Discovery of BAY-298 and BAY-899: Tetrahydro-1,6-naphthyridine-Based, Potent, and Selective Antagonists of the Luteinizing Hormone Receptor Which Reduce Sex Hormone Levels in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.9b01382

The human luteinizing hormone receptor (hLH-R) is a member of the glycoprotein hormone family of G-protein-coupled receptors (GPCRs), activated by luteinizing hormone (hLH) and essentially involved in the regulation of sex hormone production. Thus, hLH-R

Full text of DOI:10.1021/acs.jmedchem.9b01382

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Article
Discovery of BAY-298 and BAY-899: Tetrahydro-1,6-naphthyridine-
Based, Potent and Selective Antagonists of the Luteinizing
Hormone Receptor Which Reduce Sex Hormone Levels In Vivo
Lars Wortmann, Bernhard Lindenthal, Peter Muhn, Alexander Walter, Reinhard Nubbemeyer,
Dieter Heldmann, Lothar Sobek, Federica Morandi, Anna K. Schrey, Dieter Moosmayer,
Judith Günther, Joachim Kuhnke, Marcus Koppitz, Ulrich Luecking, Ulrike Roehn, Martina
Schäfer, Katrin Nowak-Reppel, Ronald Kuehne, Hilmar Weinmann, and Gernot Langer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01382 • Publication Date (Web): 31 Oct 2019
Downloaded from pubs.acs.org on November 3, 2019
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Discovery of BAY-298 and BAY-899: Tetrahydro-
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,6-naphthyridine-Based, Potent and Selective
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Antagonists of the Luteinizing Hormone Receptor
Which Reduce Sex Hormone Levels In Vivo
#
⋕
⋕
⋕
Lars Wortmann,* Bernhard Lindenthal,* Peter Muhn, Alexander Walter, Reinhard
⋕
⋕
⋕
†
†
Nubbemeyer, Dieter Heldmann, Lothar Sobek, Federica Morandi, Anna K. Schrey, Dieter
⋕
⋕
⋕
⋕
⋕
Moosmayer, Judith Günther, Joachim Kuhnke, Marcus Koppitz, Ulrich Lücking, Ulrike
⋕
⋕
⋕
†
⋕
Röhn, Martina Schäfer, Katrin Nowak-Reppel, Ronald Kühne, Hilmar Weinmann, and
Gernot Langer⋕
⋕Bayer AG, Research & Development, Pharmaceuticals, 13353 Berlin, Germany
†Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle Strasse 10,
Campus Berlin-Buch, 13125 Berlin, Germany
This publication is dedicated to the memory of Heiner Fritzemeier, a great scientist, colleague and
friend who passed away in 2017.
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KEYWORDS: GPCR, luteinizing hormone receptor, LH, contraception, cancer, polycystic
ovarian syndrome, PCOS, chemical probe, hLH receptor antagonist, BAY-298, BAY-899, HPG
axis, sex hormones
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ABSTRACT:
The human luteinizing hormone receptor (hLH-R) is a member of the glycoprotein hormone family
of GPCRs, activated by luteinizing hormone (hLH) and essentially involved in the regulation of
sex hormone production. Thus, hLH-R represents a valid target for the treatment of sex hormone-
dependent cancers and diseases (polycystic ovary syndrome, uterine fibroids, endometriosis) as
well as contraception. Screening of the Bayer compound library led to the discovery of
tetrahydrothienopyridine derivatives as novel, small molecule (SMOL) hLH-R inhibitors and to
the development of BAY-298, the first nanomolar hLH-R antagonist reducing sex hormone levels
in vivo. Further optimization of physicochemical, pharmacokinetic and safety parameters led to
the identification of BAY-899 with an improved in vitro profile and proven efficacy in vivo. BAY-
1
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98 and BAY-899 serve as valuable tool compounds to study hLH-R signaling in vitro and to
interfere with the production of sex hormones in vivo.
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INTRODUCTION
The hypothalamic–pituitary–gonadal (HPG) axis is the key pathway for the production of the
1
sex hormones estradiol and progesterone in females, as well as testosterone in males. Sex
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hormone synthesis is initiated via binding of gonadotropin-releasing hormone (GnRH) to its
cognate receptor (GnRH-R) leading to receptor activation and the expression and secretion of two
gonadotropins, namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH).
Binding of these glycoprotein hormones to their specific downstream receptors, the FSH and LH
receptors (FSH-R, LH-R), then stimulates the production of sex hormones in both ovaries and
testes. The actual sex hormone levels produced are tightly controlled by several negative feedback
loops.2,3 Given the essential role of sex hormones in a wide variety of developmental and
physiological processes, an impressive number of strategies aimed at the modulation of their actual
levels and function have been developed to treat hormone-dependent diseases such as certain types
4
5
6
7
of cancers, uterine fibroids, polycystic ovary syndrome (PCOS) and endometriosis, as well as
8
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for female and male contraception. Thus, interfering with hLH-R-dependent hormone production
with small molecule (SMOL) antagonists represents a valid approach not only to study sex
hormone physiology but also to tackle sex hormone dependent disease conditions or contraception.
Based on several characteristic features, the hLH-R has been assigned to the -group of the
rhodopsin family of G-protein-coupled receptors (GPCRs)10 and belongs to the glycoprotein
hormone receptor cluster, together with the hFSH-R and thyroid-stimulating hormone receptor
(TSH-R). Whereas most rhodopsin receptor ligands bind to a rhodopsin-like binding cavity within
1
1
the 7-transmembrane region (7TMR), the glycoprotein hormone receptors are activated by high
affinity agonists binding to a large, glycosylated extracellular domain. Besides FSH, gonadal sex
hormone production is predominantly regulated by pituitary-derived LH, a non-covalently linked,
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heterodimeric glycoprotein consisting of an - and a -subunit, the -subunit being common to
all glycoprotein hormones. Whereas agonist–receptor binding first and foremost induces cAMP
1
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13
second messenger signaling, it also results in Ca release as a secondary coupling mechanism.
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Receptor activation has also been linked to additional downstream signaling processes and high
1
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organ responsiveness, adding further to complexity.
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Two tool compound classes for the LH receptor have been described. Heitman et al. reported
in vitro studies with the LH receptor agonist Org 4355317 and the LH receptor antagonist
1
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LUF5771 (Figure 1).
Agonist
Antagonist
H
N
N
O
O
NH2
O
O
O
N
N
H
S
N
H
S
N
Org 43553
LUF5771
Figure 1. Reported agonist and antagonist for the hLH-R.
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These workers convincingly demonstrated that Org 43553 reversibly binds to an allosteric
binding site in the hLH-R without interfering with the binding of the natural ligand LH.
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Furthermore, follow-up studies by the same group indicated the existence of a second, distinct
allosteric binding site (i.e., different from the Org 43553 binding site) to which the antagonist
LUF5771 binds, blocking Org 43553 agonist activity. However, no SMOL hLH-R antagonist
suitable for in vivo application has been described so far. Thus, we decided to initiate a lead finding
program for this target and now report the identification of the tetrahydro-1,6-naphthyridine-based
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compounds BAY-298 and BAY-899 as potent and selective antagonists of the hLH-R with a good
pharmacokinetic profile and proven activity in several in vivo models.
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RESULTS AND DISCUSSION
High-throughput screening and identification of screening hit 1. High-throughput screening
(HTS) was used to identify SMOL antagonists of the hLH receptor. A total of 1.19 million
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compounds was screened using frozen cells expressing the hLH-R, quantifying the changes in
agonist-induced cAMP levels using time-resolved fluorescence resonance energy transfer (TR-
FRET) technology21–23 (see Supporting Information for full details). After removal of compounds
interfering with the readout format, the remaining hits were checked for the absence of any
unspecific cAMP modulation via interference with adenylyl cyclase activity using forskolin-
stimulated HEK293 membranes (see Supporting Information for assay details). Following a
thorough hit-to-lead examination adressing additional off-target, selectivity and species specificity
properties and the removal of compounds with pan-assay interference motifs,24
tetrahydrothienopyridine-based compound 1 (Table 1) was identified as lead structure.
Early profiling of 1 further revealed no residual hLH-R agonist activity as well as no antagonist
activity in a panel of closely related glycoprotein hormone and leucine-rich-repeat-containing
GPCRs (hFSH-R, hTSH-R, hRXFP1/hLGR7-R). In addition, 1 was active at the rat LH receptor
(rLH-R) with a similar potency, yet inactive at the corresponding murine LH receptor (mLH-R,
see Table 1). Compound 1 was of moderate potency (IC (hLH) = 378 nM) and exhibited
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borderline Lipinski rule of five properties, being suboptimal for an oral route of administration.
In particular, the lipophilicity of 1 (logD = 5, TPSA = 42) was considered to be high. At the same
time the Caco-2 permeability was low (< 1 nm/s for P (A-B)). Therefore, we carefully monitored
app
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logD during compound optimization, even though reduction of lipophilicity while simultaneously
improving potency was considered challenging as we assumed binding of our ligand to the
lipophilic 7TMR.
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Table 1. Analysis of the Role of the Absolute Configuration of 1a
O
Cl
O
*
N
N
H
Cl
S
IC [nM]
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Absolute
Compound
configuration
hLH
rLH
212
mLH
hFSH
hTSH
1
racemate
378
403
>30000 >16500
>30000 >16500
>30000
1
a
(S)-(+)-enantiomer
412
>30000
>30000
1b
(R)-(–)-enantiomer 6790
4890 >30000 >16500
a
IC values are arithmetic means of at least two independent experiments run in duplicates.
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0
Synthesis of screening hit 1. Compound 1 was synthesized as outlined in Scheme 1. Following
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a literature procedure, key intermediate 4 was prepared in racemic form via a Pictet–Spengler
reaction from commercially available 2-(thiophen-2-yl)ethan-1-amine (2) and 4-
methoxybenzaldehyde (3). Subsequent reaction of 4 with 1,3-dichloro-5-isocyanatobenzene
furnished urea 1. This short and efficient route allowed the introduction of a broad scope of
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variations by using different benzaldehydes and different capping reactions to functionalize the
secondary amine in compound 4.
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Scheme 1. Synthesis of HTS Hit 1
O
O
O
Cl
O
NH2
a, b
c
+
N
N
H
Cl
NH
S
O
S
S
2
3
4
1
aReagents and conditions: (a) triethylamine, EtOH; (b) TFA; (c) 1,3-dichloro-5-
isocyanatobenzene, triethylamine, DMSO, 26%.
Structure–Activity Relationship and Profiling of the hLH-R Antagonists BAY-298 and
BAY-899. We first investigated the role of the absolute configuration of 1 (Table 1). Thus,
racemate 1 was separated into its enantiomers 1a and 1b, with subsequent tests demonstrating that
1a is the physiologically relevant enantiomer (hLH-R ~17 times, rLH-R ~12 times more active
than 1b). We were able to obtain diffracting crystals of a closely related derivative of eutomer 1a,
namely compound 5 (Figure 2) with the (S)-configuration determined by X-ray analysis.
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Figure 2. X-ray analysis of compound 5 determined the (S)-configuration of the eutomer.
Next, we investigated the structure-activity relationship of the substituent at the 4-position of
tetrahydrothienopyridines (see Table 2). Removal of the p-methoxy group of 1, which could be a
potential metabolic weak spot, led to significantly reduced activity (see compound 6).
Investigation of other para substituents (compounds 11–14, 19), however, clearly demonstrated
that target activity was retained upon replacement of the methoxy by a chloro substituent
(compound 14). All attempts to lower logD values by the introduction of a polar group were
detrimental to activity (compare compounds 7–9, 18, 19). Investigation of selected compounds in
a human liver microsome assay revealed low to moderate metabolic stability (for classification
of the results, the following criteria were used: Fmax > 70% (= CL <0.4 L/h/kg) was classified as
high metabolic stability, Fmax 30–70% (CL 0.4–0.9 L/h/kg) as moderate stability and Fmax < 30%
(CL > 0.9 L/h/kg) as low metabolic stability). A halogen substituent at the para position of the 4-
phenyl group seemed to increase metabolic stability significantly (see compounds 13–15).
Table 2. SAR Investigation of the Substituent at the 4-Positiona
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IC (hLH)
exp.
logD
calc.
logD
CL hLM Fmax hLM
5
0
Compound
R
[
nM]
[L/h/kg]
[%]
O
1
378
5.0
n.d.
n.d.
4.2
4.2
4.9
1.3
3
6
7
8
9
1620
>16500
7470
4.9
4.1
3.8
3.7
n.d.
n.d.
1.2
n.d.
n.d.
11
N
N
N
5180
n.d.
n.d.
10
>16500
n.d.
5.3
n.d.
n.d.
1
1
1
2
2610
5.5
5.3
6.2
1.2
8
>16500
n.d.
n.d.
n.d.
F
F
1
1
3
4
3650
486
n.d.
n.d.
5.7
5.4
0.7
0.7
51
48
F
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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
F
17
H
N
18
2310
3.7
3.7
2.8
3.3
n.d.
n.d.
n.d.
n.d.
O
O
19
>16500
NH2
a
IC values are arithmetic means of at least two independent experiments run in duplicates.
5
0
The results of our SAR investigation of the urea capping group at the 5-position are depicted in
Table 3. Both chloro atoms in 1 could be replaced either by methyl (compound 20) or by methoxy
compound 21), reducing logD values while maintaining target activity. Removal of one (compare
(
compounds 1 and 22) or both (compare compounds 14 and 23) chloro atoms led to a significantly
lower logD value while retaining LH antagonism. A sterically constrained para substituent
(
compound 24) or a combination of two lipophilic meta and para substituents (compounds 25, 26)
were tolerated without loss of target activity. In the case of polar substituents, the target potency
was dramatically reduced (compounds 27, 28). To our surprise, even a sterically bulky lipophilic
substituent at the para or meta position was tolerated (compounds 29, 30) but led to a significant
increase in the logD value. With regard to metabolic stability in human liver microsomes, a
1
2
combination of R = Cl and a para-substituted R group led to increased stability of 26 and 30
(Table 3).
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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
Table 3. SAR Investigation of the Substituent at the 5-Positiona
1
R
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
O
2
R
N
N
H
5
S
IC (hLH)
exp.
logD
calc.
logD [L/h/kg]
CL hLM Fmax hLM
50
Compound
R1
R2
[nM]
[%]
Cl
Cl
1
OMe
378
580
5.0
4.1
4.9
4.5
1.3.
n.d.
3
2
2
0
1
OMe
OMe
OMe
n.d.
3
O
O
207
275
3.6
4.2
4.0
4.4
1.3
1.2
22
7
Cl
2
3
Cl
95
4.0
3.9
4.2
4.2
1.0
1.1
26
18
Cl
24
25
OMe
107
F
Cl
174
5.1
4.7
n.d.
n.d.
Cl
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Journal of Medicinal Chemistry
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6
7
8
9
F
F
26
27
Cl
81
5.2
3.1
4.9
3.1
0.8
42
O
N
F
F
F
S
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
OMe
>16500
n.d.
n.d.
O
O
F
2
2
8
9
OMe
Cl
1680
332
2.8
5.4
2.7
5.1
n.d.
n.d.
n.d.
n.d.
NH2
O
O
O
3
0
Cl
127
5.3
5.1
0.6
56
F
a
IC values are arithmetic means of at least two independent experiments run in duplicates.
5
0
The SAR data compiled in Table 4 underscored the importance of the urea moiety, indicating
that both the carbonyl and the NH group are essential for target activity. Thus, 31 with a thiourea
moiety is about 16 times less active than 21, implying that the carbonyl group might be
incorporated in a binding interaction with the hLH-R. Replacement of the urea NH group by CH2
(
compound 32) led to a complete loss of activity (compare compound 20, Table 3).
Table 4. SAR Investigation of Urea Capping Moiety Aa
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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
O
A
N
R
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
S
IC (hLH)
exp.
logD
calc.
logD
50
Compound
A
R
[nM]
O
O
S
31
3320
3.7
4.8
4.2
4.5
N
H
O
3
2
>16000
a
IC values are arithmetic means of at least two independent experiments run in duplicates.
5
0
2
7
As thiophenes are known to carry a high risk for formation of reactive metabolites and to
further decrease metabolic stability in human liver microsomes, we investigated potential
replacement options for the thiophene moiety (see ‘A’ ring, Table 5). In doing so, we observed
that replacement of the thiophene in 30 (see Table 3) by a pyridine (see compound 33, Table 5)
resulted in retained target activity while significantly improving the metabolic stability in human
liver microsomes.
Table 5. SAR Investigation of Hetaryl Ring ‘A’ and Identification of 33 (BAY-298) and
Chemical Probe 36 (BAY-899)a
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9
R
Y
O
O
Z
N
N
H
F
A
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
N
IC50
(hLH)
[nM]
exp.
logD
calc.
logD
CL hLM Fmax hLM
Compound
R
Y
Z
[L/h/kg]
[%]
3
3
3 (BAY-298) Cl CH CH
96
4.7
3.9
3.2
2.8
4.5
4.2
3.5
3.0
0.2
0.3
0.2
0.1
85
75
83
89
34
F
F
F
CH CH
102
312
185
3
5
CH
N
N
N
6 (BAY-899)
a
IC values are arithmetic means of at least two independent experiments run in duplicates.
5
0
Given these results, we elected to further profile 33 (BAY-298) as a potential chemical probe28
for the hLH-R both in vitro and in vivo (with the results obtained summarized in Figures 3 and 4).
Compound 33 showed potent antagonism at the human, rat and cynomolgus monkey LH-R (see
Figure 3) and was expected to be inactive at the respective mouse receptor as previously shown
for 1. Likewise, 33 did not show any residual hLH agonism, was inactive in a hFSH-R assay and
exhibited only marginal (i.e., ~24-fold selective) hTSH-R antagonist activity.
Compound 33 is characterized by good in vitro pharmacological, physicochemical, safety and
DMPK property profiles (Figure 3). With regard to safety, 33 was moderately active in the hERG
assay (IC = 3 µM) and had a mixed CYP inhibition profile (no effect on isoforms 1A2, 2D6 and
5
0
3
A4 but significant inhibition of 2C8 and 2C9). Compound 33 performed particularly well in both
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Journal of Medicinal Chemistry
Page 16 of 79
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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
human and rat liver microsome and also rat hepatocyte assays, indicative of a low clearance. In
addition, it showed good permeability and no efflux in a Caco-2 assay system. In vivo these
properties translated to a low-dose pharmacokinetic profile in rats with low clearance, long half-
life and a high volume of distribution resulting in a good oral bioavailability and high exposure
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
(see Figure 4).
Figure 3. Summary of chemical, physicochemical, safety and in vitro DMPK properties of 33
BAY-298).
(
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Page 17 of 79
Journal of Medicinal Chemistry
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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
Figure 4. Low-dose pharmacokinetic profiles of 33 (BAY-298) and 36 (BAY-899) in rats.
Our continued efforts to further improve the properties of 33 (BAY-298) by lowering the logD
value led to the discovery of 36 (BAY-899, see Table 5). The logD value could be lowered by
almost two log units; in turn, compound 36 was about ten times more soluble than 33, had better
permeability in the Caco-2 assay and showed an improved CYP inhibition as well as hERG profile
(compare Figures 3 and 5). In addition, the selectivity versus the TSH receptor could be further
increased to >100-fold. Investigation of 36 in a low-dose pharmacokinetic study in rats also
revealed a low clearance, a long half-life and a high volume of distribution resulting in a good oral
bioavailability and high exposure (see Figure 4). Both compounds (33 and 36) were investigated
in a cell-based panel of 25 GPCRs (see SI) and none of which activated or inhibited a GPCR >
7
0% at 10 µM compound concentration indicating an excellent selectivity within the target family.
3
3 and 36 both exhibited an appealing in vitro and in vivo profile with 36 bearing superior
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Page 18 of 79
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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
properties in several assays tested as discussed above. Thus, 36 (BAY-899) meets the criteria for
2
8
29
chemical probes established by the Structural Genomics Consortium (SGC) and was handed
over to the SGC as a ‘donated chemical probe’. The less active enantiomer (data not shown) might
serve as a negative control compound for future pharmacological experiments.
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
Figure 5. Summary of chemical, physicochemical, safety and in vitro DMPK properties of 36
(BAY-899).
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Page 19 of 79
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Synthesis of 33 (BAY-298) and Chemical Probe 36 (BAY-899). The synthesis of 33 (BAY-
98) is outlined in Scheme 2. Piperidone intermediate 40 was synthesized in two steps from
2
commercially available 4-chlorophenylmagnesium bromide (37), benzyl chloroformate (38) and
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
30
4
-methoxypyridine (39), following a method described by Di Fabio et al., with subsequent zinc
reduction31 of the resulting double bond. A gold-catalyzed reaction32 with propargylamine
furnished naphthyridine 41 as a mixture with the undesired regioisomer 42. The mixture of
compounds 41 and 42 could be converted into 33 via deprotection of the carboxybenzyl protecting
group, subsequent urea formation using 4-nitrophenyl chloroformate as a reagent, and separation
of the regioisomers and enantiomers. The synthesis of chemical probe 36 (BAY-899) followed a
similar route.
Scheme 2. Synthesis of 33 (BAY-298)
Cl
Cl
O
O
O
a, b
Cl
O
+
+
N
O
N
O
MgBr
3
7
38
39
40
c
Cl
Cl
Cl
O
O
O
d, e, f
O
+
N
O
N
N
H
F
N
O
N
N
N
3
3
41
ratio ~ 3:1
42
BAY-298
aReagents and conditions: (a) THF, 81%; (b) zinc, acetic acid, quantitative; (c) propargylamine,
sodium tetrachloroaurate dihydrate, EtOH, 27% (as a mixture of regioisomers); (d) TFA, 76% (as
a mixture of regioisomers); (e) 4-nitrophenyl chloroformate, 4-(4-fluorophenoxy)aniline, THF,
1
7%; (f) HPLC separation of regioisomers, ~ 60%; separation of enantiomers, 39 %.
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Journal of Medicinal Chemistry
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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
Mode of Action Studies.
Antagonist Dose–Response Studies in the Presence of Multiples of the EC Concentration
8
0
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
of either hLH or the SMOL hLH-R Agonist Org 43553. To shed light on the mode of action,
we performed antagonist dose–response studies with 33 (BAY-298) in the presence of four
different, single concentrations of the natural ligand hLH (Figure 6A) or the SMOL hLH-R agonist
Org 43553 (Figure 6B) using the results obtained at 1 × EC as control.
8
0
Figure 6. Antagonist dose–response studies with 33 (BAY-298) in the presence of integral
multiples of the EC concentration of hLH-R agonists: (A) the glycoprotein hLH and (B) the
8
0
SMOL agonist Org 43553.
In both cases (Figure 6A and 6B), the antagonist efficacy of 33 remained largely unaffected and
potency values clearly showed a limited rightward shift in dose–response indicative of a saturation
in antagonist activity not being affected by agonist. Thus, we conclude that 33 binds to a site
different from the actual agonist binding sites of hLH and Org 43553. In both cases, 33 behaved
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Page 21 of 79
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like a negative allosteric modulator of agonist-induced hLH-R activity. The finding that the
antagonist activity of 33 is neither affected by the binding of hLH nor the SMOL hLH-R agonist
Org 43553 is well in line with previous results. Heitman et al.18,19 not only demonstrated reversible
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
11
binding of Org 43553 to a putative rhodopsin-like SMOL binding cavity but also validated the
existence of a second, independent SMOL binding site for both positive and negative modulators
of Org 43553 hLH-R agonist activity in close vicinity. Future studies will be necessary to finally
clarify whether 33 binds in a similar way18,33 or to an unrelated, non-overlapping site within the
‘
ligand binding landscape’ of the -subgroup of rhodopsin receptors.34
Short- and Long-Term Effects of the Treatment of Female and Male Rats with 33 (BAY-
2
98) and 36 (BAY-899). On the basis of the promising pharmacokinetic results obtained, we
further analyzed the effects of our SMOL LH-R antagonists in vivo. LH receptor signaling rapidly
induces specific gene expression cascades, initiates ovulation in the female and regulates
testosterone synthesis in the male. Therefore, the effects of 33 (BAY-298) both on testosterone
synthesis in male rats and ovulatory marker genes as well as progesterone plasma concentrations
in female rats were evaluated. LH-induced gene expression was studied in female rats following
treatment with cetrorelix (a competitive GnRH-R antagonist) which completely blocks the
animal’s HPG axis and thus endogenous FSH and LH synthesis and secretion. Intravenous
administration of hLH (0.5 µg/animal, 2.27 µg/kg) led to a significant increase in the mRNA levels
3
5
of known LH-dependent marker genes (i.e., COX-2, Egr-1 and RUNX-1) whereas treatment with
3, applied simultaneously iv, led to a dose-dependent decrease in their respective mRNA levels
Figure 7).
3
(
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2
2
2
2
2
2
2
3
3
3
3
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3
3
3
4
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4
4
4
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0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Suppression of hLH-induced ovarian marker gene expression in cetrorelix-treated
female rats after 2h of iv administration of hLH and 33 (BAY-298). Control animals received
either hLH or vehicle treatment only. Data obtained from 4 animals (i.e., 8 ovaries) per group.
3
6
In accordance with published data, administration of hLH also reduced the expression of
aromatase (CYP19A1) mRNA in the same animal model. This effect was counteracted by
treatment with 33 in a dose-dependent manner (Figure 8A). In addition, a marked reduction in
plasma progesterone concentrations in comparison to control animals treated with hLH only was
demonstrated (Figure 8B).
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Figure 8. (A) Dose-dependent reversal of hLH-induced suppression of aromatase gene expression
and (B) suppression of plasma progesterone levels in cetrorelix-treated female rats in the presence
of 33 (BAY-298) (iv administration of both, hLH and 33 (BAY-298) 2h bevor sample collection,
*
hLH: 0.5 µg/animal, 2.27 µg/kg). Data obtained from 4 animals (i.e., 8 ovaries) per group; p <
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.05 compared to hLH treatment.
Likewise, iv administration of hLH (0.1 µg and 0.3 µg/animal, i.e. 0.45 and 1.36 µg/kg,
respectively) led to a rapid, dose-dependent increase in plasma testosterone levels in cetrorelix-
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treated male rats 2 hours after treatment (Figure 9). These levels were significantly lowered in
the presence of 33 (BAY-298), simultaneously applied iv at 3 mg/animal (i.e., 14 mg/kg).
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Figure 9. Treatment with 33 (BAY-298) counteracts the hLH-induced increase in plasma
testosterone levels in male rats 2 hours after iv application of hLH and 33 (BAY-298). Data
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obtained from 4 animals per group; p < 0.01 compared to hLH treatment.
Taken together, the results demonstrate that treatment with 33 rapidly blocks short-term effects
of LH signaling with respect to the expression of known LH receptor induced genes and by
lowering plasma hormone levels in male and female rats in a dose-dependent manner.
Therefore, we proceeded to an investigation of long-term effects in naturally cycling female rats
upon treatment with 33. Checks for changes in the estrus cycle as well as effects on estradiol levels
were made. In contrast to a fertile cycle of 28 days in women, the estrus cycle of female rats lasts
only 4 days and can be split into four stages, all of which can be routinely monitored via the
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examination of vaginal smears (i.e., rapid follicle development (diestrus), ovulation (proestrus),
formation of corpora lutea and progesterone production (estrus) and the regression of unfertilized
corpora lutea (metestrus)). Cycle-staged rats were treated orally with 33 for 8 days and monitored
for two cycles (Figure 10). Whereas control animals cycled normally, animals receiving 33 once
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daily showed cycle arrest in diestrus and metestrus in a dose-dependent manner, in analogy to the
results obtained with LH-R knock-out mice39 and in line with known hLH-R mutations in
women.40
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Figure 10. Treatment with 33 (BAY-298) led to interrupted folliculogenesis in the 4-day cycle of
adult female rats. Cycle stages were determined by vaginal smears and characterized as follows: 1
= diestrus, 2 = proestrus, 3 = estrus, 4 = metestrus. Data are shown for the 4 animals per treatment
group on the 8 treatment days. Green color indicates that the expected cycle stage was detected
whereas red coloring shows arrested cycle stages.
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Serum estradiol levels were measured after the 8-day treatments and found to be dose-
dependently lowered compared to vehicle-treated rats in proestrus, the day of highest estradiol
concentrations (Figure 11A). Plasma concentrations on day 8 were in the range of 4-39 µM,
increased according to the different dose groups (4.5 – 72 mg/kg). Given a fraction unbound of
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.24% a plasma concentration of 10 µM results of an unbound plasma concentration of 24 nM,
which is close to the IC value of 23 nM. Therefore the obtained plasma concentrations fit to the
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general rules for dose selections, which can be summarized as follows: Based on in vitro potency,
PK data and protein binding the dosing for in vivo studies should result in free plasma
concentrations in the range of the in vitro IC values. Higher and lower doses are then selected
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for dose response studies. Collectively, these experimental data demonstrate that 33 is a potent
antagonist of LH-R-mediated physiological functions in vivo, both in the ovary and testis.
Compound 33 is equally suited for use in long-term studies addressing the physiological effects of
LH-R blockade in reproductive biology and/or pathophysiological conditions.
In an additional study, 36 (BAY-899) was tested for its ability to reduce serum estradiol levels
in intact female rats (oral application for 8 d, 12.5 mg/kg, q.d.), and showed an efficiency
comparable to 33 (BAY-298) (Figure 11B). It is also worth noting that two animals of the BAY-
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99-treated group and three animals of the BAY-298-treated cohort again showed signs in vaginal
smears of disturbed estrus cycles. However, given typically high individual variations in natural
cycling rats adding to the variability between studies in this model, results should only compared
within one experiment. Plasma concentrations on day 8 were 7 ± 2 µM for compound 33 (BAY-
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98) and 34 ± 19 µM for compound 36 (BAY-899), which is in accordance to the pharmacokinetic
properties of both compounds. Thus, 36 (BAY-899) and 33 (BAY-298) can be regarded as being
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of comparable efficacy in in vivo studies, as expected from in vitro and pharmacokinetic data. No
adverse events or undesired effects (e.g. loss of body weight or lower activity) were observed in
in vivo studies with cmpd 33 and 36.
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Figure 11. Reduction in serum estradiol levels in female rats following treatment for 8 days (q.d.,
po) with either 33 (BAY-298) (A, B) or 36 (BAY-899) (B). Data obtained from 4–5 animals per
*
**
group; p < 0.05¸ p < 0.01.
CONCLUSION
Comprehensive hit-to-lead activities led to the identification of compound 33 (BAY-298) and
chemical probe 36 (BAY-899) and their characterization as the first potent and selective LH
receptor antagonists suitable for in vivo administration. Both compounds are active at the human,
rat and cynomolgus monkey LH receptors in vitro. They act in an allosteric manner, do not
compete with the binding of agonists to the hLH-R and most likely bind to a putative rhodopsin-
like SMOL binding cavity located within the 7-transmembrane region of the hLH-R. Both
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compounds have drug-like properties. In comparison to BAY-298, BAY-899 does not inhibit
hTSH-R signaling (>100-fold selectivity), and is significantly less lipophilic with an improved
water solubility, CYP inhibition and hERG profile. In vivo pharmacokinetics of both compounds
revealed a low clearance, a high volume of distribution and a long half-life resulting in good oral
bioavailability and a high exposure.
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Compound 33 (BAY-298) demonstrated efficacy in different animal models in female and male
rats. In short-term experiments, for example, 33 dose-dependently reduced LH-induced gene
expression in the ovary and testosterone production after LH administration in male rats.
Furthermore, once daily oral application was sufficient in long-term studies in female rats to
demonstrate dose-dependent effects on plasma estradiol concentrations and the estrus cycle.
Recent pipeline news indicates an unchanged high medical need for novel anti-hormonal
treatment options. For example, Arvinas has moved ARV-110, the first androgen receptor targeted
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PROTAC, to phase 1 clinical trials, AstraZeneca is testing AZD9496, a new oral, selective
estrogen receptor degrader, in advanced breast cancers,42 and Bayer has been investigating
vilaprisan, a selective progesterone receptor modulator, in phase 3 clinical trials for the treatment
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of gynecological diseases such as endometriosis and uterine fibroids. Thus, BAY-298 (33) and
BAY-899 (36) represent valuable chemical tool compounds to study LH receptor mediated effects
on sex hormones and can be used in long-term experiments to examine the receptor’s involvement
in reproductive biology or pathophysiological conditions.
EXPERIMENTAL SECTION
Chemistry. General Methods. All reagents and solvents were used as purchased, unless
otherwise specified. All final products were at least 95% pure, as determined by UPLC.
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Compound names. ICS software was used to generate IUPAC compound names. NMR spectra
were recorded as solutions in DMSO-d or CDCl on Bruker Avance III HD spectrometers
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operating at 300 or 400 MHz for H and 101 MHz for C acquisitions. Chemical shifts () are
given in parts per million (ppm), and coupling constants (J) are in hertz (Hz). Spin multiplicities
are reported as: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. HR-MS
spectra were recorded on a MS Waters XEVO G2XS with Acquity i-Class UPLC, MS spectra on
a MS Waters SQD 2 coupled to a Acquity UPLC and optical rotations on a JASCO P2000
polarimeter.
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rac-N-(3,5-Dichlorophenyl)-4-(4-methoxyphenyl)-6,7-dihydrothieno[3,2-c]pyridine-
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(4H)-carboxamide (1). A solution of 4-(4-methoxyphenyl)-4,5,6,7-tetrahydrothieno[3,2-
c]pyridine (4; 2.00 g, 8.15 mmol; commercially available, CAS-RN: [213462-19-2]), 1,3-dichloro-
-isocyanatobenzene (1.53 g, 8.15 mmol; CAS-RN: [34893-92-0]) and triethylamine (3.4 mL) in
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DMSO (30 mL) was stirred at 70 °C overnight. The reaction mixture was poured into water and
filtered (brown slime). The residue was dissolved in EtOAc, dried over Na SO and the solvent
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was removed under reduced pressure. Column chromatography (silica gel, hexanes/EtOAc) gave
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1. Yield 920 mg (26%). H NMR (300 MHz, DMSO-d ):  = 8.96 (s, 1H, NH), 7.59 (d, J = 1.88
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Hz, 2H, dichlorophenyl), 7.35 (d, J = 5.27 Hz, 1H, thiophene), 7.06–7.14 (m, 3H, methoxyphenyl,
dichlorophenyl), 6.86 (d, J = 8.85 Hz, 2H, methoxyphenyl), 6.74 (d, J = 5.27 Hz, 1H, thiophene),
6.44 (s, 1H, CHN), 4.13–4.24 (m, 1H, CH N), 3.68 (s, 3H, OMe), 2.81–3.12 (m, 3H, CH CH ).
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C NMR (101 MHz, DMSO-d ):  = 158.7, 153.9, 143.1, 134.5, 134.3, 133.7, 133.3, 129.2, 126.4,
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23.7, 120.9, 117.5, 113.8, 55.1, 54.3, 37.8, 24.9. MS (ESI): m/z = 433.4 [M + H]+.
(4S)-(+)-N-(3,5-Dichlorophenyl)-4-(4-methoxyphenyl)-6,7-dihydrothieno[3,2-c]pyridine-
(4H)-carboxamide (1a). Obtained via HPLC separation of the enantiomers of 1 (column:
5
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