Discovery of substituted benzamides as follicle stimulating hormone receptor allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2014.03.018

Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of follicular development and maturation. Multiple injections of protein formulations are used during clinical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of assisted reproductive technologies. In order to increase patient convenience and compliance several research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines. We report here the discovery of a series of substituted benzamides as positive allosteric modulators (PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR) and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising in vitro and pharmacokinetic profiles.

Full text of DOI:10.1016/j.bmcl.2014.03.018

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2168–2172
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of substituted benzamides as follicle stimulating hormone
receptor allosteric modulators
c
b
a
a
Henry N. Yu a,b, , Thomas E. Richardson , Selva Nataraja , David J. Fischer , Venkataraman Sriraman ,
Xuliang Jiang a, Pandi Bharathi c, Robert J. Foglesong c, Thomas F. N. Haxell c, Brian H. Heasley c,
Mathew Jenks c, Jane Li a, Melanie S. Dugas a, Regina Collis a, Hui Tian a, Stephen Palmer b,
Andreas Goutopoulos a
⇑
a EMD Serono Research Institute, 45A Middlesex Turnpike, Billerica, MA 01821, United States
b TocopheRx, Inc., Suite 1077, 15 New England Executive Park, Burlington, MA 01803, United States
c Scynexis, Inc., 3501 C Tricenter Boulevard, Durham, NC 27713, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of
follicular development and maturation. Multiple injections of protein formulations are used during clin-
ical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of
assisted reproductive technologies. In order to increase patient convenience and compliance several
research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines.
We report here the discovery of a series of substituted benzamides as positive allosteric modulators
(PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa
cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR)
and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising
in vitro and pharmacokinetic profiles.
Received 3 January 2014
Revised 6 March 2014
Accepted 10 March 2014
Available online 20 March 2014
Keywords:
GPCR
PAM
Agonist
Antagonist
Gonadotropin
Glycoprotein hormone
Infertility
Reproductive
Ovary
Ó 2014 Elsevier Ltd. All rights reserved.
Gonadotropins are a family of structurally-related heterodimer
glycoprotein hormones that serve important functions in the
reproductive process. This family consists of three members: two
pituitary and one placenta hormones. Follicle stimulating hormone
(FSH) and luteinizing hormone (LH), are released from the anterior
pituitary under the control of gonadrotropin-releasing hormone
from the hypothalamus, and chorionic gonadotropin (CG) is pro-
duced in the placenta. These three gonadotropins are also structur-
ally related to another glycoprotein hormone, thyroid stimulating
hormone (TSH).1 All four glycoprotein hormones are marketed
therapeutic drugs. FSH is used clinically in women for the treat-
ment of anovulatory infertility and for controlled ovarian stimula-
tion in in vitro fertilization (IVF) and in men for hypogonadotropic
hypogonadism.2
ovarian and testicular differentiation and steroidogenesis. FSHR,
together with the other glycoprotein hormone receptors (LHR
and TSHR), are members of the G-protein coupled receptor (GPCR)
superfamily. The superfamily transduces extracellular signals into
G
protein activation via their seven-helical transmembrane
(7TM) domain.3 Ligand-stimulated FSHR leads to the activation of
a cascade of kinases in a cAMP-dependent manner. Glycoprotein
hormone receptors are unique among 7TM GPCRs for their large
ectodomains at the N-terminus and short cytoplasmic tails at the
C-terminus. While the nature of hormone–receptor interaction
has been mapped and the structure of FSH binding to the ectodo-
main of FSHR has been solved,4–6 the crystal structure of the
full-length FSHR, however, has not been available. Consequently,
atomic details about how the protein hormone activates the 7TM
domain remain unknown.
FSH exercises its physiological function by acting on its receptor
(FSHR) on specific gonadal somatic cells that leads to initiation of
Although FSH is highly effective and has been heralded as a use-
ful drug for couples who undergo fertility treatment,7 more than
two-thirds of infertile women discontinue infertility treatments
before FSH therapy is initiated. The number of patients dropping
out of fertility treatment can be as high as 70%.8 FSH, a protein mol-
⇑
Corresponding author. Tel.: +1 978 294 1422.
E-mail address: henry.yu@emdserono.com (H.N. Yu).
http://dx.doi.org/10.1016/j.bmcl.2014.03.018
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

H. N. Yu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2168–2172
2169
O
N
North
O
HN
HN
O
N
O
East
West
N
1
Figure 2. Schematic illustration of the SAR exploration of different regions of the
compound series.
Figure 1. Chemical structure of compound 1.
Synthesis of 1, depicted in Scheme 1, is the general synthetic
scheme used for derivatives.29 Displacement of the aryl fluoride
of 2 with piperazine 3 in an SNAr reaction followed by hydrogena-
tion of the nitro group of 4 rendered aniline 5 in an overall yield of
35%. Acylation of 5 with furan-2-carbonyl chloride followed by
amide coupling of 6 afforded 1 in 50% yield.
In order to investigate the structure-activity relationship (SAR)
around the central aromatic ring, analogs were prepared in a fash-
ion similar to Scheme 1. Efficacy of these derivatives to stimulate
cAMP in CHO-hFSHR cells was monitored. We first explored the
SAR in the northern part (Fig. 2) of the molecule (Table 1). The fur-
an regio-isomer (7a) was less potent than compound 1. Blocking
ecule, must be administrated daily or twice daily by injections for
two weeks. Although the convenience of FSH injections have been
improved with long lasting versions of FSH,9–12 women report the
daily injections are unbearable, and are one of the main reasons
that patients who seek fertility treatment decide to drop out.13
Small molecule drugs, on the other hand, are advantageous, as they
can be taken orally.14 Thus, there is an unmet medical need for bio-
available FSH mimetics that would be more convenient for the pa-
tients and potentially improve their treatment compliance. To
date, several chemical series of low-molecule-weight (LMW) FSH
mimetics have been reported.15–21 Two have been demonstrated
by mutagenesis studies to bind the 7TM domain of FSHR,19,22 but
a defined domain of the receptor where the small molecules binds
is unknown and awaits structural studies of the small-molecule/
FSHR complex. In addition to being developed for drug candidates,
LMW ligands have been useful tools for obtaining GPCR crystals23
and deciphering the target disulfide pairs.24 As biased signaling is a
general phenomenon in GPCR functions, including FSHR actions,25
it is advantageous to have several chemical series available for elu-
cidation of the FSHR mechanism and for the ‘holy grail’ of drug
development targeting FSHR.26 Here we report another chemical
series of small molecules targeting FSHR.
The substituted benzamide 1 (Fig. 1) was identified as a prom-
ising starting point following screening of a small molecule library.
High-throughput screening was performed using a Chinese ham-
ster ovarian (CHO) cell line expressing the recombinant human
FSH receptor (CHO-hFSHR) in the presence of an EC20 concentra-
tion of FSH. This concentration of FSH corresponds to the basal lev-
els of hormone present in the circulation of a great majority of
women seeking infertility treatments.27 Cyclic adenosine mono-
phosphase (cAMP), the second messenger stimulated by FSH, was
measured as the readout in this assay.28 These compounds were
also counter-screened in CHO cells expressing human TSHR
(hTSHR), human LHR (hLHR) and parental CHO cells. Compounds
specific for hFSHR were selected. The EC50 of compound 1 in the
CHO-hFSHR assay is 164 nM.
the N–H with a methyl group rendered ꢀ8.8
lM potency in analog
7b, which indicates that the hydrogen donor is critical for the po-
tency. Sulfonamide replacement of the amide gave little agonist
activity (7c). 5-Methyl furan analog (7d) retained potency, but its
isomer 7e was reduced in potency by about three fold. Replace-
ment of the furan with other five-member rings (7f, 7g, 7h, 7i) gave
a range of results, while 2-cyclopropyloxazole (7h) provided the
most promising compound (EC50 = 99 nM). Interestingly, the sim-
ple cyclopropane analog (7j) maintained potency of 199 nM. The
phenyl analog (7k) showed a reduced potency (554 nM). Replace-
ment of the aryl ring in 7k with a 2-pyridine (7i) retained most of
the potency (184 nM). The importance of the N position in the pyr-
idine ring is shown by little agonist activity shown in the two pyr-
idine isomers (7m and 7n).
The SAR in the western region of compound 1, maintaining
either a furan or a 2-cyclopropyloxazole in the northern section,
quickly demonstrated a steep SAR around the piperazine phenyl
moiety (Table 2). A minor change of the 2-methyl group resulted
in a loss of potency. For instance, substitution of the 2-methyl with
an electron withdrawing group 2-CF3 (8a) leads to a 6-fold de-
crease in EC50. The electron donating group 2-OMe (8b) analog dis-
played little agonist activity. Isomers of o-tolyl showed reduced
potencies as well, with an EC50 of 1.4 lM for the m-tolyl analog
(8c). The p-tolyl analog (8d) loses most of its agonist activity,
and replacement of the o-tolyl group with 2-pyridine (8f) or H
N
NH
O2N
H2N
O2N
3
F
N
CO2H
CO2H
N
N
N
CO2H
a
b
4
5
2
furan-2-carbonyl
chloride
O
HN
O
O
H2N
N
COCl
O
1
CO2H
N
N
c
d
6
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 36%; (b) Pd/C, H2, EtOH/MeOH, 94%; (c) Et3N, DMAP, DCM, 56%; (d) HOBt, EDC, DCM, 84%.

2170
H. N. Yu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2168–2172
(8e) showed a similar result. Replacement of the piperazine with
piperidine (8g) resulted in a loss of activity. Due to either steric
or electronic perturbation within the binding pocket, we were un-
able to improve on the piperidine phenyl moiety despite over one
hundred analogs that were synthesized and screened. It is interest-
ing to note that the 2-methyl piperidine (8h) analog showed an
To explore the SAR at the eastern region systematically, we syn-
thesized and tested additional compounds (Table 3). A constraint
approach to the propyl linker (9a) led to loss of activity; however,
shorter linkers, such as 9b, 9d, 9e, 9k, proved to be more tolerated,
and so did the replacement of the pyrrolidin-2-one with other five-
(9e, 9f, 9h, 9i, 9j) or six-member rings (9g and 9k). This indicates
that this region of the protein binding pocket accommodates dif-
ferent fragments although it is still critical for potency, as the com-
plete removal of the pyrrolidin-2-one (9c) shifted the EC50 to
antagonistic activity (À36%) at a concentration of 50
lM, suggest-
ing a switch from an agonist to an antagonist with a small struc-
ture change. Although the assay format is not designed to
identify antagonists, it is not an uncommon finding that GPCR allo-
steric modulators can switch from agonists to antagonists through
substitution.30–32
1.8
methylene, 9l switches to a potential antagonist, with -91% activity
at 50 M. Similar to 8h above, a proper assay format is needed to
lM. More interestingly, when the carbonyl was replaced with
l
validate the antagonist property of the compound. An FSHR antag-
onist has the potential to be a novel, non-steroidal contraception
treatment as well as a useful tool for characterization of the full-
length crystal structure of FSHR. We are currently carrying out
more research on the antagonist mode with 9l, 8h and their related
analogs. These results will be disclosed in future publications.
Compound 1 was tested in in vitro rat microsomal stability
assays15 and it showed high rates of clearance in human and rat
Table 1
R
Y
X
HN
N
N
O
N
O
microsomes at 312
ll/min/mg and 233 ll/min/mg, respectively.
Likewise, an in vitro metabolic liability was observed in 7h, with
Clint values in human and rat microsomes of 260
l
l/min/mg and
a
Compound
X
Y
R
EC50 (nM)
7a
NH
C(O)
494
Table 2
O
O
R1
O
7b
7c
NMe
NH
C(O)
8820
HN
HN
R2
O
S(O)2
O
10%@12b
129
lM
N
O
a
Compound
R1
R2
EC50 (nM)
7d
NH
C(O)
O
N
N
O
8a
1001
7e
7f
NH
NH
C(O)
C(O)
O
508
N
CF3
N
N
N
N
4900
HN
20%@50b
1440
l
M
O
8b
8c
O
O
7g
7h
NH
NH
C(O)
C(O)
250
99
O
O
N
N
N
O
N
N
N
8d
13%@50b
20%@50b
l
M
M
S
7i
NH
C(O)
184
N
N
N
8f
l
O
O
O
N
7j
NH
NH
C(O)
C(O)
199
554
HN
N
8e
8g
1%@50b
1%@50b
lM
7k
N
N
7l
NH
C(O)
185
lM
N
7m
7n
NH
NH
C(O)
C(O)
37%@12b
34%@12b
l
M
M
O
N
N
8h
À36%@50b
lM
N
N
l
a
a
Values are means of at least two experiments.
% Response of the compounds compared to maximal FSH response.
Values are means of at least two experiments.
% Response of the compounds compared to maximal FSH response.
b
b

H. N. Yu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2168–2172
2171
Table 3
R1
O
HN
A
B R2
N
N
a
Compound
R1
A
B
R2
EC50 (nM)
Microsomal stability (human, rat)
l/min/mg protein)
(l
O
9a
9b
9c
O
O
C(O)
C(O)
C(O)
B + R2
NH
NH
=
40%@12b
30
lM
N
N
O
N
NH2
O
O
1850
9d
C(O)
NH
46
54, 31
O
NH2
9e
9f
O
O
C(O)
C(O)
NH
NH
129
88
O
O
N
9g
9h
9i
C(O)
C(O)
C(O)
NH
NH
NH
153
28
N
O
O
O
59, 25
33, 38
N
N
O
O
184
O
N
O
O
O
NH
N
N
9j
C(O)
C(O)
CH2
NH
NH
NH
89
20, 18
72, 13
N
N
N
O
O
9k
9l
53
O
N
4.5%@12b
lM
a
Values are means of at least two experiments.
% Response of the compounds compared to the maximal FSH response.
b
225
l
l/min/mg, respectively. Improved in vitro metabolic stability
partial-agonist modulators could be used to selectively target spe-
cific signaling pathway of FSHR and have the potential for better
control and prevention of unwanted effects like ovarian hyper-
stimulation in IVF treatment.30 Interestingly, the compounds re-
quired EC20 FSH concentration for stimulating the primary granu-
losa cells, thus behaving as PAM. Thus, women undergoing
infertility treatment in the clinic have basal levels of FSH,27 so
the compounds can potentiate this low level of FSH to achieve
the desired clinical effect. Both compounds were advanced into
was achieved with substitution of the pyrrolidin-2-one moiety
(Table 2, 9d, 9h–9k).
Compound 9j and 9k were chosen to be further profiled in a rat
granulosa cell assay33 and evaluated for in vivo pharmacokinetics
(Table 4). Both compounds induced estradiol (E2) production in
the rat granulosa cells with EC50 values of 170 nM and 5 nM,
respectively. Both compounds achieved an Emax ꢀ80% as compared
to the maximum normalized FSH response of 100%. These

2172
H. N. Yu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2168–2172
Table 4
In vitro and in vivo data for selected compounds
Compound
Rat granulosa cell EC50
,
Immature rat pharmacokinetic parametersb
(% efficacy compared to FSH)a
F%
CL (mL/min/kg)
T1/2
Vdss (L/Kg)
9j
9k
170 nM (78%)
5 nM (81%)
21
23
13.2
3.41
1.95
4.4
5.46
1.65
a
Values are the means from at least two experiments.
Clearance (CL), and volume of distribution were generated following a 0.2 mg/kg iv dose in immature Sprague-Dawley rats. Oral bioavailability (%F) and half-life (T1/2
b
)
were determined following a 0.5 mg/kg po dose in immature Sprague-Dawley rats.
17. Pelletier, J. C.; Rogers, J.; Wrobel, J.; Perez, M. C.; Shen, E. S. Bioorg. Med. Chem.
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13226.
PK studies. The in vivo PK study showed that both compounds have
a distinct PK profile; compound 9j exhibited high clearance and a
short half-life, while 9k had medium clearance and a longer half-
life. The additional advantage of 9j was the aqueous solubility
(160 lM at pH 7.4). In addition, both compounds showed
significant in vitro selectivity over the related luteinizing hormone
receptor (LHR) (>1000-fold selectivity) and thyrotropin receptor
(TSHR) (>1000-fold selectivity). Short acting FSHR compounds with
surge-like PK properties have been reported to have an inhibitory
effect in an animal model;34 thus molecules with distinct PK pro-
files make both compounds valuable research tools in animal mod-
el studies. Both compounds are being advanced into PK/PD studies
via various routes of administration. The complicated in vivo bio-
logical result will be reported in due course.35
20. van Straten, N.; Timmers, M. Annu. Rep. Med. Chem. 2009, 44, 171.
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H. J.; van Achterberg, T. A.; van Amstel, M. G.; Brands, J. H.; van Doornmalen, E.
J.; Wat, J.; Mulder, S. J.; Raafs, B. C.; Verkaik, S.; Hanssen, R. G.; Timmers, C. M.
Biochem. Pharmacol. 2013, 85, 1162.
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In summary, we have identified a series of benzamides from
high-throughput screening, represented by compound 1, as FSHR
PAMs. Development of these novel FSHR PAMs was prepared using
compound 1 as a starting point for SAR development. Although the
western region had a particularly steep SAR, the western side
chains were useful in modulating both potency and microsomal
stability. Compounds 9j and 9k displayed good potency on CHO
cells expressed with human FSHR and also in primary granulosa
cell assays. These partial agonist modulators have the potential
for better control and prevention of ovarian hyper-stimulation in
IVF treatment. Compounds 9j and 9k had acceptable PK properties
and are candidates for future PK/PD studies.
27. de Ziegler, D.; Fraisse, T.; de Candolle, G.; Vulliemoz, N.; Bellavia, M.;
Colamaria, S. Reprod. Biomed. Online 2007, 15, 507.
28. In Cho-hFSHR-cells (2500 cells/well) were plated in 5 ll of phenol red free
DMEM/F12 + 1% FBS. Cells were plated in 384 well, solid white low volume
plates (Greiner 784075) by Multidrop. Varying concentrations of stimulants
(FSH and compounds with 200 lM IBMX) was added in 5 ll of media.
Compounds were generally stimulated in the presence of EC20 FSH (0.265 pM).
The plates were incubated at 37 °C for 1 h. Following stimulation, cAMP
produced was measured using HTRF reagents (CisBio #62AM4PEC), as
described by the manufacturer. In brief, 10 ll of mixed HTRF reagents were
added per well and incubated at room temperature for 1 h. The plates were
read on EnVisionmultilabel plate reader (Perkin Elmer, CA). The readout was
the calculated fluorescence ratio (665 nm/620 nm). Dose response curves for
FSH and compounds were generated using Genedata (Genedata, Basel,
Switzerland). EC50 value and
maximal FSH were calculated.
% response of the compounds compared to
29. Yu, H.; Li, J.; Richardson, T. E.; Bharathi, P.; Heasley, B.; Goutopoulos, A. US
20130023522, 2013.
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dilution of compound starting 10 lM at 8 different concentrations was tested.
At 24 h after treatment supernatant was collected and the estradiol
concentration was determined by ELISA (DRG, Mountainside, NJ). The optical
densities of the ELISA were measured by Spectramax, (Sunnyvale, CA). The
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