Synthesis and pharmacology of a novel pyrrolo[2,1,5-cd] indolizine (NNC 45-0095), a high affinity non-steroidal agonist for the estrogen receptor

Article abstract of DOI:10.1016/S0960-894X(00)00015-9

1-Ethyl-2-(4-hydroxyphenyl)pyrrolo[2,1,5-cd]indolizine (NNC 45-0095) is a novel compound which represents the parent pharmacophore structure of a series of pyrrolo[2,1,5-cd]indolizine derivatives with mixed estrogen agonist/antagonist properties. NNC 45-0095 binds with high affinity to the estrogen receptor (IC₅₀ = 9.5 nM) and exhibits full protection of bone loss in the ovariectomized mouse model for post-menopausal osteoporosis. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00015-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 399±402
Synthesis and Pharmacology of a Novel Pyrrolo[2,1,5-cd]
Indolizine (NNC 45-0095), a High Anity Non-Steroidal Agonist
for the Estrogen Receptor
A. S. Jùrgensen, a P. Jacobsen, a L. B. Christiansen, a P. S. Bury, a A. Kanstrup, a
S. M. Thorpe, a S. Bain, b L. Nñrum a and K. Wassermann a,*
a
Ê
Health Care Discovery & Preclinical Development, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Malùv, Denmark
bSkeleTech Inc., Seattle, WA, USA
Received 21 October 1999; accepted 5 January 2000
AbstractÐ1-Ethyl-2-(4-hydroxyphenyl)pyrrolo[2,1,5-cd]indolizine (NNC 45-0095) is a novel compound which represents the parent
pharmacophore structure of a series of pyrrolo[2,1,5-cd]indolizine derivatives with mixed estrogen agonist/antagonist properties.
NNC 45-0095 binds with high anity to the estrogen receptor (IC50=9.5 nM) and exhibits full protection of bone loss in the
ovariectomized mouse model for post-menopausal osteoporosis. # 2000 Elsevier Science Ltd. All rights reserved.
The central role of endogenous estrogen in the develop-
ment and maintenance of the female reproductive
organs and other sexual characteristics has long been
acknowledged. More recently 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-meno-
pausal degenerative changes, particularly osteoporosis
and coronary heart disease.6 Among the various treat-
ment 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 a real or per-
ceived increase in risk of endometrial hyperplasia and
breast cancer which, in turn, has reduced patient com-
pliance.7,8 This realisation 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 Here we report for the ®rst time on the
synthesis of the novel non-steroidal full estrogen agonist,
1-ethyl-2-(4-hydroxyphenyl)pyrrolo[2,1,5-cd]indolizine
(NNC 45-0095), a compound which represents the
parent pharmacophore structure of a series of pyr-
rolo[2,1,5-cd]indolizine derivatives with mixed estrogen
agonist/antagonist properties for the prevention or
treatment of estrogen related diseases or symptoms
(manuscript in preparation). Herein, we describe the
compound's synthesis, the in vitro e€ects in receptor
binding and cell proliferation assays, and the e€ects on
bone and uterine tissue in an ovariectomized (OVX)
mouse model for post-menopausal disorders.
Synthesis
The synthesis of NNC 45-0095 was performed as
outlined in Scheme 1. 2-Propylpyridine 1 and the halo-
ketone 2 were reacted under Tschitschibabin condi-
tions12 to a€ord the indolizine 3. Reaction of the latter
with dimethyl acetylenedicarboxylate (DMAD), fol-
lowed by oxidation with 2,3-dichloro-5,6-dicyanoqui-
none (DDQ) gave the diester 4, which was hydrolysed
to the diacid 5. Decarboxylation of 5 a€orded the pyr-
roloindolizine 6, which was debenzylated to the ®nal
product NNC 45-0095.13
Pharmacological Evaluation
The estrogen receptor (ER) is traditionally regarded as a
ligand-inducible transcription factor that regulates the
*Corresponding author. Fax: +45-4443-4537; e-mail: kwa@novo.dk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00015-9

400
A. S. Jùrgensen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 399±402
Scheme 1. Synthesis of 45-0095: (a) acetone, re¯ux and isolate intermediate quaternary pyridinium salt; (b) NaHCO3, H2O, re¯ux; (c) DMAD,
toluene, 0 ꢀC; (d) DDQ, rt; (e) KOH, H2O, MeOH, re¯ux then dilute HCl; (f) Cu, quinolene, 170 ꢀC; (g) H2, Pd/C, EtOH.
transcription of estrogen responsive genes by virtue of
its binding as a homodimer to imperfect palindromic
sequences, estrogen responsive elements, on DNA.
Consequently, the biological activity of ER-bound
ligand is considered genomic and the initial identi®ca-
tion of estrogen-like compounds is often based on ER
anity for the ligand in question. Hence, the ability of
NNC 45-0095 to compete with [3H]-17b-estradiol for
receptor binding was determined in ER-rich cytosol
from rabbit uterine skeletal muscle tissue.14 As shown in
Table 1, the concentrations of NNC 45-0095 and 17b-
estradiol, which displaced 50% of the maximal binding
of [3H]-17b-estradiol to ER (IC50), were 9.5 and 0.7 nM,
respectively.
maximal agonist activity of 105% relative to moxestrol
and an EC50-value of 13 nM. Also, this in vitro agonism
of NNC 45-0095 correlated with observations of utero-
trophic activity using changes in uterine weight in the
immature mouse (Table 1) and in the mature ovar-
iectomized (OVX) mouse (Fig. 1B) as a measure for
estrogenicity.16,17
In addition to uterotrophic activity, estrogen also has
positive therapeutic e€ects on various other tissues; for
example in the skeleton, where estrogen restrains the
bone resorption seen in post-menopausal women.18,19
To determine if NNC 45-0095 retained estrogenic
activity in bone remodelling, we investigated the anti-
resorptive properties of this new pyrroloindolizine ana-
logue in an OVX mouse model of post-menopausal
osteoporosis.20 Measures of total bone mineral density
(BMD) were determined by the Archimedes principle21
in the distal femur following 5 weeks of daily sub-
cutaneous administration of compound and were com-
pared to the e€ects of 17b-estradiol. Analysis showed
Whether the high receptor binding anity of NNC 45-
0095 is re¯ected in intrinsic estrogenic activity was ®rst
assessed in vitro based on stimulation of alkaline phos-
phatase activity in the Ishikawa line of human endo-
metrial adenocarcinoma cells.15 As shown in Table 1,
NNC 45-0095 was fully agonistic in this model with a
Table 1. Estrogen receptor binding anity (ER-LBA), endometrial activity in vitro and uterotrophic activity in vivo for NNC 45-0095, moxestrol
and 17b-estradiol
ER-LBAa
IC50 (nM)
Ishikawa
Immature mouse
b
EC50 (nM)
Emax (% max. stimulation
relative to moxestrol)
EC50 (nmol/g)
% maximal relative
agonism
NNC 45-0095
Moxestrol
17b-estradiol
9.5
2.5
0.7
13
0.1
Ð
105
100
Ð
0.96
Ð
Ð
76
Ð
Ð
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.
bEmax of moxestrol is 903% of basal proliferation. Values are presented as mean from 2±4 separate studies.

A. S. Jùrgensen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 399±402
401
Figure 1. E€ects of NNC 45-0095 and 17b-estradiol (17b-E) on total BMD in the distal femur of the OVX mouse (A) and uterine wet weight (B).
NNC 45-0095 (0.1±10 nmol/g body weight) and 17b-estradiol (0.5 nmol/g body weight) were administered by the subcutaneous route ®ve and three
times weekly, respectively, for 5 weeks. Data represent mean and SEM for 6±8 animals per group. *P<0.05 and **P<0.01 versus OVX vehicle
(ANOVA and Dunnett's multiple comparison test).
2. Carani, C.; Qin, K.; Simoni, M.; Faustinifustini, M.; Serpente,
signi®cant di€erences in mouse distal femur BMD
S.; Boyd, J.; Korach, K. S.; Simpson, E. R. N. Engl. J. Med.
1997, 337, 91.
3. Cosman, F.; Dempster, D.; Lindsay, R. In Estrogen and
Antiestrogens. Basic and Clinical Aspects; Lindsay, R.; Dempster,
D. W.; Jordan, V. C., Eds.; Lippincott-Raven: Philadelphia,
1997, pp 151±164.
(Fig. 1A). Vehicle treated mice OVXed for 5 weeks
su€ered signi®cant decreases in BMD. Administration
of NNC 45-0095 in the range of 0.1±10 nmol/g body
weight at ®ve times per week, completely prevented the
loss of total BMD of the distal femur at 5 nmol/g.
Likewise, 17b-estradiol at 0.5 nmol/g body weight
administered three times weekly, signi®cantly main-
tained total BMD at the level of sham-operated animals.
4. Sherwin, B. B. In Estrogen and Antiestrogens. Basic and
Clinical Aspects; Lindsay, R.; Dempster, D. W.; Jordan, V. C.,
Eds.; Lippincott-Raven: Philadelphia, 1997, pp 75±87.
5. Farhat, M. Y.; Lavigne, M. C.; Ramwell, P. W. FASEB J.
1996, 10, 615.
6. Lobo, R. A. In Estrogen and Antiestrogens. Basic and Clin-
ical Aspects; Lindsay, R.; Dempster, D. W.; Jordan, V. C.,
Eds.; Lippincott-Raven, Philadelphia, 1997, pp 63±72.
7. Colditz, G. A.; Hankinson, S. E.; Hunter, D. J.; Willett, W.
C.; Manson, J. E.; Stampfer, M. J.; Hennekens, C.; Rosner,
B.; Speizer, F. E. N. Engl. J. Med. 1995, 332, 1589.
8. Stanford, J. L.; Weiss, N. S.; Voigt, L. F.; Daling, J. R.;
Habel, L. A.; Rossing, M. A. JAMA 1995, 274, 137.
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In conclusion, these data strongly suggest that this novel
pyrroloindolizine, NNC 45-0095 possesses profound
estrogenic activity. Moreover, this new high anity
non-steroidal ER ligand appeared as a full estrogen
agonist when evaluated in classical estrogen responsive
tissues such as the endometrium and the skeleton. The
pharmacological pro®le of this parent pharmacophore
may provide the basis for rational design of tissue
selective ER ligands with mixed estrogen agonist/
antagonist pro®le.
10. Nuttall, M. E.; Bradbeer, J. N.; Stroup, G. B.; Nadeau, D.
P.; Ho€man, S. J.; Zhao, H.; Rehm, S.; Gowen, M. Endocri-
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Acknowledgements
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Owen, T. A.; Smock, S. L.; Chen, H. K.; Jee, W. S. S.;
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12. Tschitschibabin, A. E. Chem. Ber. 1927, 60, 1607.
13. Compound NNC 45-0095 was fully characterised: mp 104±
We thank Minna Susanne Mùller, Bent Hùegh, Aase
Vinterby, Susanne Dam Hùiberg and Marianne Barfoed
for excellent technical assistance.
105 ꢀC. H NMR (DMSO-d6, 200 MHz) d: 1.41 (t, 3H), 3.22
1
References and Notes
(q, 2H), 6.98 (d, 2H), 7.29 (d, 1H), 7.67 (d, 2H), 7.69 (d, 1H),
7.72 (t, 1H), 8.05 (d, 1H), 8.10 (d, 1H), 9.68 (s, 1H). Elemental
analysis: calcd for C18H15NO: C, 82.73; H, 5.79; N, 5.36%.
Found: C, 83.03; H, 5.88; N, 5.16%.
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