Substituted terphenyl compounds as the first class of low molecular weight allosteric inhibitors of the luteinizing hormone receptor

Article abstract of DOI:10.1021/jm801561h

The luteinizing hormone (LH) receptor plays an important role in fertility and certain cancers. The endogenous ligands human chorionic gonadotropin (hCG) and LH bind to the large N terminal domain of the receptor. We recently reported on the first radiola

Full text of DOI:10.1021/jm801561h

2036
J. Med. Chem. 2009, 52, 2036–2042
Substituted Terphenyl Compounds as the First Class of Low Molecular Weight Allosteric
Inhibitors of the Luteinizing Hormone Receptor
Laura H. Heitman,* Rajeshwar Narlawar, Henk de Vries, Milou N. Willemsen, Dieter Wolfram, Johannes Brussee, and
Adriaan P. IJzerman
DiVision of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden UniVersity, P.O. Box 9502,
2300 RA Leiden, The Netherlands
ReceiVed December 10, 2008
The luteinizing hormone (LH) receptor plays an important role in fertility and certain cancers. The endogenous
ligands human chorionic gonadotropin (hCG) and LH bind to the large N terminal domain of the receptor.
We recently reported on the first radiolabeled low molecular weight (LMW) agonist for this receptor, [3H]Org
43553, which was now used to screen for new LMW ligands. We identified a terphenyl derivative that
inhibited [3H]Org 43553 binding to the receptor, which led us to synthesize a number of derivatives. The
most potent compound of this terphenyl series, 24 (LUF5771), was able to increase the dissociation rate of
[3H]Org 43553 by 3.3-fold (at 10 µM). In a functional assay, the presence of 24 resulted in a 2- to 3-fold
lower potency of both Org 43553 and LH. Thus, the compounds presented in this paper are the first LMW
ligands that allosterically inhibit the LH receptor.
Introduction
Recently, we reported on the first radiolabeled LMW agonist
for the LH receptor, [3H]Org 43553.8 Here, we used this
radioligand to screen for new LMW ligands at the LH receptor.
Initially, 50 compounds were screened for displacement of
[3H]Org 43553. Subsequently, the same library was screened
in a kinetic radioligand binding assay, where a change in
dissociation rate is indicative for allosteric modulation of the
radioligand used.9 The latter resulted in a few hits including
the terphenyl compound 4, which we anticipated to be an
allosteric inhibitor as it increased the dissociation rate of [3H]Org
43553. Subsequently, several analogues of 4 were synthesized
and tested for their effect on the dissociation rate of [3H]Org
43553. This yielded an even more potent allosteric inhibitor,
compound 24, which was further characterized in radioligand
dissociation experiments and functional assays. As a conse-
quence, the present study is the first to report LMW allosteric
inhibitors of the LH receptor.
The luteinizing hormone (LH) receptor is a member of the
glycoprotein hormone receptor family within the class A subfamily
of G protein-coupled receptors (GPCRsa).1 While most class A
GPCRs recognize low molecular weight (LMW) endogenous
ligands that bind in the seven transmembrane (7-TM) domain, the
LH receptor has two high molecular weight endogenous ligands,
human chorionic gonadotropin (hCG) and LH. Both hormones bind
with high affinity and selectivity to the N terminus of the LH
receptor and thereby activate the receptor.2 In the clinic, these so-
called gonadotropins are currently used in infertility treatment. The
hormones need to be administered by parenteral (subcutaneous or
intramuscular) injection.3
To increase patient convenience and compliance, efforts are
made to develop nonpeptide orally active gonadotropins as drugs.
For the LH receptor only a few compound classes have been
described as LMW receptor agonists, such as the thienopyrimidine
Org 43553. This compound was shown to have in vivo efficacy
upon oral administration.4 Recently the first high molecular weight
antagonist was reported for the LH receptor, two fused beta-
subunits of hCG.5 However, LMW antagonists have not been
reported so far.6 Antagonists for the LH receptor may be novel
contraceptive agents. In addition, antagonists could be used against
ovarian cancer related to menopause.7 Therefore, next to LMW
agonists, antagonists would be very beneficial as well.
Results and Discussion
Chemistry. A series of symmetrical and unsymmetrical
terphenyl carbamates was synthesized as depicted in Scheme
1. The synthesis was started from commercially available 3,5-
dibromophenol 5. The microwave assisted Suzuki-Miyaura
cross coupling of phenol 5 with phenylboronic acid and
substituted phenylboronic acid using the catalyst [(Ph3P)4]Pd
gave terphenylphenols 6-10.10 These compounds (6-10) were
then treated with various isocyanates and Et3N in anhydrous
dichloromethane to furnish the terphenyl carbamates (4, 16-25,
35, 37, 39, 41). The esterification of terphenylphenol 6 was
achieved using EDAC and HOBt to afford the cyclopentyl ester
26. The unsymmetrical terphenylphenols (12-15) were syn-
thesized via the sequential Suzuki-Miyaura cross coupling of
phenol 5 with phenylboronic acid followed by substituted
phenylboronic acid. The intermediates were treated with cy-
clopentyl isocyanate and Et3N in anhydrous dichloromethane
to afford the terphenyl carbamates (34, 36, 38, 40).
* To whom correspondence should be addressed. Phone: +31-(0)71 527
4558. Fax: +31-(0)71 527 4565. E-mail: l.h.heitman@lacdr.leidenuniv.nl.
a Abbreviations: BSA, bovine serum albumin; cAMP, cyclic adenosine-
5′-monophosphate; CHO, Chinese hamster ovary; CHOhLHr_luc, human
LH receptor and luciferase reporter gene transfected in CHO cells; CRE-
luc, cAMP-response-element luciferase reporter gene; DCM, dichlo-
romethane; DMF, dimethylformamide; EDAC, N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide hydrochloride; EDTA, ethylene diamine tetraacetic
acid; EtOAc, ethyl acetate; GPCR, G protein-coupled receptor; HOBt,
1-hydroxybenzotriazole; log D, logarithm of octanol-water distribution
coefficient; Org 43553, 5-amino-2-methylsulfanyl-4-[3-(2-morpholin-4-yl-
acetylamino)-phenyl]-thieno[2,3-d]pyrimidine-6-carboxylic acid tert-buty-
lamide; (Ph3P)4Pd, tetrakis(triphenylphosphine)palladium; PBS, phosphate-
buffered saline; rec-hCG, recombinant human chorionic gonadotropin;
recLH, recombinant luteinizing hormone; SEM, standard error of the mean;
TLC, thin-layer chromatography.
Terphenyl amide derivative 31 and terphenyl urea derivative
32 were synthesized as outlined in Scheme 2. Deamination of
commercially available 2,6-dibromo-4-nitroaniline 27 was
10.1021/jm801561h CCC: $40.75
2009 American Chemical Society
Published on Web 03/18/2009

LMW Allosteric Inhibitors of the LH Receptor
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2037
Scheme 1. Synthetic Route to Compounds 4, 6, 16-26, 34-41a
Figure 1. Chemical structures of the first hits (1-4) resulting from
screening in the [3H]Org 43553 dissociation assay. The presence of 10
µM of compounds 1-4 resulted in 42, 16, 29 and 79% enhanced
radioligand dissociation compared to control conditions, respectively.
a Reagents and conditions: (a) PhB(OH)2, aq Na2CO3, toluene, MW, 10
min, 70-90%; (b) EDAC, HOBt, cyclopentanecarboxylic acid, Et3N,
CH2Cl2, RT, 24 h, 40%; (c) R5NCO, Et3N, CH2Cl2, RT, overnight, 70-90%;
(d) PhB(OH)2, aq Na2CO3, toluene, reflux, 3 h, 30%; (e) substituted
phenylboronic acid, aq Na2CO3, toluene, MW, 10 min, 35-52%; (f) R5NCO,
Et3N, CH2Cl2, RT, overnight, 60-82%.
diphenylnitrobenzene 29. The nitro group was reduced using
anhydrous SnCl2 in EtOH at 70 °C to afford amine 30, which
subsequently was treated with cyclopentanecarboxylic acid, ethyl
chloroformate and Et3N in anhydrous dichloromethane to afford
the amide 31. Terphenyl urea 32 was synthesized from amine
30 using cyclopentyl isocyanate and Et3N in anhydrous dichlo-
romethane. Biphenyl carbamate 33 was synthesized by treating
commercially available 3-phenylphenol with isopentyl isocy-
anate and Et3N in anhydrous dichloromethane.
Scheme 2. Synthetic Route to Compounds 30-33a
Structure-Activity Relationships. In an initial screen, 50
diverse low molecular weight compounds were tested for their
ability to either increase or decrease the dissociation rate of
[3H]Org 43553 from the human LH receptor stably expressed
on CHO cell membranes. Most compounds did not change the
dissociation rate compared to control conditions. However, some
hits were obtained that significantly increased the dissociation
rate of the radioligand, indicative for allosteric inhibition. In
Figure 1, four of these hits (1-4) are depicted that showed some
resemblance in their chemical structures. The presence of these
compounds resulted in an increase of the dissociation by 42,
16, 29 and 79% when compared to the dissociation of [3H]Org
43553 by unlabeled Org 43553 alone, respectively.
Subsequently, the structure-activity relationships around
compound 4 were further explored for two reasons: (1) this was
the most potent allosteric inhibitor (79% increased dissociation
compared to control), and (2) compounds 1-3 have been
reported as adenosine receptor antagonists, where 1 and 2
showed low affinity for the A1 receptor subtype,12,13 while 3
displayed nanomolar affinity for both the A1 and A2A receptor.14
Notably, 4 did not show any affinity for the adenosine receptor
subtypes (data not shown). Compound 4 also caused a displace-
ment of [3H]Org 43553 in equilibrium radioligand binding
studies (see also Table 1). This probably results from noncom-
petitive (allosteric) inhibition, as shown for other allosteric
inhibitors, e.g. 5-(N,N-hexamethylene)amiloride (HMA) on the
human GnRH receptor.15
Analogues of 4 were synthesized for further exploration of the
structure-affinity relationships of this prototype allosteric inhibitor
of the luteinizing hormone receptor; their behavior in radioligand
binding studies is reported in Tables 1 and 2. First the phenyl
carbamic acid of 4 was removed yielding a phenol analogue (6)
that had low modulating potency and did not cause any displace-
ment. Addition of a 4-chloro (16) or 4-methoxy (17) also resulted
in a lower modulating potency. Apparently substitution of the
phenyl is not tolerated in that binding pocket. In addition, the
a Reagents and conditions: (a) NaNO2, H2SO4, CH3COOH, CuO, EtOH,
overnight, 30%; (b) (Ph3P)4Pd, PhB(OH)2, aq Na2CO3, toluene, reflux, 16 h,
42%; (c) anhydrous SnCl2, EtOH, 70 °C, 30 min, 40%; (d) cyclopentan-
ecarboxylic acid, EtOCOCl, Et3N, CH2Cl2, RT, 4 h, 50%; (e) cyclopentyl
isocyanate, Et3N, CH2Cl2, RT, overnight, 70%.
achieved using NaNO2 and CuO to yield 3,5-dibromonitroben-
zene 28.11 The Suzuki-Miyaura cross coupling of 28 with
phenylboronic acid using the catalyst [(Ph3P)4]Pd gave 3,5-

2038 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Heitman et al.
Table 2. Displacement and Allosteric Modulation of [3H]Org 43553
Binding at the Human Luteinizing Hormone Receptor by 10 µM
Concentrations of Compounds 33-41
Table 1. Displacement and Allosteric Modulation of [3H]Org 43553
Binding at the Human Luteinizing Hormone Receptor by 10 µM
Concentration of Compounds 4, 6, 16-26, 30-32
% allosteric
% displacementa inhibitionb
compd
R1
R2
phenyl
4-Cl-phenyl
4-Cl-phenyl
33
H
81 (77/84)
77 (71/82)
55 (50/60)
69 (64/71)
7 (6/8)
75 (70/79)
71 (65/77)
ndc
64 (57/72)
nd
34 phenyl
35 4-Cl-phenyl
36 phenyl
37 3,4-diCl-phenyl 3,4-diCl-phenyl
38 phenyl 4-MeO-phenyl
39 4-MeO-phenyl 4-MeO-phenyl
3,4-diCl-phenyl
67 (58/76)
17 (7/26)
72 (67/74)
62 (58/66)
83 (82/83)
31 (27/34)
102 (97/106)
59 (57/61)
40 phenyl
41 4-Me-phenyl
4-Me-phenyl
4-Me-phenyl
a % displacement of specific [3H]Org 43553 binding from human
luteinizing hormone receptors stably expressed on CHO-K1 cell membranes
at 10 µM concentrations (n ) 2, duplicate). b % enhanced dissociation
[3H]Org 43553 from human luteinizing hormone receptors stably expressed
on CHO-K1 cell membranes in the absence (control; 0%) or presence of
10 µM of the compounds (n ) 2, duplicate). c Not determined.
carbamic acid was substituted with several alkyls, where an ethyl
(18) and isopropyl substituent (22) were less potent than 4, but
compounds bearing a propyl (19), butyl (20) or pentyl (21)
substituent were equally potent in modulating [3H]Org 43553
binding. Remarkably, compound 18 was not able to displace the
radioligand. The first compounds that showed an increased potency
possessed a tert-butyl (23) or cyclopentyl (24) substituent. The latter
compound was able to increase the dissociation of [3H]Org 43553
by 88%. In addition, 10 µM 24 caused a similar amount of
displacement. The more bulky cyclohexyl group (25) resulted in a
loss of potency, which indicated some steric hindrance in the
binding pocket. Then the carbamic acid linker between the
cyclopentyl and the terphenyl scaffold was examined. Replacement
with an ester (26) or amide (31) resulted in a moderate potency.
To examine whether this was due to the length of the linker, a
urea derivative was introduced (32). This compound had only a
slightly decreased potency indicating that at least the length or the
location of the nitrogen in the linker was important. The free amine
(30) did not increase the dissociation rate, which was similar to
the hydroxyl (6) analogue.
It follows from Table 1 that a carbamate linker with a
cyclopentyl substituent results in the most potent allosteric inhibitor.
Therefore, other analogues were prepared where the terphenyl
scaffold was substituted (Table 2). First, one of the phenyl rings
was removed (33), still yielding an allosteric inhibitor, but much
less lipophilic (Figure 2). Apparently, the third phenyl ring is not
a prerequisite for high modulating potency. Subsequently, either
one or both phenyl rings were substituted. As the molecule has a
symmetry axis, it does not matter where the single substituent is
introduced. Introduction of one 4-chloro (34) results in a slightly
reduced potency that is reflected in the displacement and modulat-
ing potency. Substitution of the second phenyl ring with a 4-chloro
atom (35) decreases its ability to displace the radioligand even
further. The initial concentration of a compound in a dissociation
assay is 10-fold higher than in a displacement assay. For certain
compounds (35 and 37) this resulted in solubility problems, and
their modulating potency could therefore not be determined.
However, extrapolation of the other results indicates that the
modulating potency of 35 will probably also be decreased. A
similar observation was done for the single (36) and double 3,4-
dichloro (37) substituted compounds. Compound 36 had an
a % displacement of specific [3H]Org 43553 binding from human
luteinizing hormone receptors stably expressed on CHO-K1 cell membranes
at 10 µM concentrations (n ) 2, duplicate). b % enhanced dissociation of
[3H]Org 43553 from human luteinizing hormone receptors stably expressed
on CHO-K1 cell membranes in the absence (control; 0%) or presence of
10 µM concentrations of the compounds (n ) 2, duplicate).

LMW Allosteric Inhibitors of the LH Receptor
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2039
Table 3. Dissociation (koff) Rate Constants of [3H]Org 43553 in the
Presence of Buffer (Control) or 1 µM or 10 µM 24a
b
condition
koff (min-1
)
shiftc
control
+1 µM 24
+10 µM 24
0.026 ( 0.0007
0.054 ( 0.004
0.087 ( 0.002
2.1
3.3
a Values are means ((SEM) of three separate assays performed in
duplicate. b The values of the kinetic dissociation rate constants were
obtained by analysis of the exponential dissociation of [3H]Org 43553 bound
to human luteinizing hormone receptors. c The shift is defined as the ratio
of koff values in absence (control) and presence of compound 24, respec-
tively.
Figure 2. Log D of compounds 1-4, 6, 16-26, 34-41 plotted against
their allosteric effect on [3H]Org 43553 binding. Analysis of the plot
by linear regression resulted in a poor correlation (r2 ) 0.05791).
Compounds 35 and 37 did not dissolve in the methanol-based eluent
and are therefore not included in the graph.
intermediate modulating potency, while that was almost completely
lost for 37 (based on the displacement values due to solubility
problems). The first substituted compound that did not lose a
significant amount of potency was compound 38 with a 4-methoxy
group. A second 4-methoxy substituent (39), however, resulted in
a substantial loss of potency. Introduction of a smaller 4-methyl
substituent (40) resulted in a significant gain of modulating potency,
to the extent that in the presence of 40 the radioligand had fully
dissociated from the receptor after 30 min in comparison to control
conditions. Double substitution with a 4-methyl group (41) resulted
in a loss of potency. In short, although a single substituent only
results in a small loss or even a gain of potency, double substitution
resulted in a significant loss of potency when compared to
compound 24. Apparently, only one of the pockets that accom-
modate a phenyl ring has some space for a substituent, where the
smallest substituent, a 4-methyl (40), results in the highest potency.
Due to the terphenyl scaffold, these ligands are highly
lipophilic and hard to dissolve in an aqueous buffer at high
concentrations. Therefore, the log D of these compounds was
determined to assess whether a correlation existed between the
lipophilicity (log D) of these compounds and their modulating
behavior (Figure 2), which could indicate a nonspecific effect.
However, a poor correlation between the log D and the allosteric
effect was found for the terphenyl compounds. The observed
effect is therefore most probably truly LH receptor-mediated
(see also the functional assays described below). This selectivity
was further corroborated by the observation that compound 24
did not allosterically modulate another class A GPCR, the
adenosine A3 receptor (data not shown).
Further studies were undertaken to investigate the pharma-
cological characteristics of these novel allosteric inhibitors of
the LH receptor. Based on the results of the first series of
compounds (Table 1), the kinetic behavior of [3H]Org 43553
was studied by performing full dissociation experiments in the
absence (control) and presence of 1 µM or 10 µM 24 (Table 3
and Figure 3). The dissociation rate of the radioligand obtained
by the addition of unlabeled ligand alone was 0.026 ( 0.0007
min-1. This was slightly higher than the rate reported previously
(0.021 min-1),8 due to the presence of a higher concentration
of DMSO (also applied in the control experiment) that was
necessary to dissolve these highly lipophilic compounds. In the
presence of 1 µM 24 the dissociation rate was increased 2.1-
fold to 0.054 ( 0.004 min-1. In addition, allosteric inhibition
by 24 was concentration-dependent, as the presence of 10 µM
of 24 increased the dissociation even further, by 3.3-fold (Table
3). As shown in the single point experiments 24 also displaced
[3H]Org 43553 in an equilibrium binding experiment by 91%
Figure 3. (a) Dissociation kinetics of [3H]Org 43553 binding to human
luteinizing hormone receptors stably expressed on CHO-K1 cell
membranes. Dissociation was initialized by the addition of 10 µM Org
43553 mixed with buffer (control) or 1 µM or 10 µM (final concentra-
tions) of 24. Representative graphs are shown from one experiment
performed in duplicate (see Table 3 for kinetic parameters). (b)
Dissociation kinetics of 125I-hCG binding to human luteinizing hormone
receptors stably expressed on CHO-K1 cell membranes. Dissociation
was initialized by the addition of 70 nM recLH mixed with buffer
(control) or 10 µM (final concentrations) Org 43553 or 24. In addition,
dissociation was initialized by infinite dilution (i.d.) in absence (control)
or presence of 70 nM recLH, 10 µM Org 43553 or 24. The amount of
125I-hCG dissociation was determined after 4 h, where total binding
(at t ) 0) was set to 0% dissociation. Values are means ((SEM) of at
least two separate assays performed in triplicate (* p < 0.05, ** p <
0.005, *** p < 0.001 versus control).
at 10 µM (Table 1). Therefore, displacement of [3H]Org 43553
equilibrium binding at different concentrations of 24 was
determined (Figure 4). The obtained inhibition curve was best
described by a one-site receptor model and yielded an IC50 value
of 2.3 ( 0.4 µM with a pseudo-Hill coefficient of 1.1 ( 0.06.

2040 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Heitman et al.
Table 4. Receptor Activation by recLH, rec-hCG or Org 43553 in the
Presence or Absence of 10 µM 24, Expressed as EC50 and Emax Valuesa
activity in luciferase assayb
compound
EC50 (nM)
shiftc
Emax (%)d
recLH
+10 µM 24
Rec-hCG
+10 µM 24
Org 43553
+10 µM 24
0.056 ( 0.008
0.15 ( 0.02**
0.097 ( 0.01
0.33 ( 0.05*
1.9 ( 0.04
100 ( 1
108 ( 4
74 ( 2
69 ( 3
85 ( 4
92 ( 5
2.7
3.4
2.4
4.6 ( 0.8*
a Values are means ((SEM) of at least three separate assays performed
in duplicate (* p < 0.05, ** p < 0.005 versus control). b cAMP-mediated
luciferase activity in CHO-K1 cells that stably express the human luteinizing
hormone receptor and CRE-luciferase reporter gene. c The shift is defined
as the ratio of EC50 values in the presence or absence of 24, respectively.
d Maximal effect of either recLH or Org 43553 in the absence or presence
of 10 µM 24, where recLH in the absence of 24 was set at 100%.
Figure 4. Displacement of [3H]Org 43553 from human luteinizing
hormone receptors stably expressed on CHO-K1 cell membranes by
24. The IC50 value was 2.3 ( 0.4 µM. Representative graph is shown
from one experiment performed in duplicate.
The question arose if the LH receptor could possibly contain
two allosteric sites in the 7-TM domain, as its orthosteric ligand
binding site is located on the large N terminal domain of the
receptor. For several class A GPCRs two binding sites, one
orthosteric and one allosteric, have been reported in the 7-TM
domain. The adenosine A1 and A3 receptors and the cannabinoid
CB1 receptor, where PD81,723, LUF6000 and Org27569 are
selective allosteric enhancers, respectively, are typical examples.16-18
Therefore, the effect of 24 on the equilibrium binding of the
iodinated endogenous ligand, 125I-hCG, was examined. Compound
24 was not able to displace the hormone (data not shown), similar
to Org 43553’s behavior.8 This indicates that 24 probably also binds
to the 7-TM domain like Org 43553 does.19 In addition, the
dissociation kinetics of 125I-hCG under several conditions were
examined (Figure 3b). An excess (70 nM) of recLH was able to
induce 40% dissociation of 125I-hCG after 4 h. In the presence of
10 µM Org 43553 the dissociation was significantly increased, in
line with a recent observation by Van Koppen and co-workers.19
However, when dissociation was induced by recLH in the presence
of 10 µM 24, no significant increase was observed. We also studied
125I-hCG dissociation induced by infinite dilution (Figure 3b). In
this case, the presence of recLH did not alter the amount of
dissociation, while the presence of Org 43553 or compound 24
significantly increased radioligand dissociation. Taken together, this
suggests that the high molecular weight ligand, hCG (and most
likely recLH), and the low molecular weight ligands, Org 43553
and 24, bind at three distinct sites, where both LMW ligands induce
a conformational change that (negatively) modulates hCG binding
to the receptor.
Figure 5. (a) Concentration-effect curves of recLH (squares), hCG
(circles) and Org 43553 (triangles) in the absence (closed) or presence
(open) of 10 µM 24 and (b) of 24 itself for cAMP-mediated luciferase
production through human luteinizing hormone receptors. Representa-
tive graphs from one experiment performed in duplicate (see Table 4
for EC50 and Emax values).
Finally, the effect of compound 24 on the activation of the LH
receptor by both of its endogenous hormones was examined in
cAMP-induced luciferase assays (Table 4 and Figure 5). RecLH
had an EC50 value of 56 ( 8 pM in this functional assay, which is
comparable to previously published data.8 The other endogenous
ligand, rec-hCG, had a similar potency as recLH (EC50 ) 97 (
10 pM), while it had an approximately 25% lower efficacy in our
hands. The presence of 10 µM 24 did not affect the efficacy, while
an approximately 3-fold decrease in potency was observed for both
recLH and rec-hCG. This indicates that compound 24 induces a
conformational change in the receptor that is disfavored by both
endogenous hormones. As reported previously, Org 43553 was a
highly efficacious partial agonist in the cAMP-induced luciferase
assay (Emax ) 85 ( 4%).19 Org 43553’s efficacy was not affected
by the presence of compound 24, while its potency was decreased
over 2-fold (EC50 ) 4.6 ( 0.8 nM). The effect of two other potent
compounds (33 and 40) from the second series (Table 2) was also
investigated in a luciferase assay. Both 33 and 40 decreased the
potency of recLH and Org 43553, similar to 24 (data not shown).
Moreover, from Figure 5a it follows that 10 µM 24 alone was
able to partially activate the LH receptor by 31 ( 4%. This
agonistic behavior was further analyzed, and it was shown that 24
had an EC50 value of 1.6 ( 0.1 µM (Figure 5b). Some of the other
terphenyl ligands also showed (low) intrinsic efficacy similar to
compound 24 (data not shown). It is noteworthy that the intrinsic
activity of some of these terphenyl compounds might indicate that
the second allosteric site is located close to or partially overlapping
with the Org 43553 binding site. Interestingly, most allosteric
modulators reported so far do not have an intrinsic efficacy at the
receptor in the absence of an orthosteric agonist. However, there
are some examples of (positive) allosteric modulators that can act
as agonists by themselves.20 For example, PD81,723 is an allosteric
enhancer at the adenosine A1 receptor as mentioned above, but it
can also activate the receptor.21 For the GABAB receptor CGP7930
was reported as an allosteric enhancer that is able to activate the
receptor by interacting with the 7-TM domain of the GABAB2
subunit.22 Another example is AC-42, which was shown to activate
the receptor in absence of the orthosteric agonist, but it is also a

LMW Allosteric Inhibitors of the LH Receptor
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 2041
modest allosteric enhancer of the muscarinic M1 receptor.23 For
compounds that show allosteric agonism besides allosteric modula-
tion of the orthosteric ligand, the term ago-allosteric modulator
was proposed by Schwartz and co-workers..24,25 Compound 24
(LUF5771) could therefore be characterized as the first ago-
allosteric inhibitor of the human LH receptor.
127.9, 127.6, 127.4, 123.0, 119.4, 119.3, 53.3, 33.5, 23.8, 21.4 ppm.
Anal. (C25H25NO2) C, H, N.
Biology. Materials and Methods. Org 43553, recLH and rec-
hCG were provided by Schering Plough (Oss, The Netherlands); Org
43553 was synthesized as described previously.26 Bovine serum
albumin (BSA, fraction V) was purchased from Sigma (St. Louis, MO),
whereas BCA protein assay reagent was from Pierce Chemical
Company (Rockford, IL). [3H]Org 43553 (16.6 Ci/mmol) was labeled
as described previously.8 125I-hCG (4408 Ci/mmol) was purchased from
Perkin-Elmer Life Sciences Inc. (Boston, MA). Chinese Hamster Ovary
(CHO-K1) cells stably expressing the human luteinizing hormone (LH)
receptor and cAMP-response-element luciferase reporter gene (CRE-
luc) were kindly provided by Schering Plough (Oss, The Netherlands).
All other chemicals and cell culture materials were obtained from
standard commercial sources.
Cell Culture and Membrane Preparation. CHO cells with
stable expression of the human LH receptor and CRE-luc (CHOhL-
Hr_luc) were grown in culture medium consisting of Dulbecco’s
modified Eagle’s medium (DMEM) and Ham’s F12 medium (1:1)
supplemented with 7.5% normal adult bovine serum, streptomycin
(100 µg/mL), penicillin (100 IU/ mL) at 37 °C in 5% CO2. The
cells were subcultured twice weekly at a ratio of 1:20. Cell
membranes were prepared as described previously.8
Conclusion
This paper describes the first series of allosteric inhibitors of
[3H]Org 43553 binding at the human LH receptor. In particular
24 and 40 are highly potent. In addition, 24 inhibited the
activation of the receptor by the endogenous ligand recLH and
by Org 43553 in a functional assay. Although 24 is an allosteric
inhibitor of recLH and Org 43553, it was also able to partially
activate the LH receptor with low efficacy. The presence of a
second allosteric site in the 7-TM domain, as demonstrated in
this paper, may provide novel targets at the human luteinizing
hormone receptor for low molecular weight allosteric modulators
and allosteric agonists.
Radioligand Displacement Assays. 125I-hCG displacement assays
were performed as described previously.8 For [3H]Org 43553, mem-
brane aliquots containing 50 µg of protein were incubated in a total
volume of 100 µL of assay buffer (25 mM Tris-HCl, pH 7.4,
supplemented with 2 mM MgCl2 and 0.1% BSA) at 30 °C for 90
min. Displacement experiments were performed using 10 µM or a
range of concentrations of competing ligand in the presence of 4.5
nM [3H]Org 43553. Nonspecific binding was determined in the
presence of 10 µM Org 43553 and represented approximately 35% of
the total binding. [3H]Org 43553 did not bind specifically to membranes
prepared from CHO cells lacking the LH receptor. Total binding was
determined in the presence of buffer and was set at 100% in all
experiments, whereas nonspecific binding was set at 0%. Incubations
were terminated by dilution with 1 mL of ice-cold Tris-HCl buffer.
Bound from free radioligand was immediately separated by rapid
filtration through Whatman GF/B filters using a Millipore manifold.
Filters were subsequently washed three times with ice-cold wash buffer
(25 mM Tris HCl, pH 7.4, supplemented with 2 mM MgCl2 and 0.05%
BSA). Filter-bound radioactivity was determined by scintillation
spectrometry (Tri-Carb 2900TR; PerkinElmer Life and Analytical
Sciences) after addition of 3.5 mL of PerkinElmer Emulsifier Safe.
Radioligand Dissociation Assays. Dissociation assays with 125I-
hCG were performed as described previously.8 The amount of
radioligand still bound to the receptor was measured after 4 h of
dissociation. The total amount of radioligand binding determined
at t ) 0 was set to 0%, while nonspecific binding was set at 100%.
For [3H]Org 43553, dissociation experiments were performed by
preincubating membrane aliquots containing 50 µg of protein in a
total volume of 100 µL of assay buffer (25 mM Tris HCl, pH 7.4,
supplemented with 2 mM MgCl2 and 0.1% BSA) with 4.5 nM
[3H]Org 43553 at 30 °C for 90 min. After preincubation, dissocia-
tion was initiated by addition of 10 µM Org 43553 in the absence
(control) or presence of allosteric modulators in a total volume of
5 µL of which was 50% (v/v) DMSO. The amount of radioligand
still bound to the receptor was measured after 30 min of dissocia-
tion. The obtained amount of radioligand binding determined at
control conditions was set at 100%. In addition, the amount of
[3H]Org 43553 still bound to the receptor was measured at various
time intervals for a total of 120 min in the absence (control) and
presence of 1 and 10 µM 24. Incubations were terminated and
samples were obtained and analyzed as described under Radioligand
Displacement Assays.
Experimental Section
Chemistry. Materials and Methods. All reagents used were
obtained from commercial sources, and all solvents were of
1
analytical grade. H and 13C NMR spectra were recorded on a
Bruker AC 400 (1H NMR, 400 MHz; 13C NMR, 100 MHz) and
Bruker AC 200 (1H NMR, 200 MHz; 13C NMR, 50 MHz)
spectrometer with tetramethylsilane as an internal standard. Chemi-
cal shifts are reported in δ (ppm). Melting points were determined
by Bu¨chi melting point apparatus and are uncorrected. Elemental
analyses were performed by Leiden Institute of Chemistry and are
within 0.4% of theoretical values unless otherwise stated. Reactions
were routinely monitored by TLC using Merck silica gel F254
plates. Microwave reactions were performed on an Emrys Optimizer
(Biotage AB). Wattage was automatically adjusted to maintain the
desired temperature.
General Procedure for Carbamate Synthesis. Triethylamine
(1.5 equiv) was added to a stirred solution of phenol (1 equiv) in
anhydrous dichloromethane and stirred at ambient temperature for
15 min. Isocyanate (1.5 equiv) was added and stirred for another
12 to 18 h at ambient temperature. Upon completion (TLC), the
reaction mixture was diluted with dichloromethane (100 mL),
washed with water, washed with brine, dried and evaporated under
reduced pressure. The crude compound was purified by flash column
chromatography (dichloromethane/petroleum ether, 4:1) to get the
carbamate. The column purified carbamate was crystallized using
dichloromethane and petroleum ether.
Phenyl-carbamic Acid [1,1′,3′,1′′]Terphenyl-5′-yl Ester (4).
Yield: 259 mg, 87%. Mp: 165-166 °C. 1H NMR (200 MHz,
CDCl3): δ ) 7.68-7.61 (m, 5H), 7.51-7.31 (m, 12H), 7.17-7.05
(m, 2H), 4.08 (brs, 1H), 3.85 (s, 3H) ppm. 13C NMR (50 MHz,
CDCl3):δ ) 164.1, 151.1, 143.0, 137.2, 129.1, 128.7, 127.6, 127.2,
123.9, 123.3, 119.1, 118.8 ppm. MS (ES+): 366 (MH+). Anal.
(C25H19NO2) C, H, N.
Cyclopentyl-carbamic Acid [1,1′,3′,1′′]Terphenyl-5′-yl Ester
(24). Yield: 197 mg, 68%. Mp: 136 °C. 1H NMR (200 MHz,
CDCl3): δ ) 7.67-7.64 (m, 4H), 7.51-7.38 (m, 9H), 5.18-5.14
(m, 1H), 4.16-4.10 (m, 1H), 2.07-2.04 (m, 2H), 1.70-1.30 (m,
6H) ppm. 13C NMR (100 MHz, CDCl3):δ ) 153.9, 151.7, 142.8,
140.3, 128.7, 127.6, 127.2, 122.9, 119.2, 53.0, 33.1, 23.9 ppm. Anal.
(C24H23NO2) C, H, N.
4-Methyl-1,1′,3′,1′′-terphenyl-5′-yl Cyclopentylcarbamate (40).
Yield: 140 mg, 75%. 1H NMR (400 MHz, CDCl3): δ ) 7.65-7.58
(m, 3H), 7.52 (d, J ) 8.0 Hz, 2H), 7.43 (t, J ) 7.6 Hz, 2H),
7.37-7.30 (m, 3H), 7.24 (d, J ) 8.0 Hz, 2H), 5.06 (d, J ) 7.6 Hz,
1H), 4.12-4.05 (m, 2H), 2.38 (s, 3H), 2.08-1.99 (m, 2H),
1.75-1.59 (m, 4H), 1.54-1.46 (m, 2H) ppm.13C NMR (100 MHz,
CDCl3): δ ) 156.5, 152.0, 143.1, 140.8, 137.8, 137.7, 129.8, 129.0,
Luciferase Assays. CHOhLHr_luc cells were grown as described
above. On the day of the assay, cells were washed with PBS and
then harvested using trypsol (0.25% (w/v) in PBS containing 4.4
mM EDTA). Cells were resuspended in assay medium consisting
of DMEM and F12 (1:1) supplemented with 1 µg/mL insulin and
5 µg/mL apo-transferrin. Typically, a well contained 30 µL of

2042 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Heitman et al.
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Wiegerinck, P. H. G.; IJzerman, A. P. [3H]Org 43553, the First Low-
Molecular-Weight Agonistic and Allosteric Radioligand for the Human
Luteinizing Hormone Receptor. Mol. Pharmacol. 2008, 73, 518–524.
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ligand, 30 µL of assay medium with or without 10 µM 24 and 30
µL of cell suspension containing 7.5 × 105 cells/mL. Luciferase
assays were performed using ten concentrations of recLH, rec-hCG
or Org 43553. Basal activity was determined in the presence of
assay medium and represented approximately 10% of the maximal
activity. Maximal receptor activity was determined in the presence
of 1 nM recLH and was set at 100% in all experiments, whereas
basal activity was set at 0% in all experiments. After 4 h of
stimulation, 45 µL of Britelite (PerkinElmer, Groningen, The
Netherlands) was added to each well for detection of luciferase
protein. Finally, the luminescence signal was quantified on a
Microbeta Trilux 1450 Luminescence Counter (PerkinElmer,
Groningen, The Netherlands).
Log D Determination by HPLC. Distribution coefficients
(log D) were determined as described by Lombardo and co-
workers.27 In short, retention times of the compounds were
determined in an HPLC system with three different methanol
percentages. These retention times were converted to k′ values by
using the formula k′ ) tr - t0/t0 in which tr is the retention time
and t0 the retention time of a “nondelayed” compound (pure
methanol). The calculated k′ values were plotted against the
methanol percentage and extrapolated to a 0% methanol situation
which yielded the k′w value (y axis cutoff). In a standard curve,
the known log D values of the reference compounds were plotted
against their k′w values found in the HPLC system used. From this
standard curve the log D values of the compounds described in
this paper were determined.
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adenosine receptor. J. Med. Chem. 2006, 49, 3354–3361.
Data Analysis. All binding data were analyzed using the nonlinear
regression curve-fitting program GraphPad Prism v. 5.00 (GraphPad
Software Inc., San Diego, CA). Dissociation constants, koff, were
obtained by computer analysis of the exponential decay of [3H]Org
43553 bound to the receptor. All values obtained are means of at least
three independent experiments performed in duplicate.
(18) Price, M. R.; Baillie, G. L.; Thomas, A.; Stevenson, L. A.; Easson,
M.; Goodwin, R.; McLean, A.; McIntosh, L.; Goodwin, G.; Walker,
G.; Westwood, P.; Marrs, J.; Thomson, F.; Cowley, P.; Christopoulos,
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cannabinoid CB1 receptor. Mol. Pharmacol. 2005, 68, 1484–1495.
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S.; van de Lagemaat, R.; Smit, M. J.; Hanssen, R. A signaling-selective,
nanomolar potent allosteric low molecular weight agonist for the
human luteinizing hormone receptor. Naunyn Schmiedeberg’s Arch.
Pharmacol. 2008, 378, 503–514.
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modulation of G protein-coupled receptors. Annu. ReV. Pharmacol.
Toxicol. 2007, 47, 1–51.
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adenosine receptors increase receptor-G protein coupling and coun-
teract guanine nucleotide effects on agonist binding. Mol. Pharmacol.
2003, 64, 1557–1564.
Acknowledgment. The authors would like to thank Harold
Darwinkel and Peter Wiegerinck (Schering-Plough Research
Institute, Oss, The Netherlands) for technical support with the
log D determinations and for providing [3H]Org 43553,
respectively. This study was performed within the framework
of Top Institute Pharma project: number D1-105.
Supporting Information Available: Experimental details of the
synthesis of the compounds described in this paper, their 1H NMR
and 13C NMR spectroscopic data, and their elemental analyses. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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