Synthesis and estrogen receptor binding affinities of novel pyrrolo[2,1,5-cd]indolizine derivatives

Article abstract of DOI:10.1016/S0960-894X(00)00474-1

A series of pyrrolo[2,1,5-cd]indolizine derivatives has been synthesized and evaluated as ligands for the estrogen receptor. Properly substituted mono- and di-hydroxy derivatives showed binding in the low nanomolar range in accordance with their structural resemblance to estrogen. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00474-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2383±2386
Synthesis and Estrogen Receptor Binding Anities of Novel
Pyrrolo[2,1,5-cd]indolizine Derivatives
Anker Steen Jùrgensen,* Poul Jacobsen, Lise Brown Christiansen, Paul S. Bury,
Anders Kanstrup, Susan M. Thorpe, Lars Nñrum and Karsten Wassermann
Ê
Health Care Discovery and Preclinical Development, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Malùv, Denmark
Received 15 May 2000; revised 13 June 2000; accepted 17 August 2000
AbstractÐA series of pyrrolo[2,1,5-cd]indolizine derivatives has been synthesized and evaluated as ligands for the estrogen receptor.
Properly substituted mono- and di-hydroxy derivatives showed binding in the low nanomolar range in accordance with their
structural resemblance to estrogen. # 2000 Elsevier Science Ltd. All rights reserved.
The steroid hormone estrogen has a powerful in¯uence
on the development and maintenance of the female
reproductive organs and other sexual characteristics. In
recent years estrogen's involvement in the biological
function and sustenance of other tissues, such as the
skeleton, the cardiovascular system, and the central
nervous system, in both males and females has also been
recognised.1 5 Subsequently, the decreased production
of ovarian steroids which occurs after the climacteric
has been linked to a number of post-menopausal
degenerative changes, particularly osteoporosis and
coronary heart disease.6 Among the various treatment
modalities available, estrogen replacement therapy has
proven e€ective in reducing the risks associated with
these degenerative changes. Even though the bene®cial
e€ects of estrogen replacement on a wide variety of
organ systems and tissues appear indisputable, these
bene®ts are achieved at the expense of an increase in the
risk of endometrial hyperplasia and breast cancer
which, in turn, has reduced patient compliance.7,8 This
realization has created the need to develop non-steroidal
compounds, which interact with the estrogen receptor
(ER) and antagonise the e€ects of estrogen on uterine
and mammary tissue, whilst mimicking the e€ects of
estrogen on other tissues such as bone.9 11 Recently we
reported on the synthesis and pharmacological activity
of a novel non-steroidal tissue selective antagonist 1-
ethyl - 2 - (4 - hydroxyphenyl)pyrrolo[2,1,5 - cd]indolizine
(NNC 45-0095, 6c), which binds with high anity
(IC50=9.5 nM) to the estrogen receptor and exhibits full
protection against bone loss in the ovariectomized mouse
model for post-menopausal osteoporosis.12 Herein, we
report on the synthesis and estrogen receptor binding
properties of a series of novel pyrrolo[2,1,5-cd]indolizine
derivatives, which were prepared with the aim of further
exploring the ER binding activity relationship found
when varying the pattern of hydroxy-group substitution
seen in NNC 45-0095 and highlight, by means of mole-
cular modeling, the structural resemblance of the pyr-
rolo[2,1,5-cd]indolizine compounds to estrogen.
Chemistry
Preparation of the non-commercially available starting
materials 2a±c is outlined in Schemes 1±3. The synthesis
of 4-methoxy-2-n-propylpyridine 2a (Scheme 1) proceeded
via the intermediate 4-nitro-2-propylpyridine-N-oxide,
which was reacted with methanolic sodium methoxide fol-
lowed by reduction with zinc in aqueous sulphuric acid.
Attempts were made to prepare 4-benzyloxy-2-n-pro-
pylpyridine in a similar fashion13,14 but the reaction of
4-nitro-2-n-propylpyridine-N-oxide with sodium benzyl
alcoholate in benzyl alcohol failed. The synthesis of 5-
benzyloxy-2-n-propylpyridine 2b was performed by a
modi®ed literature procedure15 and is outlined in
Scheme 2. 2-Chloro-5-nitropyridine was reacted with
the sodium salt of diethyl ethylmalonate followed by
hydrolysis and decarboxylation to give the correspond-
ing 2-n-propylpyridine. The latter was reduced, diazo-
tized and hydroxylated to give the corresponding 5-
hydroxypyridine, which was alkylated to a€ord 2b. The
synthesis of 3-hydroxymethyl-2-n-propylpyridine 2c
(Scheme 3) was performed by analogy to the method
*Corresponding author. Tel.: +45-444-34811; fax: +45-444-34547;
e-mail: asj@novo.dk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00474-1

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A. S. Jùrgensen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2383±2386
stitution on the benzene ring (6c, 9.5 nM) was preferential
to meta-hydroxy substitution (6d, 370 nM). Despite a
high degree of symmetry of the parent molecule, it was
shown from 6a (1300nM) and 6b (180nM) that hydroxy
substitution in the pyridine ring was less favored com-
pared to para-hydroxylation of the pendant phenyl group
of 6c. Introduction of an additional hydroxy group on
the pyrroloindolizine moiety represented by compounds
6f and 6g, further improved the ER anities, and led to
binding anities (both 0.9 nM) close to that of 17b-estra-
diol itself (0.7 nM). Variation of the alkyl-substitution
pattern at the R2 position revealed that optimum ER
binding was obtained when small alkyl chains were
introduced. When R2 is ethyl (6c), n-propyl (6j) or i-pro-
pyl (6k) sub-nanomolar binding anities were obtained;
whereas binding anities for the corresponding n-butyl
(6n) and 2-pentyl (6p) were much poorer, 200 nM and
950 nM, respectively. Introduction of a hydroxymethyl
substituent in the R3 position of 6c revealed that this
modi®cation (6e, 12 nM) had no e€ect on ER binding.
The above results clearly demonstrate that the phenolic
group of 6c should best be aligned with the phenolic
moiety of 17b-estradiol and the most favoured 3D-
structures of the R2 substituents correspond well to the
volume de®ned by the B-ring in 17b-estradiol, as shown
in Figure 1. The missing mimetic of the B-ring in 6h can
explain the poor activity of this compound.
Scheme 1. Synthesis of 4-methoxy-2-n-propylpyridine (2a). Reagents
and conditions: (a) 35% H2O2/AcOH, 75 ꢀC, 13 h; (b) H2SO4/HNO3,
70 ꢀC, 22 h; (c) NaOMe/MeOH, 25 ꢀC; (d) Zn/1 M H2SO4, 75 ꢀC, 12 h.
Scheme 2. Synthesis of 5-benzyloxy-2-n-propylpyridine (2b). Reagents
and conditions: (a) Diethyl ethylmalonate/Na; (b) H2SO4 (aq), re¯ux
18 h; (c) Pd/C, EtOH; (d) 1. HCl (aq)/NaNO2, 0 ꢀC, 0.5 h, 2. 70 ꢀC, 2 h,
3. NaHCO3; (e) NaH/THF 0 ꢀC; (f) benzyl bromide/DMSO, 25 ꢀC, 3 h.
An alternative alignment of the molecules in Figure 1
could be to place R2 next to position 11b of 17b-estra-
diol, because this position generally is known to tolerate
large substituents.23 However, this alignment would
reduce the common volume overlap of the two mole-
cules and therefore this molecular ®t is considered to be
less favorable. Additionally, it can be observed that the
3D position of the second hydroxy group within 17b-
estradiol corresponds to both the R4 and R5 substitu-
tion positions of the NNC 45-0095 series of compounds.
This explains the very strong anities observed for the
di-hydroxy substituted pyrroindolizines 6f and 6g. If we
further compare the best common molecular volume
overlap between the series of compounds, the degree of
molecular volumes overlap corresponds to the observed
ER binding properties (data not shown).
Scheme 3. Synthesis of 3-hydroxymethyl-2-n-propylpyridine (2c).
Reagents and conditions: (a) NH3/ether, 25 ꢀC, 8 h; (b) acrolein/
piperidin/EtOH re¯ux, 3 h; (c) sulphur, 150 ꢀC, 3 h; (d) LiAlH4/THF,
25 ꢀC, 3 h.
described for the corresponding 3-hydroxymethylpico-
line.16 The pyrrolo[2,1,5-cd]indolizines 6 were prepared
in a straightforward fashion as outlined in Scheme 4. A
halo-ketone 1 and the 2-alkylpyridine 2 were reacted
under Tschitschibabin conditions17 to a€ord the indoli-
zine 3. Reaction of the latter18 with dimethyl acet-
ylenedicarboxylate (DMAD), followed by oxidation
with 2,3-dichloro-5,6-dicyanoquinone (DDQ) gave the
diester 4, which was hydrolyzed and decarboxylated to
a€ord compound 5. Removal of the O-protection of 5
gave the ®nal product 6 (see Table 1).
Results and Discussion
Estrogen receptor (ER) binding data of the pyr-
rolo[2,1,5-cd]indolizines 6 are summarized in Table 1.
The receptor binding was determined in ER-rich cytosol
from rabbit uterine tissue.19 The IC50-value for a parti-
cular compound represents the concentration at which
[3H]-17b-estradiol is displaced by 50% of the maximal
binding. We systematically modi®ed various regions of
the parent compound, 2-phenylpyrrolo[2,1,5-cd]indoli-
zine, to elucidate important features for activity. The ®rst
approach was to synthesize the mono-hydroxy com-
pounds 6a±d, which illustrated that para-hydroxy sub-
Figure 1. NNC 45-0095 (6c, black) compared to 17b-estradiol (grey)
and represented by the alignment derived from the pharmacophoric
22
pattern recognition and superimposition program GASP.20

A. S. Jùrgensen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2383±2386
2385
Scheme 4. Synthesis of mono- and di-hydroxy substituted pyrrolo[2,1,5-cd]indolizines (6). Reagents and conditions: (a) Acetone, re¯ux and isolate
intermediate quaternary pyridinium salt; (b) NaHCO3/H2O, re¯ux; (c) DMAD/toluene, 0 ꢀC. When R2 is CH2OH in 3 this substituent is protected by
reaction with t-butyl-dimethylsilylchloride. Protecting group is removed in step (e); (d) DDQ, 25ꢀC, then re¯ux; (e) 1. KOH/H2O/MeOH, re¯ux, 2. HCl
(aq); (f) Cu/quinoline, 170 ꢀC, (g) Deprotecting methods: A: H2, Pd/C, EtOH/THF, 25ꢀC. B: BBr3/CH2Cl2, 65±25 ꢀC overnight. C: 1. AlCl3/EtSH/
CH2Cl2, 25 ꢀC, 2. THF/6 M HCl, 0 ꢀC, 3. Extraction with EtOAc. See Table 1
Table 1. Synthesis and estrogen receptor binding anity (ER-LBA) of pyrrolo[2,1,5-cd]indolizines 6
Substituents of 6
O-Protecting of 5
5!6
Methodb
ER-LBAa
IC50 (nM)
R2
R3
R4
R5
R1
R1
R4
R5
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
Et
Et
Et
Et
Et
Et
Et
H
Me
n-Propyl
i-Propyl
H
H
H
H
OH
H
H
H
OH
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
Ð
H
H
Ð
Ð
OMe
Ð
Ð
Ð
Ð
Ð
OBn
Ð
Ð
Ð
OBn
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
B
A
A
B
A
A
B
A
B
A
C
B
B
C
B
B
Ð
1300
180
9.5
370
12
0.9
0.9
700
28
p-OH
m-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
Ð
OBn
OMe
OBn
OBn
OMe
OBn
OMe
OBn
OMe
OMe
OMe
OMe
OMe
OMe
Ð
H
CH2OH
H
H
OH
H
H
H
H
H
H
H
H
H
Ð
H
H
H
H
H
H
Ð
OMe
Ð
Ð
Ð
Ð
Ð
Ð
Ð
Ð
5
8
30
±(CH2)3±
±(CH2)4±
25
n-Butyl
2-Phenylethyl
2-Pentyl
H
H
H
Ð
200
180
950
0.7
Ð
Ð
Ð
Ð
17ꢀ-Estradiol
aEstrogen receptor binding anity expressed as IC50, i.e. the concentration of compound required to displace 50% of the maximal binding for [3H]-
17b-estradiol.
bFor details see Scheme 4.
Conclusions
binding and structural features closely related to 17b-
estradiol. Further work is underway to apply the pyr-
A novel series of pyrrolo[2,1,5-cd]indolizine derivatives
has been synthesized and evaluated in vitro as ligands
for the estrogen receptor. The di-hydroxy derivatives 6e
and 6f are highly potent compounds, which exhibit
rolo[2,1,5-cd]indolizine ring system as a sca€old for
further derivatization in order to obtain compounds
with mixed agonist/antagonist estrogen activity (anti-
estrogens).

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A. S. Jùrgensen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2383±2386
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Christensen, Alice Ravn, Alice Jùrgensen, and Mar-
ianne Barfoed for excellent technical assistance.
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Reaction of 3 with DMAD and DDQ was inspired by
Noguchi et al. with modi®cations as follows: DMAD (1.15
equiv) was added at 0 ꢀC and only a slight excess of DDQ
(1.04 equiv) was used. Further reaction details can be obtained
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available within the Sybyl 6.5.2 software package (Tripos Inc.)
was used to establish the alignment shown in the ®gure.
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(Molecular Simulations Inc.) was used to display the 3D
alignment.
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