Thyrotropin-releasing hormone (TRH) analogues that exhibit selectivity to TRH receptor subtype 2

Article abstract of DOI:10.1021/jm0505462

Thyrotropin-releasing hormone (TRH) analogues in which the C-2 position of the imidazole ring of the centrally placed histidine residue is substituted with various alkyl groups were synthesized and studied as agonists for TRH receptor subtype 1 (TRH-R1) a

Full text of DOI:10.1021/jm0505462

6162
J. Med. Chem. 2005, 48, 6162-6165
Brief Articles
Thyrotropin-Releasing Hormone (TRH) Analogues That Exhibit Selectivity to
TRH Receptor Subtype 2
Navneet Kaur,† Xinping Lu,‡ Marvin C. Gershengorn,‡ and Rahul Jain*,†
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S.
Nagar - 160 062, Punjab, India, and Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
Received June 10, 2005
Thyrotropin-releasing hormone (TRH) analogues in which the C-2 position of the imidazole
ring of the centrally placed histidine residue is substituted with various alkyl groups were
synthesized and studied as agonists for TRH receptor subtype 1 (TRH-R1) and subtype 2 (TRH-
R2). Several analogues were found to be selective agonists for TRH-R2 exhibiting no activation
of TRH-R1. For example, analogue 4 (R) c-C3H5) was found to activate TRH-R2 with a potency
(EC50) of 0.41 µM but did not activate TRH-R1 (potency > 100 µM). This study describes the
first discovery of TRH-R2-specific agonists and provides impetus to design predominately CNS-
effective TRH peptides.
Introduction
activity have not been successful. Although, analogues
with potent CNS activity were synthesized, none were
completely devoid of endocrine side effects. Moreover,
in common with other potential drugs that are peptides,
the therapeutic efficacy of TRH is compromised by its
instability and hydrophilic nature. Thus, the high-
dosage regimes needed to obtain neuropharmacological
effects often result in adverse side effects arising from
the endocrine actions of TRH.
Thyrotropin-releasing hormone (TRH, L-pGlu-L-His-
L-ProNH2), a simple tripeptide amide, is the first neu-
ropeptide whose chemical structure was elucidated.1,2
TRH is synthesized in the hypothalamus and acts in
the anterior pituitary to control levels of TSH (thyrotro-
pin, thyroid stimulating hormone) and prolactin. This
peptide is the first hypothalamic releasing factor char-
acterized, establishing the fundamental proof for the
existence of neuroendocrine regulation of pituitary
functions by hypothalamic neuronal structures.3,4 TRH
plays a central role in regulating the pituitary-thyroid
axis by stimulating TSH release and de novo synthesis
in all mammalian species. The peptide also acts as an
equally potent stimulator of prolactin secretion and
synthesis. Though the neuroendocrine action of TRH on
the anterior pituitary (PIT) dominated the earlier
research, it was discovered that the effects of TRH are
not limited to the anterior pituitary but that TRH exerts
profound effects on the central nervous system (CNS).5
Thus, TRH has been shown to (a) alter neuronal
excitability, (b) enhance transmitter release and turn-
over, (c) increase CNS arousal, (d) increase blood
pressure, body temperature and respiration rate, (e)
alter body water and food intake, (f) enhance locomotor
activity, and (g) produce antinociception. Interestingly,
the potential therapeutic applications of TRH that have
attracted the most attention are not based on its
endocrine properties but on its broad spectrum of
stimulatory actions within the CNS. These CNS-medi-
ated effects provides the rationale for the use of TRH
in the treatment of brain and spinal injury and certain
CNS disorders, including Alzheimer’s disease and motor
neuron disease (MND). Research efforts toward devel-
opment of potent TRH analogues with selective CNS
TRH initiates its effects by interacting with receptors
on the surface of cells. Receptors for TRH belongs to the
rhodopsin/â-adrenergic receptor subfamily of seven
transmembrane (TM)-spanning, G protein-coupled re-
ceptors (GPCRs).6 The first TRH receptor (TRH-R1) was
cloned from a mouse pituitary tumor, and then ortholo-
gous receptors were cloned from different species,
including rat, chicken, Catostomus commersoni, and
human.7-9 Recently, a second subtype of TRH receptor
(TRH-R2) was identified in rats, mouse, and Catostomus
commersoni.10-13 Amino acid sequences of the two
subtypes of TRH receptor from the same species reveal
a 51% overall identity.14 Subsequent comparison of
TRH-R1 and TRH-R2 has been made regarding their
tissue distribution. TRH-R1 is highly expressed in the
anterior pituitary and is mainly involved in the signal-
ing of TRH within neuroendocrine brain regions, the
autonomic nervous system, and the visceral brainstem
regions. TRH-R1 exhibits only a very limited mRNA
expression pattern in other regions of the CNS. In
contrast, TRH-R2 is strongly expressed in rat brain and
spinal cord, but is not detectable in the pituitary.14 The
specific expression of TRH-R2 in brain areas that are
important for the transmission of somatosensory signals
and higher CNS functions indicates that in the CNS this
receptor subtype may be of major functional importance.
Therefore, we believe that a therapeutically useful CNS
selective TRH analogue (mediated by TRH-R2) should
exert weak or no TSH-releasing effects (mediated by
TRH-R1). However, analogues reported to date exhibit
* Corresponding author. E-mail: rahuljain@niper.ac.in, Tel no.: 91-
0172-221-4682, Fax: 91-0172-221-4692.
† National Institute of Pharmaceutical Education and Research.
‡ National Institutes of Health.
10.1021/jm0505462 CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/19/2005

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6163
Scheme 1a
Figure 1.
strong TSH-releasing effects while at the same time
produce “nonendocrine” CNS effects.15 Therefore, if
analogues were developed with high selectivity for TRH-
R2 receptor subtype, they could probably demonstrate
more specific and selective profiles of CNS effects.
a Reagents and conditions: (i) (CF3CO)2O, rt, 24 h; (ii) RCO2H,
AgNO3, 10% H2SO4, (NH4)2S2O8, 70-80 °C, 15 min; (iii) 6N HCl,
reflux, Dowex ion-exchange resin (50 × 2-200, H+ form); (iv) a.
Boc2O, 4 N NaOH, dioxane-H2O (2:1), rt, 24 h; b. MeOH, 12 h; c.
KHSO4, pH ) 3.75.
All three residues in TRH contribute to its binding
to TRH receptors.16 Removal of key amino acids, pGlu
by proteolytic pyroglutamyl aminopeptidase II, and
histidine by the histidyl-proline iminopeptidase have
restricted the therapeutic utility of this important
hormonal/CNS peptide.17 With a paradigm shift in
peptide research, it is now established that synthetic
and modified R-amino acids play a significant role in
the area of peptide-based drug design and discovery
research. These important building blocks are being
extensively incorporated into bioactive peptides to re-
strict their conformational flexibility, enhance pro-
teolytic stability, improve drug-like properties, and most
importantly increase selectivity. Upon the basis of these
observations, our current research efforts are in the
direction of synthesizing novel TRH analogues with
particular emphasis on the replacement of the pGlu and
His residues with their modified counterparts and
scaffolds.18,19 In continuation of our research program,
we report herein synthesis and receptor binding studies
for a set of six TRH analogues where the central
histidine residue is modified by incorporating an alkyl
group at the C-2 position of the imidazole ring (Figure
1). Hydrophobic alkyl substituents, such as propyl,
isopropyl, tert-butyl, cyclobutyl, cyclohexyl, and an
adamantyl group, were selected for this initial study.
In addition, to observe the effect of their placement on
receptor subtype selectivity, the choice of these groups
was also made upon the assumption that their incor-
poration may slightly reduce the hydrophilicity of the
synthesized peptides thereby making them amenable
for drug development.
mechanism and offers an unparalleled procedure for
functionalization of the electron-deficient imidazole
ring.22,23 Reaction involves nucleophilic addition of an
alkyl radical (generated by the silver-catalyzed oxidative
decarboxylation of alkylcarboxylic acid with ammonium
persulfate) to a protonated imidazole ring followed by
rearomatization leading to direct and selective imidazole
C-2 alkylation. Complete deprotection of 10-15 by
refluxing a solution of them in 6 N HCl and evaporation
of the acidic hydrolysis solution under reduced pressure
produced amino acid dihydrochloride salts. The free
amino acids 16-21 were obtained by elution with 25%
NH4OH solution from Dowex ion-exchange resin (50 ×
2-200, H+ form) column. Compounds 16-21 upon
reaction with di-tert-butyl dicarbonate in the presence
of aqueous 4 N NaOH solution at ambient temperature
in dioxane-water (2:1) mixture for 24 h followed by
stirring with MeOH for 12 h led to the formation of N-R-
Boc-2-alkyl-L-histidines 22-27, suitable for the synthe-
sis of TRH analogues.24 At the same time, L-pyroglutam-
ic acid (L-pGlu) upon reaction with 2,4,5-trichlorophenol
in the presence of 1,3-dicyclohexylcarbodiimide (DCC)
in DMF for 12 h gave 2,4,5-trichlorophenyl active ester
(L-pGlu-OTcp) in excellent yields as described earlier.19
The C-terminal to N-terminal solution phase peptide
synthesis of the intermediate dipeptides 29-34 was
accomplished by the reaction of commercial L-prolina-
mide (28) with N-R-Boc-2-alkyl-L-histidines 22-27 in
the presence of coupling reagents endo-N-hydroxy-5-
norbornene-2,3-dicarboximide (HONB) and 1,3-diiso-
propylcarbodiimide (DIC) at 4 °C for 36 h. Subsequently,
cleavage of the N-R-Boc group with TFA (40% solution
in DCM) produced dipeptide trifluoroacetate salts,
which were not isolated and immediately subjected to
next reaction. The free dipeptides were generated in situ
by reaction of the latter compounds with 7 N NH3 in
MeOH. Complete removal of the solvent followed by
coupling of the free dipeptides with L-pGlu-OTcp in
DMF at 4 °C for 36 h produced TRH analogues 2-7 in
satisfactory yields (Scheme 2).
Results and Discussion
Chemistry. 2-Alkyl-L-histidines 16-21 required for
the preparation of N-R-Boc-2-alkyl-L-histidines 22-27
were synthesized in three convenient steps (Scheme
1).20,21 Thus, commercially available L-histidine methyl
ester dihydrochloride (8) upon reaction with trifluoro-
acetic anhydride for 24 h at ambient temperature
provided N-R-trifluoroacetyl-L-histidine methyl ester (9).
The latter compound 9 upon reaction with various
commercially available alkylcarboxylic acids in the
presence of 10% H2SO4, catalytic silver nitrate, and
ammonium persulfate at 70-80 °C for 15 min provided
the N-R-trifluoroacetyl-2-alkyl-L-histidine methyl esters
10-15. The reaction proceeds through a free radical
Pharmacology at TRH-R1 and TRH-R2. Synthe-
sized TRH analogues 2-7 were examined for their
affinity for TRH-R1 and TRH-R2 and their ability to
serve as agonists for the receptor.25 Affinities, reported
as Ki (µM) values, were determined by measuring the

6164 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Brief Articles
Scheme 2a
(R ) c-C3H5) exhibited the highest potency (EC50 ) 0.41
µM) for TRH-R2 of the analogues that did not activate
TRH-R1. Since analogues 3, 5, and 6 did not activate
TRH-R1, they are specific agonists of TRH-R2 also. In
contrast, analogue 7 (R ) adamantan-1-yl) was inactive
at both TRH receptors. These results are highly satisfy-
ing and represent the first report of the discovery of
TRH analogues that exhibit high selectivity as agonists
for TRH-R2. As discussed above, TRH-R2 is predomi-
nantly and selectively expressed in brain areas that are
important for the higher central nervous system func-
tions, whereas TRH-R1 is known to mainly mediate
endocrine functions. Therefore, high agonist selectivity
produced by compound 2-6 is of considerable signifi-
cance and beneficial in search of additional analogues
that will not only be CNS-selective but more active than
TRH.
Conclusions
a Reagents and conditions: (i) HONB, DIC, DMF, 4 °C, 36 h;
(ii) a. 40% TFA, DCM, rt, 30 min; b. 7 N NH3/MeOH, 10 min; c.
L-pGlu-OTcp, DMF, 4 °C, 36 h.
In the present study we have attempted to determine
the effects of TRH peptides synthesized through modi-
fication at the imidazole ring of the histidine residue
on receptor subtype specificity. For the first time,
specific TRH agonists toward TRH-R2 receptor at low
micromolar concentrations are described. These com-
pounds hold great promise, and we hope that their
optimization will lead to additional new synthetic pep-
tides with even greater specificity for TRH-R2. Explora-
tion of alkyl groups of variable sizes at other feasible
position(s) on the imidazole ring of TRH and their effect
on the receptor subtype selectivity is currently under-
way in our laboratory.
concentration of the analogue required to compete with
[Nτ(1)-Me-His]-TRH for the binding of the receptor. [Nτ-
(1)-Me-His]-TRH is known to bind to TRH-R1 and TRH-
R2 with affinities approximately 7-10 times higher
than TRH. The agonist behavior of the analogues was
tested in HEK 293EM cells stably expressing TRH-R1
or TRH-R2 by incubating the cells with various doses
of the analogues being examined. The extent of agonist
behavior was then determined by measuring signaling
through a reporter gene, and the data are reported as
EC50 (µM) values.
None of the 2-alkyl-L-histidine substituted TRH ana-
logues 2-7 bound with high affinity (Ki) to TRH-R1 or
TRH-R2 (Table 1). Analogues 2-6, however, displayed
full agonist activity at TRH-R2 but only analogue 2
activated TRH-R1 whereas analogues 3-6 did not
activate TRH-R1. Analogue 2 (R ) CH2CH2CH3) was a
potent agonist for TRH-R2 (EC50 ) 0.024 µM) with >12-
fold selectivity for TRH-R2 over TRH-R1. Analogue 4
Experimental Section
Receptor Binding Assay. HEK 293EM cells stably ex-
pressing either TRH-R1 or TRH-R2 were grown in Dulbecco’s
modified Eagle’s medium containing 10% fetal bovine serum
and 200 mg/mL hygromycin. For equilibrium binding experi-
ments, HEK 293EM cells stably expressing TRH-R1 or TRH-
R2 were seeded into 24-well plates (1.5 × 105/well). After 48
h, cells were incubated at 37 °C for 1 h with [3H]Nτ(1)-Me-
His-TRH (MeTRH, 0.1 nM-20 nM) in Hank’s Balanced Salt
Table 1. Binding Affinities Ki (µM) and Signaling (activation) Potencies EC50 (µM) Produced by TRH Analogues 2-7 for TRH-R1 and
TRH-R2 Receptors
Ki (µM)a
TRH-R2
EC50 (µM)b
TRH-R2
fold select.
(TRH-R2)
fold select.
(TRH-R2)
no.
R
TRH-R1
TRH-R1
2
CH2CH2CH3
CH(CH3)2
c-C3H5
0.32 (0.27-0.37)
>100
0.17 (0.12-0.24)
>100
1.88
-
0.29 (0.16-0.51)
>100
0.024 (0.011-0.052)
1.5 (0.69-3.2)
0.41 (0.19-0.85)
1.3 (0.76-2.2)
1.1 (0.21-5.7)
>100
>12
>67
>244
>77
>91
-
3
4
>100
>100
-
>100
5
C(CH3)3
>100
>100
>100
-
>100
6
c-C6H11
>100
-
>100
7
adamantan-1-yl >100
>100
-
>100
TRH
H
0.02 (0.001-0.003) 0.01
0.003
2
0.003 (0.002-0.004) 0.003
-
[Nτ(1)-Me-His]-TRH
-
-
0.0005
-
-
a For binding, cells expressing TRH-R1 or TRH-R2 were incubated with 1 nM [3H]Nτ(1)-Me-His-TRH in the absence or presence of
various doses of unlabeled TRH analogues for 1 h at 37 °C. b For signaling, cells expressing TRH-R1 or TRH-R2 and a CREB-luciferase
reporter were incubated with various doses of TRH analogues for 6 h at 37 °C, and luciferase activity was measured. Experiments were
performed with intact HEK. All data are means (95% confidence limits) of nine doses of analogues assayed in duplicate determinations
in two experiments.

Brief Articles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 6165
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JM0505462