Discovery and Characterization of BAY 1214784, an Orally Available Spiroindoline Derivative Acting as a Potent and Selective Antagonist of the Human Gonadotropin-Releasing Hormone Receptor as Proven in a First-In-Human Study in Postmenopausal Women

Article abstract of DOI:10.1021/acs.jmedchem.0c01076

The growth of uterine fibroids is sex hormone-dependent and commonly associated with highly incapacitating symptoms. Most treatment options consist of the control of these hormonal effects, ultimately blocking proliferative estrogen signaling (i.e., oral contraceptives/antagonization of human gonadotropin-releasing hormone receptor [hGnRH-R] activity). Full hGnRH-R blockade, however, results in menopausal symptoms and affects bone mineralization, thus limiting treatment duration or demanding estrogen add-back approaches. To overcome such issues, we aimed to identify novel, small-molecule hGnRH-R antagonists. This led to the discovery of compound BAY 1214784, an orally available, potent, and selective hGnRH-R antagonist. Altering the geminal dimethylindoline core of the initial hit compound to a spiroindoline system significantly improved GnRH-R antagonist potencies across several species, mandatory for a successful compound optimization in vivo. In a first-in-human study in postmenopausal women, once daily treatment with BAY 1214784 effectively lowered plasma luteinizing hormone levels by up to 49%, at the same time being associated with low pharmacokinetic variability and good tolerability.

Full text of DOI:10.1021/acs.jmedchem.0c01076

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
Discovery and Characterization of BAY 1214784, an Orally Available
Spiroindoline Derivative Acting as a Potent and Selective Antagonist
of the Human Gonadotropin-Releasing Hormone Receptor as
Proven in a First-in-Human Study in Postmenopausal Women
Olaf Panknin, Andrea Wagenfeld, Wilhelm Bone, Eckhard Bender, Katrin Nowak-
Reppel, Amaury E. Fernández-Montalván, Reinhard Nubbemeyer, Stefan Bäurle,
Sven Ring, Norbert Schmees, Olaf Prien, Martina Schäfer, Christian Friedrich,
Thomas M. Zollner, Andreas Steinmeyer, Thomas Mueller, and Gernot Langer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01076 • Publication Date (Web): 22 Sep 2020
Downloaded from pubs.acs.org on September 22, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Discovery and Characterization of BAY 1214784,
an Orally Available Spiroindoline Derivative Acting
as a Potent and Selective Antagonist of the Human
Gonadotropin-Releasing Hormone Receptor as
Proven in a First-in-Human Study in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Postmenopausal Women
#
#
#
‡
Olaf Panknin,* Andrea Wagenfeld, Wilhelm Bone, Eckhard Bender, Katrin Nowak-
#
#§
#
#
Reppel, Amaury E. Fernández-Montalván, Reinhard Nubbemeyer, Stefan Bäurle,
#
#
#
#
#
Sven Ring, Norbert Schmees, Olaf Prien, Martina Schäfer, Christian Friedrich,
#
#
‡
#
Thomas M. Zollner, Andreas Steinmeyer, Thomas Mueller, and Gernot Langer*
#Bayer AG, Research & Development, Pharmaceuticals, Müllerstrasse 170, 13342
Berlin, Germany
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
‡Bayer AG, Research & Development, Pharmaceuticals, Aprather Weg 18a, 42113
Wuppertal, Germany
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
KEYWORDS. gonadotropin-releasing hormone receptor, luteinizing hormone
suppression, spiroindolines, structure–activity relationship, drug metabolism and
pharmacokinetics optimization, uterine fibroids, heavy menstrual bleeding
ABSTRACT. The growth of uterine fibroids is sex hormone dependent and commonly
associated with highly incapacitating symptoms. Most treatment options consist of the
control of these hormonal effects, ultimately blocking proliferative estrogen signaling
(i.e., oral contraceptives/antagonization of human Gonadotropin-Releasing Hormone
Receptor [hGnRH-R] activity). Full hGnRH-R blockade, however, results in menopausal
symptoms and affects bone mineralization, thus limiting treatment duration or
demanding estrogen add-back approaches. To overcome such issues, we aimed to
identify novel, small-molecule hGnRH-R antagonists. This led to the discovery of
compound BAY 1214784, an orally available, potent, and selective hGnRH-R
ACS Paragon Plus Environment
2

Page 3 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
antagonist. Altering the geminal dimethylindoline core of the initial hit compound to a
spiroindoline system significantly improved GnRH-R antagonist potencies across
several species, mandatory for a successful compound optimization in vivo. In a first-in-
human study in postmenopausal women, once daily treatment with BAY 1214784
effectively lowered plasma luteinizing hormone levels by up to 49%, at the same time
being associated with low pharmacokinetic variability and good tolerability.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
Uterine fibroids (also known as uterine leiomyomas) are the most common benign
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
tumors of the uterine muscle layer with a prevalence of >70% in women. Although the
precise genesis of uterine fibroids is still subject to current research,2–4 their actual
growth is unambiguously sex hormone dependent.5–7 Thus, they are frequently
associated with menses-related heavy menstrual bleeding (leading to dysmenorrhea;
i.e., pelvic pain and pressure as well as anemia) or even infertility. Yet, despite the large
number of women presenting with leiomyoma complaints (some 20–50% of women of
reproductive age), the choice of treatment options available is quite limited.8,9 The
spectrum ranges from curative surgical interventions10,11 to noncurative, symptomatic
treatment of uterine fibroid associated pain (analgesics) and medications primarily
controlling (heavy) bleeding symptoms,12 namely oral contraceptives or hGnRH-R I
functional antagonists, the latter widely used to also control the estrogen-dependent
growth of malignant ovarian and breast cancers. However, given the induction of
menopausal symptoms and effects on bone mineralization, hGnRH-R blockers currently
in use are not yet entirely suitable for long-term treatment. Full antagonization of
ACS Paragon Plus Environment
4

Page 5 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
hGnRH-R signaling results in a complete blockade of estrogen production in the vast
majority of cases owing to the central role of this receptor in the production of the
gonadotropins LH and FSH, which in turn control the conversion of androgen precursors
in the ovary, the major source of estrogens in women of reproductive age.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Actual fine-tuning and expression of both gonadotropins is thought to be achieved by
amplitude and frequency changes in GnRH secretion13,14 and thus differential, cell-type
specific hGnRH-R stimulation.15,16 Continuous, nonpulsatile peptide agonist treatment
results in a rapid development of tolerance to further stimulation, uncoupling of
downstream second messenger cascades, and a complete blockade of agonist effects
(functional antagonization).17,18 In addition to parenteral administration procedures
further complicating the matter, the high degree of suppression of ovarian function
achieved with early peptide agonist protocols often required estrogen add-back
therapies to adequately control and prevent menopausal side effects when treating
patients.
All of this highly favored the search for orally available, non-peptide, small-molecule
(SMOL) hGnRH-R antagonists to effectively improve treatment protocols and patient
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compliance. Consequently, a number of such SMOL antagonists have been
investigated.19 In particular, two SMOL GnRH-R antagonists have already attained
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
market approval, namely relugolix (TAK-385, Takeda; indication: uterine fibroids)20,21
and elagolix (NBI-56418 Na, AbbVie; indication: endometriosis-related pain),22–24 and
the very same compounds are in Phase III clinical trials with regard to the respective
other type of indication [i.e., elagolix (AbbVie) for uterine fibroids and relugolix (Myovant
25
Sciences) for endometriosis], as is linzagolix (ObsEva/Kissei Pharmaceutical) for both
indications. Furthermore, elagolix (AbbVie) is in Phase II clinical trials for the treatment
of polycystic ovary syndrome (PCOS). Table 1 gives their chemical structures and
summarizes some of their reported properties.
The adverse effects reported for these compounds so far [i.e., intermenstrual and
heavy menstrual bleeding (metrorrhagia/menorrhagia), hot flushes, headache, and
dose-dependent loss of bone mineral density with no endometrial findings for all] are
consistent with their mode of action. However, both relugolix and elagolix, when
administered at higher doses, also necessitated the use of estrogen add-back therapy
(ABT) – the main reason for this most likely resulting from the actual compounds’ PK/PD
ACS Paragon Plus Environment
6

Page 7 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
properties. Thus, a high unmet medical need still exists to identify hGnRH-R antagonists
modulating receptor signaling behavior in favor of a partial deprivation of estradiol (E2)
levels only. Therefore, we decided to initiate a comprehensive compound identification
and optimization program aimed at the detection of SMOL hGnRH-R antagonists with
improved, superior PK/PD properties. From the outset, we focused on compounds
possessing comparable multispecies activities at both the human and the rat GnRH
receptor to be able to take advantage of the use of rat animal models for further
optimization. Here, we report the identification and extensive characterization of 5a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(BAY 1214784) as a novel, orally available, potent, and selective hGnRH-R antagonist
with the desired pharmacological profile of partially lowering of LH levels only, in
preclinical as well as first-in-human studies.
RESULTS AND DISCUSSION
HTS, Identification of Screening Hit 15 and Improved Compound 1a. A cell-based
high-throughput screen of the Bayer Pharma compound library (comprising ~2.5 million
compounds) led to the identification of an indoline hit cluster with borderline activities at
the human GnRH receptor (see the Supporting Information for details of the hit-to-lead
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
6
examination and the removal of compounds with pan-assay interference motifs ).
Although the members of this cluster also possess high lipophilicity and low solubility
characteristics, this scaffold was considered the most promising starting point because
of comparable multispecies potencies at both the human and the rat GnRH receptor, a
prerequisite considered necessary for further preclinical optimization in vivo. Thus,
racemic screening hit 15 (see Table 2) was chosen for resynthesis and as the starting
point for a first round of SAR modifications. These efforts resulted in the identification of
enantiomerically pure compound 1a with improved potencies at both human and rat
GnRH receptors [IC50 = 568 and 726 nM (LHRH), respectively]. Given that routine
monitoring of 1a with functional profiling in a panel of 25 GPCRs confirmed no
significant off-target liabilities (data not shown), this compound was chosen for further
optimization even though its lipophilicity is high (logD at pH 7.5 = 3.8), its solubility
proved to be low, and compound clearance in rat hepatocytes turned out to be high [CLb
(rat female Wistar) = 3.1 L/h/kg; see the Supporting Information Table S1 for a detailed
compound profile]. An overview of key compounds synthesized in the subsequent
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
8

Page 9 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
course of the hit-to-lead and lead optimization process, along with their core properties,
is given in Figure 1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of Screening Hit 15 and Improved Compound 1a. The racemic
trimethylindoline core of 15 and 1 was prepared in two steps starting with a Fischer
indole synthesis using 4-hydrazinobenzoic acid (6) and 3-methylbutan-2-one (7) under
acidic conditions followed by reduction of the formed indolenine intermediate 8 with
sodium borohydride to give indoline 9 (see Scheme 1). The carboxyli group at C-5 was
protected as a methyl ester (intermediate 10) which was followed by sulfonamide
formation with the respective benzenesulfonyl chloride to furnish 11 and 12. Ester
saponification of 11 with aqueous lithium hydroxide and subsequent amide coupling of
13 with 1-(2-chlorophenyl)methanamine gave the racemic amide 15. For the isolation of
1a, an enantiomeric separation by chiral HPLC was performed at the final stage, after
saponification of 12 to 14 and amide coupling to provide 1.
Variations at N-1, C-2, and C-5 of the 3,3-Dimethylindoline Core. Numerous attempts
to significantly increase compound potency by exploring extensive variations at N-1, C-
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
, and C-5 of the indoline core proved unsuccessful (see Figure 2 for a brief qualitative
summary).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Incorporation of a GPCR Privileged Structure: Identification of Lead 2b. In a second
approach to improve compound potency, we turned towards the concept of GPCR
privileged structures27–33 (see Table 2). The incorporation of an N-acetylated
spiropiperidine system at C-3 of the indoline core, as in compound 2b, boosted
antagonistic potency in all species [IC50 hGnRH-R/rGnRH-R = 41/29 nM (LHRH), IC50
cynomolgus monkey GnRH-R = 205 nM (buserelin)] and improved the LLE by
approximately 2 log units relative to 1a (see the Supporting Information Table S1 for a
detailed compound profile). Therefore, compound 2b was chosen as lead compound
and starting point for further optimization with a special focus on addressing still-existing
liabilities with regard to its physicochemical (i.e., low solubility: 25 mg/L at pH 6.5) and
pharmacokinetic properties [i.e., high blood clearance in vivo: CL (rat female Wistar) =
b
4.7 L/h/kg and limited permeation in Caco-2 cells (Papp A–B = 38 nm/s, efflux ratio 2.4)].
Variations at the Spiropiperidine Nitrogen. Subsequently, a number of racemic
derivatives with variations at the spiropiperidine nitrogen atom were synthesized and
ACS Paragon Plus Environment
10

Page 11 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
tested with the aim of improving both the DMPK profile and the potency of compound
2b. However, extensive variations including amides, sulfonamides, ureas, carbamates,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and alkyl chains bearing polar groups did not lead to the desired results (data not
shown). We therefore elected to retain the N-acetyl moiety and to continue with SAR
studies at C-2, N-1, and C-5 of the spiro[piperidine-indoline] core.
Variation of the Substituent at C-2 of the Spiro[piperidine-indoline] Core. This
approach confirmed that monosubstitution at C-2 has a major influence on antagonist
potency (see Table 3), as the introduction of one methyl in racemic 16 increased the
compound’s potency in the human LHRH assay roughly fivefold compared with the C-2
unsubstituted derivative 17. Generally, sterically less demanding hydrocarbons were
preferred at C-2 and polar substituents were not well tolerated (as exemplified by the
58-fold drop in potency of hydroxyalkyl derivative 19 relative to allyl derivative 18).
Likewise, more sterically demanding residues (i.e., aryls or heteroaryls) at C-2 also led
to a decrease in potency (data not shown). Overall, compound 20 containing a
cyclopropyl moiety was considered to possess the best balance in terms of potency and
DMPK profile. As already seen for the 3,3-dimethylindoline hit cluster, the actual
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
configuration at the stereogenic center had a major impact on potency, with one
enantiomer being almost exclusively active only [e.g., compare the potency of racemic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6 (IC hGnRH-R = 87 nM) with distomer 16a (IC >20 M) and eutomer 16b (IC =
50
50
50
21 nM), LHRH assay; absolute stereochemistry not elucidated].
Variations at N-1 of the Spiro[piperidine-indoline] Core. As already observed for the
3,3-dimethyl hit cluster, quite a steep SAR was found at the indoline nitrogen atom (N-1,
see Table 4). Only aryl sulfonamides were tolerated while truncation to a methyl
sulfonamide led to an almost complete loss of potency (cf. 21 and 22). In addition,
amide and benzyl substituents were also significantly less potent than their sulfonamide
counterparts (cf. 16 with 23 and 24). Heteroaryl sulfonamide substituents (e.g., in 25)
and sterically small substituents at the meta or para position of the phenyl sulfonamide
(i.e., methoxy, fluoro, cyano) were tolerated (data not shown) while the introduction of a
para-trifluoromethyl moiety as in 26 led to a surprisingly low potency. On the other hand,
the para-fluoro substituent at the phenyl sulfonamide as in 16 improved potency roughly
two- to threefold when compared with the corresponding methoxy derivative 2 (see
ACS Paragon Plus Environment
12

Page 13 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 2) without adding additional lipophilicity to the system (clogD of 16 = 3.25 vs
clogD of 2 = 3.15).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Variations at C-5 of the Spiro[piperidine-indoline] Core. When evaluating the influence
of substituents at C-5 (see Table 5), we noticed that benzamides consisting of lipophilic
benzylic amines and incorporating an ortho substituent at the aromatic residue were
preferred. The introduction of an ortho-chloro group as in 20 improved antagonist
potency roughly 8- to 12-fold compared with the unsubstituted derivative 27.
Furthermore, benzylic amides with an additional para substituent at the aromatic
residue were also tolerated (see para-fluoro derivative 28). Blocking of the benzylic
position with a quaternary carbon shifted the preference of monosubstitution at the
aromatic residue from the ortho to the para position (cf. 29 vs 30). However, this
approach did not improve the in vitro clearance of these derivatives. Generally, NH
amides were more potent than the corresponding N-methyl amides, and anilides were
tolerated as well (data not shown). Of note, the connection of the carboxamide function
to the core could be reversed. In this subseries, meta-substituted benzamides were
preferred and heteroaryls were also tolerated (see 31 and 32). Notwithstanding the
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
excellent potencies achieved this way, we nevertheless decided to discontinue further
investigation of aniline core derivatives, having a potentially mutagenic profile upon
metabolic deacylation in mind.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Identification of 3a Suitable for In Vivo Experiments. Returning to further options for
spiro[piperidine-indoline] variations we chose to (a) remove the N-acetyl residue in
compound 2b (otherwise exhibiting favorable potencies at both the human and the rat
receptor) with the aim of improving solubility, and (b) also exchange the para-methoxy
residue at the phenyl sulfonamide in an attempt to block potential metabolism (see 3a in
Table 6). Indeed, these modifications significantly improved the solubility of the resulting
compound 3a (from 25 mg/L for 2b to 271 mg/L for 3a at pH 6.5) and led to a decrease
in in vivo blood clearance (from 4.7 L/h/kg for 2b to 1.4 L/h/kg for 3a). Along with an
acceptable antagonist potency [IC50 rGnRH-R = 27 nM (LHRH) and IC50 hGnRH-R =
172 nM (buserelin)], these data were considered reasonably sufficient to initiate a first
animal study in rats. Nevertheless, 3a had to be administered parenterally because of
its limited oral bioavailability in rats (F <2%; see the Supporting Information Table S1 for
details). To prove any blockade in GnRH/GnRH-R signaling, 3a was tested in
ACS Paragon Plus Environment
14

Page 15 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ovariectomized (OVX) rats, a well-established animal model to study compound effects
on OVX-induced increased gonadotropin release leading to elevated plasma LH levels.
A single intraperitoneal injection of 3a at 30 mg/kg lowered plasma LH levels in these
animals significantly (i.e. approximately 74% reduction after 1 h) and reversibly in
comparison with animals treated with either the peptidic GnRH antagonist cetrorelix
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(0.1 mg/kg, sc, long-lasting LH reduction) or vehicle (no effect; see the Supporting
Information Figure S6).
Towards Orally Available Compounds. Even though treatment with 3a resulted in a
successful lowering of plasma LH levels in vivo for the first time, it transpired that the
compound (apart from its already known basic and lipophilic properties as well as oral
bioavailability and Caco-2 issues) suffered from off-target effects at several ion
channels, including hERG (IC = 1.1 M), clearly requiring additional improvements to
50
reduce its basicity and lipophilicity. Thus, guided by the results of in vitro investigations
in human hepatocytes which revealed the benzylic amide as the main spot of oxidative
metabolism, we firstly aimed to decrease the lipophilicity and electron density in this part
of the molecule by introducing heteroatoms to further improve the compound’s
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
clearance profile. Although not very effective in terms of potency initially, unsubstituted
pyridylmethyl amides clearly showed a lower metabolic clearance in human hepatocytes
compared to the corresponding benzylic amides. Next, the introduction of a substituent
at C-3 of the pyridyl ring, for example chloro, led to a sufficient recovery in antagonist
potency while retaining an improved clearance profile in both human hepatocytes and
rats in vivo (see Table 7, compounds 20b and 33a).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In additional experiments aimed at improving the off-target profile, we investigated the
influence of basicity at the spiropiperidine nitrogen atom. However, all attempts to
reduce basicity and to simultaneously improve the pharmacokinetic profile by employing
extensive substituent variations at this nitrogen failed to provide the desired outcome
(see ‘variations at the spiropiperidine nitrogen’ section above). Thus, we decided to
evaluate non-nitrogen-containing structural modifications of the spiropiperidine core
itself. A broad range of different spirocarbocyclic and spiroheterocyclic systems at C-3
of the indoline core was synthesized and tested (see Table 8). Whereas secondary and
tertiary alcohols (e.g., 37, 38) and (thio)pyrans (e.g., 39, 40) were tolerated, but
somewhat less potent than 20, oxidized thiopyran derivatives (i.e., sulfoximines,
ACS Paragon Plus Environment
16

Page 17 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
sulfoxides, and sulfones; see 34a/b, 35, and 36) proved to be most promising with clear
indications of a link between the absolute configuration at the stereogenic sulfur and the
actual potency achieved.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Identification of Orally Available Compound 4a. In addition to having advantages in
terms of synthetic access (such as less stereochemistry issues than sulfoximines or
sulfoxides), sulfones in general were finally considered as resulting in the best balance
between potency, clearance, and permeation. Consequently, the combination of (a) the
chloropyridylmethyl amide at C-5 with (b) the spirocyclic sulfone at C-3 of the indoline
and (c) a cyclopropyl at C-2 resulted in compound 4a which was chosen for a first
experiment making use of po administration in OVX rats. Table 9 summarizes the
technical profile of 4a (for the full profile, see the Supporting Information Table S1; note
that oral bioavailability in this structural class is strongly dependent on the actual
formulation vehicle used).
Thus, single po administrations of 4a in a range of 1 to 30 mg/kg suppressed tested
plasma LH levels in OVX rats in a dose-dependent manner. At doses ≥10 mg/kg, a
suppression of LH levels comparable to the peptidic GnRH antagonist cetrorelix (dosed
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
at 0.1 mg/kg, sc) was achieved for 6 hours at least (calculated ED ca. 5.5 mg/kg, see
50
Figure 3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Identification of the Clinical Candidate 5a. Based on the encouraging findings with
compound 4a, we started a final round of lead optimization efforts to achieve compound
characteristics having the potential of becoming a clinical candidate. Once more, we
primarily focused on further improvements in antagonistic potency and oral
bioavailability, in addition to overcoming the CYP3A4 interactions observed for 4a (for
details regarding the CYP profile, see the Supporting Information Table S1). As most of
the structural parts of 4a had already been optimized, we returned to modifications of
the pyridylmethyl amide by synthesizing and testing various substitution patterns (see
Table 10). We found that double substitution at C-3 and C-5 of the pyridyl ring had a
beneficial effect on potency (buserelin assay), while single C-3 or C-5 substitution alone
was less effective (cf. 5 vs 4 and 41). Whereas the unsubstituted pyridyl derivative 45
had a far lower potency (buserelin assay), introduction of both a chloro at C-3 and a
trifluoromethyl moiety at C-5 of the pyridyl ring resulted in the most potent combination
(cf. 5 vs 42, 43, and 44). In summary, the additional substituents not only increased
ACS Paragon Plus Environment
18

Page 19 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
antagonist potency, but also resulted in a further improvement in the compound’s
clearance and CYP interaction profile. Upon enantiomeric separation of 5 (in our view,
with the combination of modifications best suited for thorough preclinical evaluation), we
finally managed to obtain, extensively characterize, then prepare larger quantities of
eutomer 5a which was subsequently nominated as clinical candidate.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Synthetic Access to Spiroindolines and Synthesis of Clinical Candidate 5a.
The spiroindoline analogues 53 were generally prepared according to the procedures
outlined in Scheme 2. Indolenines 48 were prepared from bromophenylhydrazine 46
and carbonyl compounds 47 under acidic conditions in a Fischer indole synthesis.34
Indolenines 48 could either be reduced with sodium borohydride to give indolines 49
2
(
R = H) or reacted with Grignard reagents under Lewis acid catalysis to introduce
2
different residues R (≠ H). Functionalization at the indoline NH was accomplished
under standard acylation or alkylation conditions to furnish aryl bromides 50. The aryl
bromides were either carbonylated in the presence of carbon monoxide, methanol, and
a palladium catalyst to give esters 51, which was followed by standard saponification to
1a
1b
carboxylic acids 52 then amide coupling with amines (R )(R )NH to give spiroindoline
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
analogues 53. Alternatively, spiroindolines 53 could be obtained directly upon
palladium-catalyzed carbonylation of aryl bromides 50 with molybdenum hexacarbonyl
in the presence of the respective amine.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 5a specifically was synthesized as outlined in Scheme 3. Starting from
commercially available thiopyran 54, a masked aldehyde moiety was introduced by
Wittig reaction to give the stable and storable enol ether intermediate 55. The indoline
system was built up via a Fischer indole synthesis as described above to give
indolenine 56. Lewis acid catalyzed Grignard reaction of indolenine 56 employing
cyclopropylmagnesium bromide furnished the spiroindoline core system 57 as a
racemic mixture. Sulfonamide formation and subsequent oxidation gave bromo sulfone
59. The amide side chain at C-5 was introduced by a three-step protocol starting with a
palladium-catalyzed carbonylation to give ester 60 followed by saponification and amide
formation. Separation of the amide enantiomers by HPLC on a chiral phase yielded 5a.
Characterization of 5a: Absolute Configuration. The separated enantiomers 5a and 5b
of racemate 5 were individually tested to clarify the role of the C-2 stereocenter. This
revealed that 5a is the (only) physiologically relevant enantiomer (5a: IC hGnRH-R =
50
ACS Paragon Plus Environment
20

Page 21 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
1 nM, 5b: IC hGnRH-R = 2.43 M, buserelin assay) (see Table 10). We were able to
50
obtain diffracting crystals of eutomer 5a (Figure 4) possessing S-configuration, as
determined by X-ray analysis [see the Supporting Information Table S6 for details].
Pharmacological, Physicochemical, Safety, and DMPK Properties of 5a. Figure 5
summarizes the major findings of our in-depth studies of 5a. Compound 5a exhibited
potent, double-digit nanomolar antagonism at the human, rat, and cynomolgus monkey
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(h/r/c)GnRH-R while it showed no agonistic activity at the human GnRH-R up to 20 M
(data not shown). Nonetheless, the aqueous solubility of 5a at pH 6.5 continued to be
limited whereas its in vitro clearance proved to be moderate to low (in human, rat, dog,
and cynomolgus monkey hepatocytes). In our view, a combination of limited solubility
and low to moderate absorptive permeability can also account for the species- (rat,
cynomolgus monkey) and formulation-dependent low to high oral bioavailabilities
observed. Total blood clearance in vivo was low in rat and low to moderate in dog and
monkey, and compound half-life was intermediate in monkey and long in rat and dog.
Then again, overlaying effects of irreversible CYP3A4 inhibition in human liver
microsomes as well as CYP3A4 induction in human hepatocytes were detected in vitro,
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
still. Of particular importance, 5a was inactive in a cell-based panel of 25 GPCRs
(‘Bayer Panel’, Millipore GPCRProfiler, now Eurofins; see the Supporting Information
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table S2), none of which was activated or inhibited >70% at 10 M compound
concentration, indicative of an excellent selectivity within the target family. Additional off-
target profiling of 5a (Ricerca, now Eurofins) confirmed the absence of relevant activities
in the respective assays [see the Supporting Information Table S5 for significant
responses (i.e., ≥50%)].
Drug–Target Residence Time of 5a. In addition to the typical focus on ligand potency
and efficacy, drug candidate optimization programs increasingly rely on data regarding
the modulation of the actual duration of ligand–receptor interactions (i.e., drug–target
35
residence time ) owing to the fact that compounds with differentiated pharmacological
profiles often display unique binding properties36,37 and very much become a focus of
attention.38-40 In GPCR research longer residence times have been demonstrated to
result in a prolonged duration of drug actions41,42 as a consequence of continued
receptor modulation.43 In other cases, transient binding behavior resulted in potent
compounds with an improved side effect profile.44-46
ACS Paragon Plus Environment
22

Page 23 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Based on the established in vivo pharmacology of 5a, we therefore asked ourselves
how its drug–target residence time would compare to other known hGnRH-R
antagonists. To address this question, we used an assay previously established in our
group47,48 to characterize the hGnRH-R binding kinetic parameters of 5a and several
reference compounds (see Table 11). In terms of target recognition, 5a is, alongside
elagolix, among the fastest associating ligands known. On the other hand, its target
residence time of 7 minutes is significantly shorter than the other SMOL antagonists
(relugolix and elagolix) or peptide antagonist (cetrorelix) tested, and within the range of
the fastest dissociating peptide antagonists evaluated so far, but still superior to the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
49
residence time of GnRH itself. The ability of 5a to saturate the GnRH receptor nearly
20 times faster than its physiological ligand, while dissociating from it at only a slightly
slower rate is a unique feature that might be linked to the compound’s in vivo activity
profile. This hypothesis deserves further investigation in follow-up studies: For instance,
50
a systems pharmacology approach similar to the one reported in could be envisioned,
taking into account hGnRH-R’s atypical desensitization properties51-53 as well as the
agonist pulse frequency and amplitude dependent nature of receptor signaling.54,55
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Reduction of LH Levels in Rat and Monkey Animal Models In Vivo. The in vivo
efficacy of 5a upon po administration was determined in the well-established rat OVX
and cynomolgus monkey orchiectomy (ORX) models reliably allowing for the study of
compound effects on increased gonadotropin release and elevated plasma LH levels as
a consequence of these surgical procedures. At doses (5a) of 10 and 30 mg/kg po
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
calculated ED ca. 4.5 mg/kg), a clear reduction of plasma LH levels was noticed, and
50
comparable to that obtained upon treatment with cetrorelix (0.1 mg/kg, sc) used as
control antagonist, in the rat OVX model (Figure 6A). These results were confirmed in a
subsequent study in the monkey ORX model where a suppression of baseline LH levels
by up to 60% was achieved with a single dose (5a) of 20 mg/kg po (Figure 6B). Taken
together, these outcomes clearly demonstrate that treatment with 5a results in effects in
line with the expected profile of a potent, efficacious, and reversible GnRH-R
antagonist.
Maximum Reduction of LH Levels Obtained in a First-in-Human Study with 5a in
Postmenopausal Women. Compound 5a was tested in a first-in-human study using a
multicenter, randomized, double-blind, parallel-group, placebo-controlled design. Single
ACS Paragon Plus Environment
24

Page 25 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
doses of 5 mg, 20 mg, 60 mg, 150 mg, 300 mg and 450 mg of 5a in a self-
microemulsifying drug delivery system (SMEDDS) formulation were administered to six
postmenopausal women each.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 5a was well tolerated and safe at the doses tested in this study. Using the
SMEDDS formulation, a low variability in pharmacokinetics in terms of AUC was
observed (mean CV for AUC: about 29%). Based on a cross-study comparison, this is
considerably better than the variability in AUC previously reported for elagolix (~40%;
p = 0.03).56 Suppression of plasma LH levels reached a maximum of about 49%
reduction at the 300 mg dose of 5a, with no further increase in effect observed with the
higher dose of 450 mg (Figure 7). Further details of the clinical study will be reported
elsewhere.
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CONCLUSION
Extensive hit-to-lead and lead optimization activities led to the identification of
spiroindoline derivative 5a (BAY 1214784) and its characterization as a potent and
selective antagonist of the human GnRH receptor, finally proving efficacious in a first-in-
human study in postmenopausal women. Spiroindolines represent a new class of
selective GnRH-R antagonists exhibiting multispecies activity and high potency in
human, rat, and cynomolgous monkey models both in vitro and in vivo, a crucial factor
for project success. By introducing a spiropiperidine moiety in the barely potent, yet
multispecies active, HTS hit 15 and controlling the stereochemistry at C-2, we were able
to achieve a major improvement in potency and LLE in lead compound 2b. Still-existing
DMPK liabilities (i.e., high clearance, inhibition of CYP isoforms, and low oral
bioavailability) of the indoline hit cluster were tackled by careful optimization of the core
towards a spirocyclic sulfone and the introduction of a chlorinated pyridyl moiety in the
amide side chain. This resulted in advanced compound 4a with a far more balanced
DMPK profile, while simultaneously retaining sufficient potency for profiling in animal
models in vivo. Single oral dosing of 4a resulted in a dose-dependent lowering of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
26

Page 27 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
plasma LH levels in OVX rats (ED ca. 5.5 mg/kg). Fine-tuning of the pyridyl amide side
50
chain finally led to the identification of potent, transiently binding and selective 5a
exhibiting a superior DMPK profile (i.e., improved oral bioavailability, potency, and CYP
interaction), along with excellent safety properties. Thus, 5a was advanced to clinical
development and shown to suppress plasma LH levels by up to 45% in a first-in-human
study in postmenopausal women. In addition, low pharmacokinetic variability and good
tolerability was noted at single doses of up to 450 mg po once daily.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
57
Finally, 5a (probe code BAY-784) meets the criteria for chemical probes established
58
by the Structural Genomics Consortium (SGC) and was thus handed over to the SGC
as a ‘donated chemical probe’ to be freely available for future studies. We trust these
studies will elucidate BAY-784’s unique binding behavior, and its potential usefulness
for the prevention of the development of a hypoestrogenic state in the long term – a
prerequisite for a substantially improved treatment of uterine fibroids. All in all, we are
confident that making BAY-784 available to the scientific community will open up a wide
range of opportunities for in vitro and in vivo studies thereby contributing to the future
progress in field of hGnRH-receptor research.
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 156
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
28

Page 29 of 156
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
EXPERIMENTAL SECTION
Chemistry
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Methods and Materials. All solvents and chemicals were used as purchased
without further purification. The progress of all reactions was monitored by TLC on
Merck precoated silica gel plates (with fluorescence indicator UV254) using EtOAc/n-
hexane or DCM/MeOH as the solvent system or by UPLC (see Methods 1 and 2). TLC
spots were visualized by irradiation with UV light (254 nm). Column chromatography
was performed on Biotage chromatography systems with the solvent mixtures specified
in the corresponding experiment. Proton ( H) NMR, 13C NMR and 19F NMR spectra
1
were recorded on Bruker Avance 300, 400, or 500 MHz instruments using CDCl or
3
DMSO-d as solvent. Chemical shifts are given in parts per million ( relative to the
6
residual solvent peak). In case of enantiomeric separations the given retention times for
each enantiomer refer to the respective analytical method.
Analytical (UP)LC-MS was performed using Methods 1–3. The masses (m/z) are
reported from electrospray ionization in the positive mode, unless the negative mode is
indicated (ESI–).
ACS Paragon Plus Environment
29