I
1. Abstract
Breast cancer is one of the most common cancers in women, accounting for almost
one-fourth of all cancers in women. Now, Estrogens are known to be an important
factor involved in the growth of breast cancer, and a logical approach for the treatment
of this disease is the use of antiestrogens that block the interaction of estrogens with
their specific receptor.
As an approach towards the development of new modified estrogens, the
preparation of some 2- and/or 4-substituted E1 and E2 derivatives is planned and such
design is considered worthwhile to evaluate the changes in endocrinological activities
caused when the 2- and/or 4-position of estrogens was blocked by various moieties. The
novel drug candidates contain alkylidene and benzylideneamino, alkyl and arylamino
moieties substituted on ring-A of the steroid skeleton. In addition, some peptidylestrone
derivatives are also designed and prepared as a means of studying the effect of
combination of steroids with other naturally occurring compounds, such as amino acids,
on their biological activity.
A discussion of the theoretical basic concepts for some already acceptable methods
for the synthesis of the designed compounds is included. Referring to the available
knowledge in the literature for the preparation of structurally related compounds, welldocumented methods of synthesis such as nitration, reduction, etherification, Schiff
bases condensation, peptide formation, protection and deprotection reactions are
adopted.
Actually, thirty five new final compounds have been synthesized along with eight
key intermediates and four reagents. These are:
a) Intermediates:
II
-
2-Nitroestrone (2) and 4-nitroestrone (3).
-
2-Aminoestrone (4) and 4-aminoestrone (5).
-
2-Nitroestrone 3-methyl ether (6) and 4-nitroestrone 3-methyl ether (7).
-
2-Aminoestrone 3-methyl ether (8) and 4-nitroestrone 3-methyl ether (9).
b) Reagents:
CBz-Glycine, CBz-Serine, CBz-Valine, CBz-Phenylalanine.
The target products are:
2-Crotonylideneaminoestrone
(10a),
2-Isovalerylideneaminoestrone
(10b),
2-
Heptylideneaminoestrone (10c), 2-(4-Bromobenzylidene)aminoestrone (10d), 2-(2,4Dichlorobenzylidene)aminoestrone (10e), 2-(4-Nitrobenzylidene)aminoestrone (10f), 2(2,4-Dinitrobenzylidene)aminoestrone (10g).
2-Isopentylaminoestradiol (11a), 2-(2,4-Dichlorobenzyl)aminoestradiol (11b), 2-(4Nitrobenzyl)aminoestradiol (11c), 2-(2,4-Dinitrobenzyl)aminoestradiol (11d).
4-Crotonylideneaminoestrone
(12a),
4-Heptylideneaminoestrone
(12b),
4-(2,4-
Dichlorobenzylidene)aminoestrone (12c), 4-(4-Nitrobenzylidene)aminoestrone (12d),
4-(2,4-Dinitrobenzylidene)aminoestrone (12e).
4-Heptylaminoestradiol
(13a),
4-(4-Nitrobenzyl)aminoestradiol
(13b),
4-(2,4-
Dinitrobenzyl)aminoestradiol (13c).
2-(N-Benzyloxycarbonylglycyl)aminoestrone 3-methyl ether (14a), 2-(N-Benzyloxy
carbonyl-L-seryl)aminoestrone 3-methyl ether (14b), 2-(N-Benzyloxycarbonyl-DLvalyl)aminoestrone 3-methyl ether (14c), 2-(N-Benzyloxycarbonyl-DL-phenylalanyl)aminoestrone 3-methyl ether (14d).
2-N-Glycylaminoestrone
(15a),
2-N-L-Serylaminoestrone
aminoestrone (15c), 2-N-DL-phenylalanylaminoestrone (15d).
(15b),
2-N-DL-Valyl-
III
4-(N-Benzyloxycarbonylglycyl)aminoestrone 3-methyl ether (16a), 4-(N-Benzyloxycarbonyl-L-seryl)aminoestrone 3-methyl ether (16b), 4-(N-Benzyloxycarbonyl-DLvalyl)aminoestrone 3-methyl ether (16c), 4-(N-Benzyloxycarbonyl-DL-phenylalanyl)aminoestrone 3-methyl ether (16d).
4-N-Glycylaminoestrone
(17a),
4-N-L-Serylaminoestrone
(17b),
4-N-DL-Valyl-
aminoestrone (17c), 4-N-DL-phenylalanylaminoestrone (17d).
The structures of the synthesized compounds are confirmed by using various
spectroscopic tools including IR, UV, 1H NMR, COSY, DEPT, HETCOR,
13
C NMR,
mass spectroscopy and for one compound by x-rays crystallography.
In an exploration of these modifications, the synthesized compounds are tested for
in vivo uterotrophic and antiuterotrophic activities and in vitro cytotoxic activities. This
enable us to make a preliminary assessment of the potential selectivity of C-2 and/or C4 substitution in steroidal compounds and the relationship between such structural
modification, affinity and degree of activity.
Some of the synthesized compounds, namely 4-NO2E1 (3), 2-NO2E1MeO (6), 4NO2E1MeO (7), 2-NH2E1MeO (8) and 4-NH2E1MeO (9) were completely devoid of
uterotrophic activity and showed good order of antiuterotrophic activity. They were
capable of antagonizing the uterotrophic effect of E1 to the extent of 69-77% with 4NO2E1 (3), 4-NH2E1 (5) and 2-NO2E1MeO (6) being the most active. Among them 4NH2E1 (5) and 2-NO2E1MeO (6) were highly cytotoxic to MCF-7 cells showing a
cytotoxic effect of 92% and 85% respectively comparatively to the reference cytotoxic
compounds, 2ME2 and echinomycin respectively.
In general, all imine derivatives were less estrogenic than the standard references E1
and E2. Some of them possessed good antiestrogenic activity while being almost devoid
of estrogenic activity. A significant reduction to low levels (0%-6%) was observed for
IV
compounds 10e, 10g, 10d, 12b, 12d and 12e. Compound 12b substituted with an
isovalerylidene moiety at position 4 was completely devoid of uterotrophic activity
(0%) while possessing fairly high antiuterotrophic activity (76%). The 4-(4nitrobenzylideneamino) derivative 12d elicited minimal estrogenic activity of only 6%
but failed to antagonize the estrogenic activity of E2 and the blockade of activity was of
the order of only 17%.
O
O
HO
N
H
HO
N
H
NO 2
12b
12d
0% uterotrophic activity
76% antiuterotrophic activity
82% cytotoxicity
6% uterotrophic activity
17% antiuterotrophic activity
42% cytotoxicity
Compound 12c, showing a uterotrophic activity as low as 18%, was highly capable
of antagonizing the effect of E2 and showed the highest antiuterotrophic activity of
81%.
O
HO
N
Cl
H
Cl
12c
18% uterotrophic activity
81% antiuterotrophic activity
93% cytotoxicity
Compounds 10b, 10c, 12b and 12c elicited a high order of cytotoxicity against
MCF-7 breast cancer cells ranging from 80% to 93%, with the most active compounds
V
being 10c and 12c substituted with 2,4-dichlorobenzylidene moiety at the 2- and the 4position.
In comparison to their parent imine derivatives, the amines 11a-c and 13a-c were
almost completely devoid of uterotrophic activity. In general, 2-substituents were less
estrogenic than 4-substituents. Compounds 11a-c and 13b were the least active
exhibiting uterotrophic activity of 1-5% with 11c, bearing a 4-nitro substituent, being
the least active. On the other hand, all amines did not antagonize the induced
uterotrophic effect of E2 and the percentage of antiuterotrophic activity ranged from 0
to 23%.
In vivo evaluation of the CBz-peptidylaminoestrone derivatives 14a-d and 16a-d
revealed that, in general, a drop in estrogenic activity was observed for compounds 16ad with regard to 14a-d relative to the references E1 and E2. Compounds substituted
with amino acid residues at position 2 were more estrogenic (58%-86%) than those
substituted at position 4 (37%-50%) with the exception of the 2-valyl derivative 14c
which showed the lowest order of uterotrophic activity (23%). However, it did not
antagonize the induced uterotrophic effect of E2 to a great extent showing a moderate
antiuterotrophic activity of only 39%. The 2-glycyl derivative 14a was highly
estrogenic showing 86% uterotrophic activity relative to E2 being even more estrogenic
than E1 (59%). On the other hand, 14a antagonized the induced uterotrophic activity of
E2 and was the most potent among the series (55%).
Results also indicated that the isobutyl carbamate ester was devoid of any
uterotrophic activity (15%) while antagonizing the induced uterotrophic activity of E2
by 40%.
VI
O
O
O
O
H
N
O
O
H3CO
H
N
N
H
O
H3CO
Carbamate ester
14c
15% uterotrophic activity;
40% antiuterotrophic activity
23% uterotrophic activity;
39% antiuterotrophic activity
20% cytotoxicity
Results of cytotoxicity evaluation against MCF-7 human breast cancer cells
indicated that the 2-glycyl derivative 14a was highly cytotoxic (98%) although being
highly estrogenic (86%) and good antiestrogenic (55%). The 4-seryl derivative 16b was
also highly cytotoxic (98%) although it showed a moderate order of uterotrophic
activity of 45% and a low order of antiuterotrophic activity of 19%.
O
H3CO
NH
O
O
O
O
CH 2OH
HN
O
O
N
H
H
N
O
H3CO
14a
86% uterotrophic activity
55% antiuterotrophic activity
98% cytotoxicity
16b
45% uterotrophic activity
19% antiuterotrophic activity
98% cytotoxicity
Finally, the deprotected peptidylaminoestrone derivatives 15a-d and 17a-d showed
a low order of uterotrophic and antiuterotrophic activity relative to E2 ranging from
19%-45% and 10-46% respectively. Among them, compounds 15c and 17a were the
least estrogenic exhibiting uterotrophic activity of 19% and 20% respectively. On the
other hand, these compounds poorly antagonized the induced uterotrophic effect of E2
to the extent of 37% and 29% respectively. The 4-phenylalanyl derivative 17d exhibited
VII
the highest order of antiuterotrophic activity (46%) while eliciting a moderate order of
uterotrophic activity (28%).
O
O
HO
NH
O
H
N
H2N
H2N
O
HO
15c
19% uterotrophic activity
37% antiuterotrophic activity
17d
28% uterotrophic activity
46% antiuterotrophic activity
These new modified estrogens should be useful for seeking novel chemical
approach to the discovery of potent steroids and, in accordance with their capabilities in
antagonizing the action of E2 at the ER, they can be used as antiestrogens and/or
cytotoxic agents with selective activity against tumor cells.