 Hz, H7),
6.44 (1H, dd, 4J = 2.7 Hz 3J = 9.6 Hz, H6), 6.64 (1H, d, 4J = 2.7 Hz),
4
3
4
4
3
3
6.73 (1H, dd, J = 2.7 Hz J 8.1 Hz), 7.16 (1H, d, J = 8.1 Hz); MS
(EI+, 70 eV) m/z (%) 171 (100), 283 (66) [M+].
17α-Cyano-estra-1,3,5(10)-triene-3,17β-diol (6)–ZnI2 (95 mg,
0.3 mmol) and estrone 3a (2 g, 7.4 mmol) were placed in dry CH2Cl2
(5 mL), and TMSCN (13.3 mmol) was added. The reaction was car-
ried out under an argon atmosphere, initially under reflux for 1h and
then at rt for 3 more hours. Thereafter, conc. HCl (2 mL) was added as
well as H2O (10 mL) and CH2Cl2 (10 mL). A white precipitate was
formed after 15 min. It was washed with CH2Cl2 (10 mL) giving 6
(1.5 g, 68%) as a colourless solid, m.p. 256–260 ºC. (Found: M+,
297.1728. C19H23O2N requires M+, 297.1729). KBr/cm−1 νmax 3414,
2924, 2850, 2236, 1649, 1610, 1450, 1220; δH (DMSO-d6) 0.90 (3H,
s, CH3), 1.38–2.54 (14H, m), 2.80–2.84 (2H, m), 4.62 (1H, s, OH),
6.56 (1H, d, 4J = 2.7 Hz), 6.63 (1H, dd, 4J = 2.7 Hz 3J = 8.3 Hz), 7.14
(1H, d, 3J = 8.3 Hz); MS (EI+, 70 eV) m/z (%) 297 (100) [M+].
17α-Cyano-3,17β-bis-tetrahydropyranyloxy-estra-1,3,5(10)-triene
(7): To a solution of the cyanohydrin 6 (1.50 g, 5.05 mmol) in CH2Cl2
(15 mL) and THF (4 mL) was added DHP (1.5 ml, 25.9 mmol) and
p-TsOH (9.6 mg, 0.05 mmol). The reaction mixture was stirred for
10 min at rt. Thereafter, pyridine (0.20 mL) and a solution of H2O
(90 mL) containing NaHCO3 (410 mg) were added, and the mixture
was extracted with CH2Cl2 (100 mL). The organic phase was washed
with H2O (100 mL), dried over anhydrous Na2SO4, and concentrated
in vacuo to dryness. The crude residue was subjected to column
chromatography on silica gel (ether/CHCl3/hexane 1:1:3) to give 17α-
cyano-3,17β-bis-O-tetrahydropyranyl-estra-1,3,5(10)-triene-3,17β-
diol, 7 (1.69g, 72%) as an amorphous solid. (Found: MH+, 466.2956.
C29H40O4N requires MH+, 466.2957). KBr/cm−1 νmax 2938, 2870, 1613,
1499, 1245, 1202, 1037; δH (DMSO-d6) 0.94 (3H, s, CH3), 1.26–1.90
(22H, m), 2.24–2.48 (3H, m), 2.82–2.87 (2H, m), 3.53–3.63 (2H, m),
3.86–3.96 (2H, m), 5.09–5.11 (1H, m), 5.37–5.40 (1H, m), 6.78 (1H,
(E)-N-(3′-O-Methyl-estra-1′,3′,5′(10′),6′-tetraen-3′-ol-17′β-yl)-[3-
(3,4-dihydroxyphenyl)]propenamide (1a): The steroidal amine 5c
(77 mg, 0.27 mmol) was added to a mixture of 12a and 13 (365 mg, ≅
0.80 mmol) in the minimum dioxane (1 mL) and the resulting mixture
was irradiated at 500 W (2450 MHz) for 4 min. The reaction was car-
ried out under an argon atmosphere. After the reaction was complete,
the mixture was subjected directly to column chromatography on
silica gel (EtOAc/CHCl3 1:1) to give 1a (70 mg, 58%) as a colourless
solid; m.p. 157–161 ºC. (Found: MH+, 446.2326. C28H32O4N requires
MH+, 446.2331). KBr/cm−1 νmax 3434, 2928, 1652, 1602, 1447, 1517,
1258, 1199; δH (CDCl3) 0.77 (3H, s, CH3), 0.85–2.40 (11H, m), 3.79
(3H, s, OCH3), 4.12–4.15 (1H, m, H17), 5.63 (1H, d, J = 7.8 Hz, NH),
5.98 (1H, d, 3J = 9.7 Hz), 6.13 (1H, b, OH), 6.27 (1H, d, EJ = 15.5 Hz),
6.46 (1H, dd, 4J = 2.4 Hz, 3J = 9.7 Hz), 6.65 (1H, d, 4J = 2.7 Hz), 6.73
(1H, dd, 3J = 8.1 Hz, 4J = 2.7 Hz), 6.87 (1H, d, 3J = 7.4 Hz), 7.02 (1H,
3
3
d, J = 7.4 Hz), 7.15 (1H, d, J = 8.1 Hz), 7.20 (1H, s), 7.59 (1H, d,
EJ = 15.5 Hz), 7.70 (1H, b, OH); δC (CDCl3) 12.1, 23.5, 24.2, 31.2,
31.5, 36.7, 38.8, 41.8, 44.3, 49.6, 55.4, 59.4, 111.8, 111.9, 112.0,
115.4, 117.4, 120.1, 124.3, 128.1, 131.4, 132.6, 135.4, 142.3, 144.2,
146.5, 158.3, 167.1; MS (FAB+, 3-nitrobenzyl alcohol) m/z (%) 446
(3.4) [MH+].
(E)-N-(3′-O-Methyl-estra-1′,3′,5′(10′)-triene-3′,17′β-diol-17′α-
ylmethyl)-[3-(3,4-dihydroxyphenyl)]propenamide (2b): The steroidal
amine 10 (110 mg, 0.35 mmol) was added to a mixture of 12a and 13
(465 mg, ≅ 1.04 mmol) in the minimum dioxane (1 mL) and the result-
ing mixture was irradiated at 500 W (2450 MHz) for 2 min. After the
reaction was complete, the mixture was subjected directly to column
chromatography on silica gel (EtOAc/CHCl3 1:1) to give 2b (120 mg,
75%) as a colourless solid; m.p. 150–154 ºC. (Found: MH+, 478.2595.
C29H36O5N requires MH+, 478.2593). KBr/cm−1 νmax 3422, 2928, 1653,
1606, 1533, 1281, 1255; δH (DMSO-d6) 0.82 (3H, s, CH3), 1.22–1.90
(11H, m), 2.15–2.34 (2H, m), 2.76 (2H, m), 3.13–3.16 (1H, m), 3.56–
3.64 (1H, m), 3.68 (3H, s, OCH3), 4.36–4.61 (1H, b, centered at 4.5,
OH), 6.58 (1H, d, EJ = 15.4 Hz), 6.59 (1H, d, 4J = 2.7 Hz), 6.66 (1H,
dd, 4J = 2.7 Hz 3J = 8.1 Hz), 6.73 (1H, d, 3J = 8.1 Hz), 6.84 (1H, dd,
4J = 1.9 Hz 3J = 8.6 Hz), 6.95 (1H, d, 4J = 1.9 Hz), 7.16 (1H, d, 3J =
8.6 Hz), 7.22 (1H, d, EJ = 15.4 Hz), 7.76–7.77 (1H, m, NH), 8.83–9.53
(2H, b, centred at 9.23, OH); δC (DMSO-d6) 14.2, 23.0, 25.9, 27.0,
29.3, 30.6, 31.0, 32.7, 43.2, 45.4, 45.8, 49.3, 54.8, 82.2, 111.4, 113.4,
114.0, 115.7, 118.8, 120.3, 126.1, 126.5, 132.1, 137.4, 139.1, 145.5,
147.2, 157.0, 166.3; MS (FAB+, 3-nitrobenzyl alcohol) m/z (%) 478
(22) [MH+].
4
3
4
d, J = 2.7 Hz), 6.84 (1H, dd, J = 8.6 Hz J = 2.7 Hz), 7.19 (1H, d,
3J = 8.6 Hz); δC (DMSO-d6) 12.7, 18.8, 19.5, 23.3, 25.2, 25.3, 26.1,
27.2, 29.6, 30.4, 31.0, 33.8, 35.2, 38.9, 43.4, 48.3, 48.6, 62.0, 63.1,
86.6, 96.4, 97.7, 114.1, 116.6, 120.7, 126.2, 133.2, 137.7, 155.0; MS
(FAB+, 3-nitrobenzyl alcohol) m/z (%) 381 (10.9), 466 (1.7) [MH+].
3-O-Methyl-17α-cyano-estra-1,3,5(10)-triene-3,17β-diol (8) ZnI2
(89 mg, 0.28 mmol) and 3-methoxy estrone 3b (2 g, 7 mmol) were
placed in dry CH2Cl2 (4 mL), and TMSCN (12.6 mmol) was added.
The reaction was carried out under an argon atmosphere for 4 hours.
The reaction mixture was then treated with conc. HCl (2 mL). H2O
(100 mL) was added to the reaction mixture, and it was extracted
with CH2Cl2 (2 × 100 mL). The organic phase was washed with H2O
(100 mL), dried over anhydrous MgSO4, and filtered. After concentra-
tion of the solvent, the crude product was submitted to column chro-
matography on silica gel (ether/hexane/CHCl3 1:1:1) to give 8 (1.8 g,
86%) as a colourless solid. (Found: MH+, 311.1894. C20H25O2N requi-
res MH+, 311.1885). KBr/cm−1 νmax 3382, 2918, 2866, 2234, 1499,
1464, 1257, 1147; δH (CDCl3) 0.90 (3H, s, CH3), 1.40–2.54 (14H, m),
2.84–2.87 (2H, m), 3.78 (3H, s, OCH3), 6.63 (1H, d, 4J = 2.6 Hz), 6.72
(1H, dd, 4J = 2.6 Hz 3J = 8.4 Hz), 7.20 (1H, d, 3J = 8.4 Hz); MS (FAB+,
3-nitrobenzyl alcohol) m/z (%) 311 (28) [MH+].
(E)-N-(3′-O-Methyl-estra-1′,3′,5′(10′)-triene-3′,17′β-diol-17′α-
ylmethyl)-[3-(4-hydroxy-3-methoxyphenyl)propenamide (2d): The
steroidal amine 10 (60 mg, 0.19 mmol) was added to 12b (210 mg,
0.60 mmol) in the minimum dioxane (1 mL) and the resulting mixture
was irradiated at 500 W (2450 MHz) for 2 min. After the reaction was
complete, the mixture was subjected directly to column chromato-
graphy on silica gel (EtOAc/CHCl3 1:1) to give 2d (60 mg, 64%) as a
colourless solid; m.p. 222–224 ºC. (Found: MH+, 492.2749. C30H38NO5
requires MH+, 492.2750). KBr/cm−1 νmax 3418, 2924, 1655, 1612,
1524, 1253, 1209; δH (CDCl3) 0.93 (3H, s, CH3), 1.25–2.37 (13H, m),
2.83–2.86 (2H, m), 3.39 (1H, dd, 4J = 2.7 Hz, gJ = 13.4 Hz), 3.68–3.73
3-O-Methyl-17α-aminomethyl-estra-1,3,5(10)-triene-3,17β-diol
(10): A solution of cyanohydrin 8 (0.72 g, 2.33 mmol) in dry THF
(7 mL) at 0 ºC was added to a suspension of LiAlH4 (0.41 g, 10.80 mmol)
in dry THF (7 mL). The reaction mixture was stirred for 24 h at rt.
The reaction mixture was then poured onto ice (20 g) and H2O (3 mL)
was added, the organic solvent was evaporated and the aluminium
salts were filtered. The aluminium salts were washed several times,
alternately with H2O and EtOAc, until no amine could be detected
(monitoring by TLCusing a 2% ethanolic sol. of ninhydrin). Conc.
HCl was added dropwise to the aqueous phase until pH ~ 1 was
reached. EtOAc (75 mL) was then added to the aqueous phase, and the
mixture was extracted. Then, the aqueous phase was extracted further
with EtOAc (2 × 50 mL). Aqueous 10% NaOH sol. was added to
the aqueous phase until basic pH was reached, and the mixture was
extracted with EtOAc (4 × 50 mL). The organic phase was washed
with H2O (50 mL), dried over anhydrous Na2SO4, and evaporated
in vacuo to dryness to give 3-O-methyl-17α-aminomethyl-estra-
1,3,5(10)-triene-3,17β-diol 10 (295 mg, 40%) as a colourless solid,
m.p. 155–161 ºC (lit. 163 ºC [17]). (Found: MH+, 316.2270. C20H30O2N
g
3
(1H, dd, J = 13.4 Hz, J = 7.4Hz), 3.77 (3H, s, OCH3), 3.92 (3H,
E
s, OCH3), 5.84 (1H, s), 6.07–6.11 (1H, m, NH), 6.31 (1H, d, J =
4
3
4
15.4 Hz), 6.63 (1H, d, J = 2.7 Hz), 6.70 (1H, dd, J = 8.5 Hz, J =
2.7 Hz), 6.91 (1H, d, 3J = 8.0 Hz), 7.01 (1H, d, 4J = 1.7 Hz), 7.07 (1H,
3
4
3
dd, J = 8.0 Hz J = 1.7 Hz), 7.18 (1H, d, J = 8.5 Hz), 7.56 (1H, d,
EJ = 15.4 Hz); δC (CDCl3, DEPT 135, DEPT 90) 14.0 (+, CH3), 23.2
(–), 26.2 (–), 27.3 (–), 29.8 (–), 31.6 (–), 34.7 (–), 39.6 (+, CH), 43.6
(+, CH), 45.7 (Cquat), 46.1 (–), 49.6 (+, CH), 55.2 (+, OCH3), 56.0 (+,
OCH3), 83.7 (Cquat), 109.6 (+, CH), 111.5 (+, CH), 113.8 (+, CH),
114.7 (+, CH), 118.2 (+, CH), 122.2 (+, CH), 126.3 (+, CH), 127.4
(Cquat), 132.4 (Cquat), 137.9 (Cquat), 141.2 (+, CH), 146.7 (Cquat), 147.4
(Cquat), 157.4 (Cquat), 167.0 (Cquat, CO); MS (FAB+, 3-nitrobenzyl
alcohol) m/z (%) 492 (18) [MH+], 177 (100).

554 JOURNAL OF CHEMICAL RESEARCH 2012
Representative procedure for the synthesis of the (E)-N-(3′-hydroxy-
estra-1′,3′,5′(10′)-triene-3′,17′β-diol-17′α-ylmethyl)-3-phenylpro-
penamide derivatives
The authors would like to thank the National Cancer Insti-
tute (CNI), Bethesda, Maryland, for performing the in vitro
study. G. Ribeiro Morais would like to thank the Fundação
para a Ciência e Tecnologia (FCT), Lisbon, for the grant
SFRH/BD/6673/2001 and the “Ciência 2008” program.
A solution of cyanohydrin 7 (1 mmol) in dry THF (4 mL) at 0 ºC was
added to a suspension of LiAlH4 (5 mmol) in dry THF (4 mL). The
reaction mixture was stirred at rt for 24 h. Thereafter, H2O (3 mL) was
then added dropwise to the solution and the solvent was evaporated.
The aluminium salts were filtered and washed several times alter-
nately with H2O and ether. The phases were separated and the organic
phase was dried over anhydrous Na2SO4, filtered and the solvent was
evaporated. The crude residue was dissolved in dioxane (1.5 mL) and
the pentafluorophenyl ester derivative, 12b, (2 mmol) was added.
The mixture was submitted to MW irradiation (two cycles of 2 min).
p-TsOH (0.15 mmol) in MeOH (10 mL) was added to the resulting
reaction mixture and the solution was stirred at rt. After 1 h, the mix-
ture was concentrated and the reaction residue was submitted directly
to column chromatography on silica gel (EtOAc/CHCl3 2:8 → 5:5
→ EtOAc 100%) to give the respective 3-phenylpropenamide
derivatives.
Electronic Supplementary Information
Tables S1, S2 and S3 have been deposited in the ESI available
through stl.publisher.ingentaconnect.com/content/stl/jcr/supp-
data.
Received 2 June 2012; accepted 2 July 2012
Paper 1201361 doi: 10.3184/174751912X13419346932954
Published online: 28 August 2012
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E
6.49 (1H, d, J = 8.1 Hz), 6.58 (1H, d, J = 15.9 Hz), 6.73 (1H, d,
3J = 8.1 Hz), 6.84 (1H, d, J = 8.1 Hz), 6.95 (1H, s), 7.03 (1H, d,
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3J = 8.1 Hz), 7.22 (1H, d, EJ = 15.9 Hz), 7.75 (1H, m, NH), 8.97 (1H,
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g
2.69 (2H, m), 3.15 (1H, dd, J = 2.7 Hz, J = 13.4 Hz), 3.79 (3H, s,
OCH3), 3.59 (1H, dd, 3J = 7.8 Hz, gJ = 13.4 Hz), 4.49 (1H, s, OH), 6.42
(1H, d, 4J = 2.3 Hz), 6.49 (1H, dd, 4J = 2.3 Hz 3J = 8.1 Hz), 6.71 (1H,
d, EJ = 15.4 Hz), 6.77 (1H, d, 3J = 8.1 Hz), 6.98 (1H, d, 3J = 8.3 Hz),
7.03 (1H, d, 3J = 8.3 Hz), 7.15 (1H, s), 7.30 (1H, d, EJ = 15.4 Hz), 7.71
(1H, dd, 3J = 2.7 Hz, 3J = 7.8 Hz, NH), 8.97 (1H, s, OH), 9.39 (1H, s,
OH); δC (DMSO-d6) 14.2, 23.0, 26.0, 27.1, 29.2, 31.1, 32.8, 43.2,
45.4, 45.8, 49.3, 55.5, 59.7, 82.2, 110.6, 112.7, 114.9, 115.6, 119.3,
121.7, 126.0, 126.5, 130.4, 137.1, 138.9, 147.8, 148.2, 154.9, 166.2;
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In vitro growth inhibition screening
The in vitro cancer screening was carried out within the
Developmental Therapeutics Program of the National Cancer
Institute, National Institute of Health, Bethesda, Md, USA.
The description of the NCI-60 DTP Human Tumor Cell Line
Screening can be obtained at http://dtp.cancer.gov/branches/
btb/ivclsp.html.

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