Synthesis, Receptor Binding, and CNS Pharmacological Studies of New Thyrotropin-Releasing Hormone (TRH) Analogues

DOI: 10.1002/cmdc.201000481

Synthesis, Receptor Binding, and CNS Pharmacological

Studies of New Thyrotropin-Releasing Hormone (TRH)

Analogues

Vikramdeep Monga,^{[a, d]}Chhuttan L. Meena,^[a]Satyendra Rajput,^[b]Chandrashekhar Pawar,^[b]

Shyam S. Sharma,^[b]Xinping Lu,^[c]Marvin C. Gershengorn,^[c]and Rahul Jain*^[a]

As part of our search for selective and CNS-active thyrotropin-

releasing hormone (TRH) analogues, we synthesized a set of 44

new analogues in which His and pGlu residues were modified

or replaced. The analogues were evaluated as agonists at TRH-

R1 and TRH-R2 in cells in vitro, and in vivo in mice for analeptic

and anticonvulsant activities. Several analogues bound to TRH-

R1 and TRH-R2 with good to moderate affinities, and are full

agonists at both receptor subtypes. Specifically, analogue 21a

(R=CH₃) exhibited binding affinities (K_ivalues) of 0.17 mm for

TRH-R1 and 0.016 mm for TRH-R2; it is 10-fold less potent than

TRH in binding to TRH-R1 and equipotent with TRH in binding

to TRH-R2. Compound 21a, the most selective agonist, activat-

ed TRH-R2 with a potency (EC₅₀value) of 0.0021 mm, but acti-

vated TRH-R1 at EC₅₀=0.05 mm, and exhibited 24-fold selectivi-

ty for TRH-R2 over TRH-R1. The newly synthesized TRH ana-

logues were also evaluated in vivo to assess their potencies in

antagonism of barbiturate-induced sleeping time, and several

analogues displayed potent analeptic activity. Specifically, ana-

logues 21a,b and 22a,b decreased sleeping time by nearly

50% more than TRH. These analogues also displayed potent

anticonvulsant activity and provided significant protection

against PTZ-induced seizures, but failed to provide any protec-

tion in MES-induced seizures at 10 mmolkg^ꢀ1. The results of

this study provide evidence that TRH analogues that show se-

lectivity for TRH-R2 over TRH-R1 possess potent CNS activity.

Introduction

Thyrotropin-releasing hormone (TRH, l-pGlu-l-His-l-ProNH₂, 1)

is generated from a large precursor protein that contains mul-

tiple repeats of the TRH progenitor tetrapeptide Gln-His-Pro-

Gly, and is the first hypothalamic hypophysiotropic neuropep-

tide to have had its sequence chemically elucidated.^[1–5]TRH

plays a central role in the endocrine system and regulates sev-

eral neurobiological activities.^[6]It exhibits antidepressant, ana-

leptic, and neuroprotective effects and is considered a promis-

ing lead for the discovery of analogues in the treatment of epi-

lepsy, motor neuron diseases, spinal cord trauma, and Alzheim-

er’s disease.^[7–9]The rhodopsin/b-adrenergic receptor subfamily

of G-protein-coupled TRH receptors (TRH-R) in brain are ubiq-

uitously distributed and found at highest densities in limbic

structures, especially the amygdala and hypothalamus and in

lower densities in brain stem and cerebellum.^[10,11]At present,

two TRH receptor subtypes (TRH-R1 and TRH-R2) have been

found in several species and exhibit an overall identity of

~50%.^[12–19]Studies conducted earlier demonstrated that TRH-

R1 is highly expressed in the anterior pituitary, the neuroendo-

crine brain regions, the autonomic nervous system, and the

visceral brain stem regions. TRH-R2 is expressed in the cerebel-

lar cortex, mesencephalon, thalamic and subthalamic regions,

and the brain stem.^[17,19]It has therefore been hypothesized

that TRH-R2 could mediate some central nervous system (CNS)

effects of TRH, whereas TRH-R1 mediates its thyroid-stimulat-

ing hormone (TSH)-releasing effects.

counting for almost all of TRH binding energy and affinity. In

our previous studies, several modified amino acids or carboxyl-

ic acid derived scaffolds were incorporated independently or

simultaneously into the sequence of TRH at pGlu and His to

[a] Dr. V. Monga, C. L. Meena, Prof. R. Jain

Department of Medicinal Chemistry

National Institute of Pharmaceutical Education and Research

Sector 67, S.A.S. Nagar, Punjab 160062 (India)

Fax: (+91)0172-221-4692

E-mail: rahuljain@niper.ac.in

[b] S. Rajput, C. Pawar, Dr. S. S. Sharma

Department of Pharmacology and Toxicology

National Institute of Pharmaceutical Education and Research

Sector 67, S.A.S. Nagar, Punjab 160062 (India)

[c] Dr. X. Lu, Dr. M. C. Gershengorn

Clinical Endocrinology Branch, NIDDK

National Institutes of Health, Bethesda, MD 20892 (USA)

[d] Dr. V. Monga

Current address: Department of Pharmaceutical Chemistry

I.S.F. College of Pharmacy, Moga, Punjab, 142001 (India)

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201000481: detailed experimental proce-

dures and spectral data for intermediates 12a–m, 13a–m, 18a–e, and

19a–e.

All three amino acid residues of TRH play a role in eliciting

its physiological response(s), with pGlu and His residues ac-

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531

R. Jain et al.

MED

synthesize a library of analogues.^[20–27]The results of these

studies showed that TRH analogues in which His is modified at

the N1(t) or C2 positions of the imidazole ring with various

bulky hydrophobic groups exhibit selectivity for TRH-R2. The

TRH analogues in which pGlu is replaced with (1R)- or (1S)-3-

oxo-1-cyclopentanecarboxylic acid (Ocp), and His is substituted

at the C2 position, bind with high affinity (K_i) to both TRH-R1

and TRH-R2, whereas TRH analogues in which pGlu is replaced

with l-pyro-2-aminoadipic acid (pAad), and His is substituted

at the N1(t) or C2 positions, exhibit good potency and selec-

tive activation of TRH-R2 over TRH-R1. These studies indicate

that independent or simultaneous change of pGlu and His resi-

dues in TRH alters its receptor binding affinity and signal trans-

ducing potency.

four steps as described earlier.^[31]Boc-1-alkyl-l-His-OH com-

pounds 17a–e were synthesized from Boc-l-His-OH in one

step as reported.^[32](1R)-3-Ocp-OH (20) was synthesized from

4-vinylcyclohexene in four steps and chemically resolved using

a well-established procedure.^[33–35]

A solid-phase peptide synthesis protocol was used for the

synthesis of targeted tripeptides 10a–g (Scheme 1). Briefly, a

4-methylbenzhydrylamine-functionalized resin (MBHA·HCl, 2)

In continuation, herein we report the synthesis of four new

series of TRH analogues that involve independent or simulta-

neous modifications at the pGlu and His residues of the parent

peptide in order to gain additional insight into the structure–

activity relationships and biological activities (Figure 1). These

Scheme 1. Reagents and conditions: a) 10% DIPEA, CH₂Cl₂, RT, 5 min; b) Boc-

l-Pro-OH (3), TBTU, DMF, 10% DIPEA in CH₂Cl₂, RT, 2 h; c) 20% TFA in CH₂Cl₂,

RT, 20 min; d) Boc-l-His(1-methyl-2-alkyl)-OH (6a–g), TBTU, DMF, 10% DIPEA

in CH₂Cl₂, RT, 2 h; e) Z-l-pGlu-OH (8), TBTU, 10% DIPEA in CH₂Cl₂, RT, 2 h;

f) TFMSA, EDT, thioanisole, TFA, 48C–RT, 2 h.

was neutralized with N,N-diisopropylethylamine (DIPEA) and

subsequently coupled with the preformed O-(benzotriazol-1-

yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU)-acti-

vated ester of Boc-l-Pro-OH (3) in N,N-dimethylformamide

(DMF) to produce a protected amino acid linked resin. The

resin-bound Boc-protected amino acid was deprotected with

trifluoroacetic acid (TFA), followed by neutralization with

DIPEA, to provide free amino acid linked resin 5. This was then

subjected to further coupling and deprotection cycles with

Boc-l-His(1-methyl-2-alkyl)-OH 6a–g and Z-l-pGlu-OH 8, re-

spectively, to yield peptide resin 9. The intermediate coupling

steps were monitored by Kaiser’s test. Cleavage of the peptide

from the resin support was carried out by using a solution of

trifluoromethanesulfonic acid (TFMSA) in TFA in the presence

of thioanisole and 1,2-ethanedithiol (EDT) as scavengers to

yield peptide amides 10a–g.

Figure 1. General structures of the four series of synthesized TRH analogues.

new series allowed a comparison of carboxylic acids at the N-

terminal residue and the effects of substitution at the His resi-

due on the binding affinity and potency of the modified pepti-

domimetics. The His residue was replaced with either N1(t)

substituted or 1,2-disubstituted-l-His, whereas the pGlu resi-

due was replaced with (1R)-3-Ocp or l-pAad. In addition to

their receptor binding studies, the synthesized TRH analogues

were also evaluated in vivo in mice for CNS activity on the an-

tagonism of pentobarbital-induced sleep duration as a primary

screen. The most promising analogues were then evaluated in

the selected models of epilepsy to assess their promise as

future drugs in the treatment of epilepsy.

All subsequent peptides were synthesized by solution-phase

synthesis to decrease consumption of the large excess of

modified amino acids required for solid-phase synthesis. Reac-

tion of l-Pro-OH 11 with Boc-l-His(1,2-dialkyl)-OH compounds

6a–m in the presence of 1,3-diisopropylcarbodiimide (DIC) and

N-hydroxy-5-norbornene-endo-2,3-dicarboximide (HONB) in

DMF yielded dipeptides 12a–m (Scheme 2). Deprotection of

the Boc group with TFA produced dipeptide salts 13a–m. The

free dipeptides were obtained in situ by reaction of 13a–m

Results and Discussion

Chemistry

Boc-1,2-dialkyl-l-His-OH compounds 6a–m were prepared

from commercially available l-His-OMe using a multistep syn-

thetic procedure as discussed previously.^[28–30]l-pAad-OPfp (15)

was synthesized from commercially available l-Lys(Z)-OH in

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ChemMedChem 2011, 6, 531 – 543

Thyrotropin-Releasing Hormone Analogues

Scheme 4. Reagents and conditions: a) 40% TFA, CH₂Cl₂, 08C, 30 min; b) 7n

NH₃in CH₃OH, 08C, 10 min; c) (1R)-3-Ocp-OH (20), DIC, HOBt, DMF, 48C,

36 h.

of the receptors.^[36]Affinities, reported as K_ivalues (mm), were

determined by measuring the concentration of the analogue

required to compete with [³H][Nt(1)-Me-His]TRH at 2 nm for re-

ceptor binding. [Nt(1)-Me-His]TRH is known to bind TRH-R1

and TRH-R2 with affinities higher than TRH. The agonist behav-

ior of the analogues was tested in HEK293EM cells stably ex-

pressing TRH-R1 or TRH-R2 by incubating the cells with various

doses of the analogues as described.^[21,37]The extent of agonist

behavior was then determined by measuring signaling through

a reporter gene, and the data are reported as EC₅₀values (mm)

in Table 1.

Scheme 2. Reagents and conditions: a) DIC, HONB, DMF, 48C, 36 h; b) 40%

TFA, CH₂Cl₂, 08C, 30 min; c) 7n NH₃in CH₃OH, 08C, 10 min; d) l-pGlu-OTcp

(14) or l-pAad-OPfp (15), DMF, 48C, 36 h.

with a solution of ammonia. The intermediate dipeptides were

not isolated; their immediate coupling with l-pGlu-OTcp (14)

or l-pAad-OPfp (15) in DMF produced the desired TRH ana-

logues (10h–n and 16a–m, respectively series 1 and 2).

We encountered low yields in using a similar strategy to

carry out the synthesis of TRH analogues 21a–e and 22a–l

(series 3 and 4). In these cases, coupling of dipeptides 12 or

19 with (1R)-3-Ocp-OH (20) using DIC and HONB provided a

complex mixture containing tripeptides in poor yields. We ob-

served that the use of 1-hydroxybenzotriazole (HOBt) as an

auxiliary nucleophile in place of HONB provided better results.

Thus, reaction of dipeptides 12 or 19 with 20 using DIC/HOBt

produced tripeptides 21a–e and 22a–l in satisfactory yields

(Scheme 3 and Scheme 4).

TRH analogues of series 1 generally exhibited weak binding

affinities for TRH-R1 and TRH-R2 (Table 1). Analogue 10a (R=

C₃H₇) displayed modest affinities for TRH-R1 and TRH-R2 (K_i=

0.32 and 0.17 mm, respectively), resulting in a ~16–17-fold de-

crease in binding affinity relative to TRH. However, 10a dis-

played promising agonist potency at TRH-R2 (EC₅₀=0.027 mm)

over TRH-R1 (EC₅₀=0.29 mm) and has 11-fold selectivity for

TRH-R2. Introduction of a bulkier and more lipophilic substitu-

ent at the C2 position of the His residue (compounds 10b–e)

yielded ligands with poor binding affinities and selectivities. In-

terestingly, analogue 10 f, with a cyclohexyl group at the C2

position of His, exhibited moderate binding affinity (TRH-R1:

K_i=0.34 mm; TRH-R2: Ki=0.14 mm) similar to that of 10a, but is

not a selective agonist (TRH-R1: EC₅₀=0.34 mm; TRH-R2: EC₅₀=

0.48 mm). To probe the steric requirements, we replaced the 1-

methyl group in analogues 10a–f with a 1-benzyl group. The

resulting TRH analogues 10h–n were found to be inactive at

both receptor subtypes. Replacement of pGlu with pAad and

attachment of a 1,2-dialkyl group on the His residue also re-

sulted in inactive analogues, 16a–m. These results provide an

initial insight into the minimum and maximum size require-

ment of the groups placed on the His residue in combination

with pGlu and its replacement by pAad in TRH peptides.

Simultaneous replacement of the His residue with 1-alkyl-

and 1,2-dialkyl-His residues, and pGlu with its bioisosteric re-

placement (1R)-3-Ocp, provided ligands with the best results

(series 3 and 4). Placement of a methyl group at the Nt(1) po-

sition of His provided analogue 21a [R=(1R)-3-Ocp, R¹=CH₃,

R²=H], which exhibits high binding affinities (K_i=0.17 and K_i=

0.016 mm at TRH-R1 and TRH-R2, respectively). Moreover, ana-

logue 21a displays potent agonist activity at TRH-R2 with high

potency (EC₅₀=0.0021 mm), which is ~1.4-fold higher than that

of TRH, and exhibits 24-fold selectivity for TRH-R2. Replace-

ment of a methyl group with an ethyl group as in analogue

21b [R=(1R)-3-Ocp, R¹=CH₃, R²=C₂H₅] resulted in higher

Pharmacology at TRH-R1 and TRH-R2

Synthesized TRH analogues were examined for their affinities

for TRH-R1 and TRH-R2 and their abilities to serve as agonists

Scheme 3. Reagents and conditions: a) DIC, HONB, DMF, 48C, 36 h; b) 40%

TFA, CH₂Cl₂, 08C, 30 min; c) 7n NH₃in CH₃OH, 08C, 10 min; d) (1R)-3-Ocp-OH

(20), DIC, HOBt, DMF, 48C, 36 h.

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Table 1. Binding affinity (K_i) and signaling (activation) potencies (EC₅₀) produced by TRH analogues (series 1–4) for TRH-R1 and TRH-R2.

K_i[mm]^[a]

TRH-R2

Fold

EC₅₀[mm]^[b]

Fold

Selectivity

Compd

R

R¹

R²

TRH-R1

Selectivity TRH-R1

TRH-R2

Series 1:

10a

10b

10c

10d

10e

10 f

10g

10h

10i

10j

10k

10l

10m

10n

l-pGlu CH₃

l-pGlu CH₂C₆H₅C₃H₇

l-pGlu CH₂C₆H₅CH(CH₃)₂

l-pGlu CH₂C₆H₅C(CH₃)₃

l-pGlu CH₂C₆H₅c-C₃H₅

l-pGlu CH₂C₆H₅c-C₅H₉

l-pGlu CH₂C₆H₅c-C₆H₁₁

C₃H₇

0.32 (0.24–0.35)

>50

11.0 (9.00–13.2)

>50

0.34 (0.28–0.38)

0.17 (0.11–0.23)

>50

0.14 (0.10–0.22)

>50

–

>50

–

>50

1.9

–

4.5

–

2.4

–

0.29 (0.18–0.47)

>50

0.34 (0.18–0.51)

>50

–

>50

–

>50

0.027 (0.015–0.050)

>50

0.48 (0.31–0.68)

>50

–

>50

–

>50

11

–

1.4

–

CH(CH₃)₂

C(CH₃)₃

c-C₃H₅

c-C₅H₉

c-C₆H₁₁

adamantan-1-yl >50

–

>50

–

>50

–

l-pGlu CH₂C₆H₅adamantan-1-yl >50

Series 2:

16a

16b

16c

16d

16e

16 f

16g

16h

16i

l-pAad CH₂C₆H₅C₃H₇

l-pAad CH₂C₆H₅CH(CH₃)₂

l-pAad CH₂C₆H₅C(CH₃)₃

l-pAad CH₂C₆H₅c-C₃H₅

l-pAad CH₂C₆H₅c-C₅H₉

l-pAad CH₂C₆H₅c-C₆H₁₁

>50

–

>50

–

>50

–

>50

–

>50

–

>50

–

>50

–

>50

–

>50

–

>50

–

>50

–

l-pAad CH₂C₆H₅adamantan-1-yl >50

l-pAad CH₃

C₃H₇

CH(CH₃)₂

C(CH₃)₃

c-C₅H₉

c-C₆H₁₁

–

16j

16k

16l

>50

–

>50

–

>50

–

>50

–

>50

–

>50

–

>50

–

>50

–

16m

adamantan-1-yl

Series 3:

21a

21b

21c

21d

21e

(R)-Ocp CH₃

(R)-Ocp C₂H₅

(R)-Ocp C₃H₇

(R)-Ocp CH(CH₃)₂

(R)-Ocp CH₂C₆H₅

H

0.17 (0.11–0.26)

0.10 (0.07–0.14)

8.1 (6.1–13.2)

>50

0.016 (0.007–0.027) 10.6

0.05 (0.03–0.09)

0.14 (0.06–0.20)

4.6 (3.7–9.1)

>50

0.0021 (0.0009–0.0035) 24

0.32 (0.28–0.37)

12.4 (6.8–16.4)

>50

3.2

1.5

–

0.030 (0.012–0.051)

1.2 (0.54–2.22)

>50

4.7

4

–

10.2 (8.1–12.2)

14.4 (10.8–20.6)

1.4

3.5 (2.5–7.8)

1.4 (0.44–2.2)

2.5

Series 4:

22a

22b

22c

22d

22e

22 f

22g

22h

22i

(R)-Ocp CH₂C₆H₅C₃H₇

0.08 (0.03–0.14)

9.6 (5.1–12.2)

–

0.35 (0.27–0.45)

10.2 (6.4–15.4)

–

4.4

1

–

1.2

–

0.10 (0.04–0.18)

3.6 (3.2–8.1)

–

0.015 (0.011–0.047)

1.1 (0.32–1.30)

–

6.6

3.2

–

2.7

–

(R)-Ocp CH₂C₆H₅CH(CH₃)₂

(R)-Ocp CH₂C₆H₅C(CH₃)₃

(R)-Ocp CH₂C₆H₅c-C₃H₅

(R)-Ocp CH₂C₆H₅c-C₅H₉

(R)-Ocp CH₂C₆H₅c-C₆H₁₁

(R)-Ocp CH₂C₆H₅adamantan-1-yl

–

7.8 (6.4–12.7)

–

>50

–

9.3 (6.5–12.8)

–

>50

–

2.7 (1.5–6.2)

–

>50

–

0.97 (0.52–1.34)

–

>50

–

>50

(R)-Ocp CH₃

C₃H₇

CH(CH₃)₂

C(CH₃)₃

c-C₆H₁₁

adamantan-1-yl >50

0.02 (0.01–0.03)

22j

22k

22l

–

>50

0.02 (0.01–0.03)

>50

0.01 (0.008–0.015)

TRH

0.003 (0.002–0.007)

[Nt(1)-Me-His]-TRH

0.003 (0.002–0.005) 0.003 (0.002–0.005)

–

0.0021 (0.002–0.005) 0.0002 (0.0002–0.0005)

–

[a] For binding, cells were incubated with 1 nm [³H]Nt(1)-Me-His-TRH in the absence or presence of various doses of unlabeled TRH analogues for 1 h at

378C. [b] For signaling, cells transfected with TRH-R1 or TRH-R2 and a CREB–luciferase reporter were incubated with various doses of TRH analogues for

6 h at 378C, and luciferase activity was measured. Experiments were performed with intact HEK293 cells. All data represent the mean ꢁSD of duplicate

determinations in three experiments; –: not determined.

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Thyrotropin-Releasing Hormone Analogues

binding affinity for TRH-R1 (K_i=0.10 mm). Analogue 21b exhib-

ited agonist potency (EC₅₀=0.030 mm) lower than that of 21a

and was moderately selective for TRH-R2 with 4.7-fold selectivi-

ty. The binding affinities and activation potencies were further

decreased with bulkier substituents placed at the Nt(1) posi-

tion of the His residue (analogues 21c and 21e). These ana-

logues also exhibited modest selectivity of 2.5–4-fold for TRH-

R2. These results are consistent with our previous findings that

the ring NH group of pGlu is not necessary for the binding of

the peptide to TRH receptors, and that modulation of the His

residue at the N1 or C2 positions imparts selectivity toward

TRH-R2.^[20–24]

control group. The observed potentiation of pentobarbital-in-

duced sleep activity may be caused by alterations in receptor

interactions due to the specific properties of the substitution

on the His residue of the TRH analogues. This altered interac-

tion may potentiate the action of pentobarbital, possibly by g-

aminobutyric acid (GABA)-mediated postsynaptic inhibition

through an allosteric modification of GABA receptors, or by en-

hancement of K⁺conductance or inhibition of Ca²⁺conduc-

tance in neurons.^[41–48]Most of the synthesized analogues of

series 3 and 4 showed positive results when evaluated in vivo

for antagonism of pentobarbital-induced sleeping time in

mice. The sleeping time after administration of analogues 21a,

21b, 21c, 22a, 22b, 22 f, and 22l were 28.2ꢁ1.7, 27.8ꢁ4.8,

34.4ꢁ1.4, 26.6ꢁ1.1, 32.6ꢁ6.1, 31.7ꢁ2.3, and 32.6ꢁ5.5 min,

respectively. The decrease in sleeping time caused by these an-

Finally, TRH analogues 22a–g (series 4) in which the His resi-

due is replaced with 1-benzyl-2-alkyl-His and pGlu is replaced

with (1R)-3-Ocp exhibited moderate binding affinities and acti-

vation potencies. For example, analogue 22a [R=(1R)-3-Ocp,

R¹=CH₂C₆H₅, R²=CH₂CH₂CH₃] exhibited K_ivalues of 0.08 and

0.35 mm to TRH-R1 and TRH-R2, respectively. Similarly, ana-

logues 22b [R=(1R)-3-Ocp, R¹=C₆H₅CH₂, R²=CH(CH₃)₂] and

22 f [R=(1R)-3-Ocp, R¹=C₆H₅CH₂, R²=c-C₆H₁₁] exhibited

modest binding affinities for TRH-R1 and TRH-R2. Analogue

22a also exhibited modest activation potency (EC₅₀=

0.015 mm) and has 6.6-fold specificity for TRH-R2. Analogues

22b and 22 f were also found to have 3.2-and 2.7-fold respec-

tive selectivity in activation potencies (EC₅₀) for TRH-R2. All TRH

analogues 22h–l, in which His is replaced with 1-methyl-2-

alkyl-His, and pGlu is replaced with (1R)-3-Ocp, were found to

be inactive at both TRH receptors (Table 1).

Table 2. Results of pentobarbital-induced sleeping time experiment in

mice for TRH analogues (series 1–4).^[a]

Compd

R

R¹

R²

Sleep

Time [min]

Decrease

in Sleep

Time [%]

Series 1:

10a

10b

10c

10d

10e

10 f

10g

10h

10i

10j

10k

10l

10m

10n

l-pGlu CH₃

C₃H₇

41.7ꢁ6.1^[b]

66.2ꢁ9.8

52.7

25.1

32.5

41.8

45.4

Antagonism of pentobarbital-induced sleeping time

CH(CH₃)₂

C(CH₃)₃

c-C₃H₅

c-C₅H₉

c-C₆H₁₁

59.6ꢁ5.5

One of the best documented CNS effects of TRH is its analeptic

action manifested by the decrease in barbiturate narcosis. TRH

is an effective analeptic agent, which reduces pentobarbital-in-

duced sleeping time by 50% or more following peripheral ad-

ministration of high doses or central injection of lower doses

in rats, rabbits, and monkeys.^[38,39]The synthesized TRH ana-

logues were evaluated in vivo by using the antagonism of a

pentobarbital-induced sleeping time model as described.^[40]

TRH analogues were injected intravenously through the tail

vein at a dose of 10 mmolkg^ꢀ1(equivalent to 3.7 mgkg^ꢀ1TRH).

Ten minutes after administration of the TRH analogue, each

animal received 50 mgkg^ꢀ1sodium pentobarbital intraperito-

neally. The sleeping time was recorded as the time elapsed

from the onset of loss of righting reflex until it returned

(Table 2).

51.4ꢁ5.1^[b]

48.2ꢁ10.1^[b]

43ꢁ8.1^[b]

51.3

24.2

adamantan-1-yl 66.9ꢁ5.3

l-pGlu CH₂C₆H₅C₃H₇

l-pGlu CH₂C₆H₅CH(CH₃)₂

l-pGlu CH₂C₆H₅C(CH₃)₃

l-pGlu CH₂C₆H₅c-C₃H₅

l-pGlu CH₂C₆H₅c-C₅H₉

l-pGlu CH₂C₆H₅c-C₆H₁₁

105.1ꢁ5.76

ꢀ18.8

ꢀ77.8

ꢀ60.5

ꢀ66.4

ꢀ84.7

ꢀ54.9

157.2ꢁ17^[b,c]

141.9ꢁ23.9^[b,c]

147.1ꢁ12.7^[b,c]

163.3ꢁ18.7^[b,c]

136.9ꢁ26.7^[b,c]

l-pGlu CH₂C₆H₅adamantan-1-yl 223.6ꢁ22.3^[b,c]ꢀ152.9

Series 2:

16a

16b

16c

16d

16e

16 f

16g

16h

16i

l-pAad CH₂C₆H₅C₃H₇

l-pAad CH₂C₆H₅CH(CH₃)₂

l-pAad CH₂C₆H₅C(CH₃)₃

l-pAad CH₂C₆H₅c-C₃H₅

l-pAad CH₂C₆H₅c-C₅H₉

l-pAad CH₂C₆H₅c-C₆H₁₁

69.3ꢁ7.3

ND

250.5ꢁ23.1

95.5ꢁ4.7

81.0ꢁ3.1

22.8

–

ꢀ178.7

ꢀ6.2

9.8

l-pAad CH₂C₆H₅adamantan-1-yl 87.5ꢁ5.0

2.6

Analogues 10a, 10b, 10d, 10e, and 10 f (series 1) showed a

significant decrease in sleeping time as compared with the

control group; however, their effects were not significantly dif-

ferent from those of the TRH-treated group. All other ana-

logues of this series were ineffective in decreasing sleep time.

Similarly, none of the analogues from series 2 showed positive

results in decreasing sleep time. Interestingly, some of the ana-

logues from series 1 and 2 were found to increase the sleeping

time significantly over control and TRH. In particular, analogues

10h, 10i, 10j, 10k, 10l, 10m, and 10n of series 1, and 16d of

series 2 increased the sleeping time significantly relative to the

l-pAad CH₃

C₃H₇

98.1ꢁ9.4

124.3ꢁ5.8

93.1ꢁ4.5

ꢀ9.1

ꢀ38.2

ꢀ3.6

ꢀ20.2

ꢀ14.8

15.4

CH(CH₃)₂

C(CH₃)₃

c-C₅H₉

c-C₆H₁₁

16j

16k

16l

108.1ꢁ14.4

103.2ꢁ9.1

16m

adamantan-1-yl 76.0ꢁ10.9

Series 3:

21a

21b

21c

21d

21e

(R)-Ocp CH₃

(R)-Ocp C₂H₅

(R)-Ocp C₃H₇

(R)-Ocp CH(CH₃)₂

(R)-Ocp CH₂C₆H₅

H

28.2ꢁ1.7^[b,c]

27.8ꢁ4.8^[b,c]

34.5ꢁ1.4^[b,c]

123.2ꢁ5.9

68.6

69

61.6

ꢀ36.8

60.1

35.8ꢁ1.9^[b,c]

ChemMedChem 2011, 6, 531 – 543

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535

R. Jain et al.

MED

Anticonvulsant activity

Table 2. (Continued)

Previous pharmacological studies have demonstrated that TRH

and its analogues inhibit pentylenetetrazole (PTZ)- or gluta-

mate-induced seizures in rat.^[17,50]Therefore, selected com-

pounds 21a–c and 22a,b were evaluated for anticonvulsant

activity against PTZ-induced seizures in mice according to a

method described earlier.^[51]The compounds were dissolved in

normal saline and administered at doses of 10 mmolkg^ꢀ1i.v.

After 10 min, animals were treated with PTZ (65 mgkg^ꢀ1i.p.)

and observed for 30 min after PTZ challenge. The results of

these experiments are listed in Table 3. Compounds 21a,b and

22a,b provided significant protection against clonic seizures

Compd

R

R¹

R²

Sleep

Time [min]

Decrease

in Sleep

Time [%]

Series 4:

22a

22b

22c

22d

22e

22 f

22g

22h

22i

22j

22k

22l

control

TRH

(R)-Ocp CH₂C₆H₅C₃H₇

26.6ꢁ1.1^[b,c]

32.7ꢁ6.1^[b,c]

91.6ꢁ3.8

104.9ꢁ5.5

ND

70.3

63.6

ꢀ1.9

ꢀ16.7

–

(R)-Ocp CH₂C₆H₅CH(CH₃)₂

(R)-Ocp CH₂C₆H₅C(CH₃)₃

(R)-Ocp CH₂C₆H₅c-C₃H₅

(R)-Ocp CH₂C₆H₅c-C₅H₉

(R)-Ocp CH₂C₆H₅c-C₆H₁₁

(R)-Ocp CH₂C₆H₅adamantan-1-yl

(R)-Ocp CH₃

Table 3. Effect of TRH and analogues on pentylenetetrazole (PTZ) and

maximal electric shock (MES)-induced seizures.^[a]

31.7ꢁ2.3^[b,c]

64.7

–

ND

C₃H₇

80.2ꢁ2.4

39.8ꢁ5.5^[b,c]

89.8ꢁ3.0

106.2ꢁ3.0

10.7

55.6

ꢀ0.01

ꢀ18.1

63.6

0.00

41.4

PTZ: Clonic Convulsions

Onset [s]

Frequency [min^ꢀ1

MES: Hind Limb

Extension

Duration [s]

CH(CH₃)₂

C(CH₃)₃

c-C₆H₁₁

adamantan-1-yl 32.6ꢁ5.5^[b,c]

Compd

]

saline

TRH

21a

21b

21c

67ꢁ5

1.3ꢁ0.16

1.3ꢁ0.49

1.3ꢁ0.33

1.3ꢁ0.21

1.60ꢁ0.06

1.20ꢁ0.20

13.6ꢁ1.10

11.2ꢁ0.50

10.0ꢁ0.60

11.3ꢁ1.4

79ꢁ7

89.8ꢁ6.8

153ꢁ7*^,**

149ꢁ8*^,**

68ꢁ3

52.6ꢁ4^[b]

[a] Pentobarbital was injected (50 mgkg^ꢀ1i.p.) 10 min after i.v. injection

of the TRH/TRH analogues. Sleep time was recorded as the time elapsed

from the onset of loss to regaining of the righting reflex. Six to eight

Swiss albino mice (body weight 20–30 g) were used in each group.

Values are expressed as the mean ꢁSEM; ND: not determined. [b] p<

0.05 relative to control using one-way ANOVA followed by post hoc

Tukey test. [c] p<0.05 relative to TRH using one-way ANOVA followed by

post hoc Tukey test.

12.3ꢁ0.90

12.0ꢁ0.40

11.0ꢁ0.70

22a

22b

179ꢁ8*^,**

224ꢁ53*^,**

[a] Mice (6–9 per group) were treated with saline, TRH, or compounds

(21a–c and 22a,b) at 10 mmolkg^ꢀ1i.v. 10 min prior to administration of

PTZ at 65 mgkg^ꢀ1i.p. or MES seizure, and were observed for 1 h. Values

are expressed as the mean ꢁSEM; statistical analysis was done with one-

way ANOVA followed by Tukey test: *p<0.05 versus saline and **p<0.05

versus TRH-treated group.

alogues was significantly different from control (89.1ꢁ4.6 min)

as well as from the TRH-treated group (52.6ꢁ3.5 min). Ana-

logues 21a and 21b of series 3 and 22a of series 4 were

found to be the most potent analogues, effecting decreases in

sleep time of 68.3, 68.8, and 70.1% relative to control. The ana-

leptic effects of these TRH analogues may be due to the en-

hancement of arousal or excitatory activity of TRH. TRH is well

known for this activity, which may be mediated through neu-

romodulation of various neurotransmitters such as acetylcho-

line, as it directly affects the septohippocampal cholinergic

pathway.^[1,2]In addition to cholinergic activity, blockade of leak-

ing K⁺current has also been proposed for this particular activi-

ty.^[49]

induced by PTZ (between 224ꢁ5 and 149ꢁ8 s, compared

with TRH: 79ꢁ7 s), whereas analogue 21c is inactive and did

not show any protection of PTZ-induced seizures. The selected

TRH analogues were also evaluated for maximal electric shock

(MES)-induced epilepsy using a protocol described earlier.^[52]

Treatment with TRH analogues at doses of 10 mmolkg^ꢀ1i.v.

was done 10 min prior to the administration of MES at 45 mA

for 0.2 s. The duration of tonic hind limb extension was record-

ed after MES, and the results are listed in Table 3. None of the

tested analogues showed protection in the MES model. The ef-

fectiveness of TRH analogues in PTZ- but not in MES-induced

seizure indicates their potential in clinical absence seizures.

TRH is known to provide protection against seizures induced

by kainic acid and glutamate, supporting the observed poten-

cy of TRH analogues in PTZ-induced seizures.^[50,53]TRH was also

shown to increase GABA release, and this may contribute to its

antiepileptic action.^[54]However, additional studies are required

to explore the exact mechanism of action of these analogues.

As evident from the receptor binding studies and prelimina-

ry in vivo screening using the pentobarbital-based sleep time

assay, TRH peptides that show good analeptic activity bind

with moderate affinities to TRH-R1 and TRH-R2, but exhibit se-

lective activation at TRH-R2, supporting our hypothesis that

TRH-R2-selective analogues exhibit potent CNS effects. The

outcome of these results show that several TRH analogues

(21a, 21b, 21c, 22a, 22b, 22 f, and 22l) are more potent than

TRH in their ability to decrease pentobarbital-induced sleeping

time in mice, which in turn prompted us to undertake further

evaluation of these compounds in various models of epilepsy.

The analogues were selected on the basis of their observed

biological activity and availability.

Conclusions

In the present study, we probed the structural requirements of

His and pGlu residues in receptor binding and biological activi-

ty by synthesizing new TRH analogues in which His residue

536

www.chemmedchem.org

ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2011, 6, 531 – 543

Thyrotropin-Releasing Hormone Analogues

phy was carried out on Merck silica gel (230–400 mesh) or neutral

alumina. IR spectra (n˜_maxin cm^ꢀ1) were recorded on a Nicolet FTIR

Impact 410 instrument either as neat or KBr pellets. ¹H and

¹³C NMR spectra were recorded on a 300 MHz Bruker FTNMR

(Avance DPX 300) spectrometer using tetramethylsilane (TMS) as

internal standard, and chemical shift values (d) are reported in

ppm. MS data were recorded on an HRMS (Finnigan Mat LCQ)

spectrometer (APCI/ESI). Elemental analyses were recorded on an

Elementar Vario EL spectrometer. The results of elemental analyses

were within ꢁ0.4% of theoretical values. Optical rotations were re-

corded on a PerkinElmer 241MC polarimeter. All final peptides

were checked for their homogeneity on a Shimadzu SPD-M20A

HPLC system using a Supelcosil^TMLC-8 column, 5 mm (25 cm ꢁ

4.6 mm i.d.). The peptides were analyzed by using an isocratic sol-

vent system of CH₃CN/H₂O/TFA (ratios as indicated) at a flow rate

was replaced with 1-alkyl-l-His/1,2-dialkyl-l-His; while pGlu res-

idue was either retained or replaced with (1R)-3-Ocp/l-pAad.

Analogues in which pGlu residue was replaced with (1R)-3-Ocp

and His was substituted at the N-1/C-2 positions displayed the

best pharmacological profile. Compounds 21a, 21b, and 22a

were all found to be full agonists with good to high functional

selectivity for TRH-R2. At the same time, analogue 21a exhibit-

ed highest binding affinity among the tested analogues. Ana-

leptic activity evaluation using pentobarbital-induced sleeping

time model revealed that some of the tested analogues were

more potent compared than TRH. Analogues 21a, 21b, 21c,

22a, 22b and 22 f produced 61 to 70% reduction in sleeping

time compared to control. The best analeptic profile was dis-

played by analogues 21a, 21b, and 22a by reducing the

sleeping time to almost 50% more than TRH. To our surprise,

some analogues like 10l, 10n and 16d increased the sleeping

time significantly. The results of this study indicate TRH-R2 as a

plausible receptor subtype mediating the analeptic effects of

TRH and its analogues. Analogues 21a, 21b, 21c, 22a, and

22b were also investigated against PTZ- and MES-induced seiz-

ures in mice, which reflect the generalized and partial seizures

that commonly occur in humans. Compounds 21a, 21b, 22a

and 22b provide significant protection against PTZ-induced

clonic seizures. However, all analogues failed to show protec-

tion against seizures induced by MES, suggesting their benefi-

cial effects in the generalized form of seizures. To what extent

the systemically active TRH analogues may have improved the

BBB permeability remains unanswered. As clear from the re-

sults of receptor binding and in vivo evaluation, TRH peptides

(21a, 21b, 22a and 22b) showing good analeptic/anticonvul-

sant activity bind with moderate affinities to TRH-R1 and TRH-

R2, but exhibited selectivities for TRH-R2, which is possibly re-

sponsible for the CNS effects of TRH. In conclusion, these ana-

logues are potent and functionally selective ligands and dis-

played potent analeptic and anticonvulsant activity when

tested in vivo in mice. For the first time, we have shown that

TRH-R2 selective analogues displayed potent CNS profile (anti-

epileptic activity), thereby making them promising candidates

for further development for the prophylactic management of

epilepsy.

of 1 mLmin^ꢀ1

.

General method for the synthesis of l-pGlu-l-His(1-methyl-

2-alkyl)-l-ProNH₂(10a–g)

4-Methylbenzhydrylamine (MBHA·HCl) resin (2, 300 mg, 0.31 mmol)

was swelled in CH₂Cl₂(5 mL) for 5 min, and then neutralized with

DIPEA (10% solution in CH₂Cl₂) for 5 min followed by washing with

DMF (2ꢁ10 mL) and CH₂Cl₂(10 mL). The reaction of the first amino

acid Boc-l-Pro-OH (3) was done in the presence of the coupling re-

agent TBTU and activator DIPEA (10% solution in CH₂Cl₂) for 2 h.

The Boc group was cleaved with TFA (20% solution in CH₂Cl₂) for

20 min. The amino acid linked resin was again neutralized with

DIPEA as described above to afford 5. Reaction of 5 with pre-

formed TBTU esters of Boc-l-His(1-methyl-2-alkyl)-OH 6a–g for 2 h

in DMF followed by removal of Boc group with TFA (20% solution

in CH₂Cl₂) and finally neutralization with DIPEA (10% in CH₂Cl₂) af-

forded dipeptides 7a–g. Upon coupling with Z-l-pGlu-OH (8) as

described above, the dipeptides 7a–g produced desired peptide

resins 9a–g. All coupling reactions were monitored quantitatively

by Kaiser’s test for completion. The dry peptide-linked resin 9a–g

was taken in a two-necked round-bottom flask equipped with a

drying tube and a rubber septum. A cleavage cocktail of EDT

(1 mL), thioanisole (2 mL), TFA (10 mL), and TFMSA (1.0 mL) were

added to the reaction vessel through the septum, and reaction

mixture was stirred for 10 min at 48C followed by stirring at ambi-

ent temperature for another 2 h. The crude peptide was separated

from the solid support by filtration, and the resin was washed with

TFA (3ꢁ4 mL). The solvent was removed under reduced pressure,

and the residue was neutralized with a saturated solution of am-

monium bicarbonate. The nonpolar impurities were removed by

extracting the aqueous layer with Et₂O (3ꢁ10 mL). The aqueous

layer was evaporated under reduced pressure to afford crude pep-

tide, which upon purification using column chromatography over

neutral alumina using CHCl₃/CH₃OH (4:1) as eluent, produced 10a–

g.

Experimental Section

Chemicals

Amino acids used in this study were purchased from Novabiochem

(Germany) or ChemImpex International (Wood Dale, IL, USA). All

other chemicals were purchased from Aldrich Chemical Ltd. (Mil-

waukee, WI, USA). Solvents used for peptide synthesis purification

were acquired from commercial sources, were of analytical or

HPLC grade, and were used without further purification unless oth-

erwise stated.

l-pGlu-l-His(1-methyl-2-propyl)-l-ProNH₂(10a). mp: 77–808C

1

(dec.); H NMR (CD₃OD): d=7.16 (s, 1H), 4.90 (m, 1H), 4.46 (m, 1H),

4.21 (m, 1H), 3.79 (m, 1H), 3.73 (s, 3H), 3.54 (m, 1H), 2.95 (m, 2H),

2.87 (t, 2H, J=7.8 Hz), 2.43–1.92 (m, 8H), 1.75 (m, 2H), 1.01 (t, 3H,

J=7.8 Hz); ¹³C NMR (CD₃OD): d=14.51, 21.72, 24.33, 25.16, 29.33,

29.38, 30.60, 31.83, 32.85, 46.81, 51.13, 55.27, 60.20, 118.47, 135.22,

147.45, 169.46, 171.39, 177.40, 177.59; MS (APCI): m/z 419 [M+1]⁺;

Anal. calcd for C₂₀H₃₀N₆O₄(418.5): C 57.40, H 7.23, N 20.08, found:

C 57.82, H 6.77, N 20.37; R_f=0.57 [CH₃OH/25% NH₄OH/CHCl₃

(20:2:78)]; HPLC: t_R=4.60 min, purity: 92.2% [CH₃CN/H₂O/TFA

(20:80:0.1%)].

Materials and methods

Peptides were routinely checked for their purity on pre-coated

silica gel G₂₅₄TLC plates (Merck), and the spots were visualized by

UV light and then by exposure to I₂vapors. Column chromatogra-

ChemMedChem 2011, 6, 531 – 543

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537

R. Jain et al.

MED

N 16.91; R_f=0.72 [CH₃OH/25% NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=

l-pGlu-l-His(1-methyl-2-isopropyl)-l-ProNH₂(10b). mp: 92–948C

(dec.); H NMR (CD₃OD): d=7.22 (s, 1H), 4.97 (m, 1H), 4.46 (m, 1H),

21.22 min, purity: 94.2% [CH₃CN/H₂O/TFA (20:80:0.1%)].

1

4.23 (m, 1H), 3.75 (m, 1H), 3.79 (s, 3H), 3.59 (m, 1H), 3.38 (m, 1H),

3.13 (m, 2H), 2.43–1.91 (m, 8H), 1.40 (m, 6H); ¹³C NMR (CD₃OD):

d=22.36, 24.33, 25.16, 29.18, 29.23, 33.11, 46.81, 51.20, 55.27,

60.20, 118.16, 136.32, 152.94, 169.66, 171.20, 177.50, 177.70; MS

(APCI): m/z 419 [M+1]⁺; Anal. calcd for C₂₀H₃₀N₆O₄(418.5): C 57.40,

H 7.23, N 20.08, found: 57.53, H 7.42, N 20.44; R_f=0.56 [CH₃OH/

25% NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=4.47 min, purity: 95.2%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

General method for the synthesis of l-pGlu-l-His(1-benzyl-2-

alkyl)-l-ProNH₂(10h–n)

The dipeptide salt (13a–g, 1 mmol) was neutralized by addition of

a solution of 7n NH₃in CH₃OH (10 mL) for 10 min at 08C. The sol-

vent was evaporated under reduced pressure to afford free dipep-

tides. A solution of free peptides (0.75 mmol) in DMF (4 mL) was

cooled to 48C, and l-pGlu-OTcp (14, 0.81 mmol) was added, and

the reaction mixture was stirred at 48C for 36 h. The solvent was

removed under reduced pressure, and the resulting residue was

purified by column chromatography over neutral alumina using

CHCl₃/CH₃OH (4:1) as eluent to provide tripeptides (10h–n).

l-pGlu-l-His(1-methyl-2-tert-butyl)-l-ProNH₂(10c). mp: 85–878C

1

(dec.); H NMR (CD₃OD): d=6.79 (s, 1H), 4.87 (m, 1H), 4.39 (m, 1H),

4.18 (m, 1H), 3.77 (m, 1H), 3.74 (s, 3H), 3.56 (m, 1H), 2.93 (m, 2H),

2.45–1.96 (m, 8H), 1.40 (m, 9H); ¹³C NMR (CD₃OD): d=24.40, 25.16,

28.35, 29.23, 29.38, 31.68, 32.56, 35.14, 46.81, 51.28, 55.27, 60.20,

117.97, 135.44, 154.42, 169.67, 170.65, 176.80, 177.21; MS (APCI):

m/z 433 [M+1]⁺; Anal. calcd for C₂₁H₃₂N₆O₄(432.2): C 58.32, H 7.46,

N, 19.43, found: C 58.77, H 7.29, N 19.76; R_f=0.60 [CH₃OH/25%

NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=4.64 min, purity: 93.8%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

l-pGlu-l-His(1-benzyl-2-propyl)-l-ProNH₂(10h). Yield: 18%; mp:

1

102–1048C (dec.); H NMR (CD₃OD): d=7.33 (m, 3H), 7.18 (m, 2H),

6.95 (s, 1H), 5.14 (s, 2H), 4.80 (m, 1H), 4.41 (m, 1H), 4.18 (m, 1H),

3.78 (m, 1H), 3.42 (m, 1H), 2.98 (m, 2H), 2.62 (t, 2H, J=7.5 Hz),

2.42–1.94 (m, 8H), 1.59 (m, 2H), 0.91 (t, 3H, J=7.2 Hz); MS (APCI):

m/z 495 [M+1]⁺; Anal. calcd for C₂₆H₃₄N₆O₄(494.6): C 63.14, H 6.93,

N 16.99, found: C 62.95, H 7.08, N 17.14; R_f=0.47 [CH₃OH/25%

NH₄OH/CHCl₃(18:2:80)]; HPLC: t_R=4.05 min, purity: 94.2%

[CH₃CN/H₂O/TFA (50:50:0.1%)].

l-pGlu-l-His(1-methyl-2-cyclopropyl)-l-ProNH₂(10d). mp: 64–

668C (dec.); ¹H NMR (CD₃OD): d=6.81 (s, 1H), 4.76 (m, 1H), 4.38

(m, 1H), 4.17 (m, 1H), 3.77 (m, 1H), 3.65 (s, 3H), 3.49 (m, 1H), 2.88

(m, 2H), 2.44–1.87 (m, 8H), 2.14 (m, 1H), 0.92 (m, 4H); ¹³C NMR

(CD₃OD): d=7.81, 8.51, 24.33, 25.16, 29.33, 29.38, 31.83, 33.27,

46.81, 51.19, 55.27, 60.20, 117.33, 136.66, 151.70, 169.66, 171.39,

177.40, 177.59; MS (APCI): m/z 417 [M+1]⁺; Anal. calcd for

C₂₀H₂₈N₆O₄(416.5): C 57.68, H 6.78, N 20.18, found: C 57.79, H 6.93,

N 20.29; R_f=0.62 [CH₃OH/25% NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=

4.23 min, purity: 97.5% [CH₃CN/H₂O/TFA (20:80:0.1%)].

l-pGlu-l-His(1-benzyl-2-isopropyl)-l-ProNH₂(10i). Yield: 19%;

1

mp: 95–978C (dec.); H NMR (CDCl₃): d=7.36 (m, 3H), 7.04 (m, 2H),

6.68 (s, 1H), 5.02 (s, 2H), 4.81 (m, 1H), 4.47 (m, 1H), 4.15 (m, 1H),

3.70 (m, 1H), 3.27 (m, 1H), 2.99 (m, 3H), 2.45–1.88 (m, 8H), 1.18 (m,

6H); MS (APCI): m/z 495 [M+1]⁺; Anal. calcd for C₂₆H₃₄N₆O₄(494.6):

C 63.14, H 6.93, N 16.99, found: C 63.38, H 7.24, N 16.82; R_f=0.45

[CH₃OH/25% NH₄OH/CHCl₃(18:2:80)]; HPLC: t_R=4.04 min, purity:

96.8% [CH₃CN/H₂O/TFA (50:50:0.1%)].

l-pGlu-l-His(1-methyl-2-cyclopentyl)-l-ProNH₂(10e). mp: 100–

1

1018C (dec.); H NMR (CD₃OD): d=6.82 (s, 1H), 4.80 (m, 1H), 4.38

l-pGlu-l-His(1-benzyl-2-tert-butyl)-l-ProNH₂(10j). Yield: 12%;

mp: 104–1068C (dec.); H NMR (CD₃OD): d=7.30 (m, 3H), 7.07 (m,

1

(m, 1H), 4.18 (m, 1H), 3.78 (m, 1H), 3.58 (s, 3H), 3.32 (m, 1H), 3.16

(m, 1H), 2.93 (m, 2H), 2.47–1.26 (m, 16H); ¹³C NMR (CD₃OD): d=

24.33, 24.76, 25.16, 29.23, 29.38, 31.68, 33.04, 33.42, 46.81, 51.25,

55.27, 60.20, 118.08, 134.20, 150.20, 168.50, 170.65, 176.90, 178.20;

MS (APCI): m/z 445 [M+1]⁺; Anal. calcd for C₂₂H₃₂N₆O₄(444.5): C

59.44, H 7.26, N 18.91, found: C 59.81, H 7.62, N 18.73; R_f=0.64

[CH₃OH/25% NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=5.80 min, purity:

99.1% [CH₃CN/H₂O/TFA (20:80:0.1%)].

2H), 6.79 (s, 1H), 5.35 (m, 2H), 4.81 (m, 1H), 4.39 (m, 1H), 4.15 (m,

1H), 3.78 (m, 1H), 3.56 (m, 1H), 2.94 (m, 2H), 2.40–1.95 (m, 8H),

1.36 (s, 9H); MS (APCI): m/z 509 [M+1]⁺; Anal. calcd for C₂₇H₃₆N₆O₄

(508.3): C 63.76, H 7.13, N 16.52, found: C 64.12, H 6.89, N 16.59;

R_f=0.45 [CH₃OH/25% NH₄OH/CHCl₃(14:2:84)]; HPLC: t_R=4.07 min,

purity: 95.1% [CH₃CN/H₂O/TFA (50:50:0.1%)].

l-pGlu-l-His(1-benzyl-2-cyclopropyl)-l-ProNH₂(10k). Yield: 13%;

mp: 84–868C (dec.); ¹H NMR (CD₃OD): d=7.29 (m, 5H), 6.96 (s,

1H), 5.24 (m, 2H), 4.78 (m, 1H), 4.39 (m, 1H), 4.16 (m, 1H), 3.76 (m,

1H), 3.43 (m, 1H), 2.93 (m, 2H), 2.40–1.93 (m, 8H), 1.83 (m, 1H),

0.90 (m, 4H); MS (APCI): m/z 493 [M+1]⁺; Anal. calcd for

C₂₆H₃₂N₆O₄(492.6): C 63.40, H 6.55, N 17.06, found: C 63.44, H 6.69,

N 16.94; R_f=0.55 [CH₃OH/25% NH₄OH/CHCl₃(14:2:84)]; HPLC: t_R=

4.02 min, purity: 95.0% [CH₃CN/H₂O/TFA (50:50:0.1%)].

l-pGlu-l-His(1-methyl-2-cyclohexyl)-l-ProNH₂(10 f). mp: 110–

112 8C (dec.); H NMR (CD₃OD): d=6.81 (s, 1H), 4.82 (m, 1H), 4.39

1

(m, 1H), 4.17 (m, 1H), 3.79 (m, 1H), 3.59 (s, 3H), 3.49 (m, 1H), 2.93

(m, 2H), 2.77 (m, 1H), 2.41–1.26 (m, 18H); ¹³C NMR (CD₃OD): d=

23.98, 25.10, 26.15, 26.85, 29.21, 32.05, 33.09, 34.07, 46.46, 51.10,

54.80, 60.60, 118.00 134.33 149.49 169.66, 171.39, 177.40, 178.10;

MS (APCI): m/z 459 [M+1]⁺; Anal. calcd for C₂₃H₃₄N₆O₄(458.6): C

60.24, H 7.47, N 18.33, found: C 60.29, H 7.78, N 17.98; R_f=0.68

[CH₃OH/25% NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=7.10 min, purity:

97.1% [CH₃CN/H₂O/TFA (20:80:0.1%)].

l-pGlu-l-His(1-benzyl-2-cyclopentyl)-l-ProNH₂(10l). Yield: 27%;

1

mp: 110–1128C (dec.); H NMR (CD₃OD): d=7.30 (m, 3H), 7.15 (m,

2H), 6.92 (s, 1H), 5.15 (s, 2H), 4.80 (m, 1H), 4.39 (m, 1H), 4.16 (m,

1H), 3.77 (m, 1H), 3.47 (m, 1H), 2.96 (m, 3H), 2.30–1.28 (m, 16); MS

(APCI): m/z 521 [M+1]⁺; Anal. calcd for C₂₈H₃₆N₆O₄(520.6): C 64.60,

H 6.97, N 16.14, found: C 64.89, H 7.11, N 15.98; R_f=0.40 [CH₃OH/

25% NH₄OH/CHCl₃(14:2:84)]; HPLC: t_R=4.58 min, purity: 98.5%

[CH₃CN/H₂O/TFA (50:50:0.1%)].

l-pGlu-l-His[1-methyl-2-(adamantan-1-yl)]-l-ProNH₂(10g). mp:

121–1238C (dec.); H NMR (CD₃OD): d=6.72 (s, 1H), 4.64 (m, 1H),

1

4.39 (m, 1H), 4.17 (m, 1H), 3.82 (m, 1H), 3.77 (s, 3H), 3.53 (m, 1H),

2.91 (m, 2H), 2.46–2.24 (m, 4H), 2.21–1.81 (m, 19H); ¹³C NMR

(CD₃OD): d=25.10, 25.1, 29.3, 29.6, 31.80, 35.06, 36.01, 39.55, 40.39,

46.81, 51.34, 55.30, 60.10, 117.94, 135.84, 149.60, 169.67, 171.45,

177.28, 177.60; MS (APCI): m/z 511 [M+1]⁺; Anal. calcd for

C₂₇H₃₈N₆O₄(510.6): C 63.51, H 7.50, N 16.46, found: C 63.89, H 7.22,

l-pGlu-l-His(1-benzyl-2-cyclohexyl)-l-ProNH₂(10m). Yield: 23%;

mp: 120–1228C (dec.); ¹H NMR (CDCl₃): d 7.34 (m, 3H), 7.06 (m,

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Thyrotropin-Releasing Hormone Analogues

2H), 6.67 (s, 1H), 5.15 (s, 2H), 4.89 (m, 1H), 4.46 (m, 1H), 4.14 (m,

1H), 3.69 (m, 1H), 3.26 (m, 1H), 2.97 (m, 2H), 2.56 (m, 1H), 2.47–

1.22 (m, 18H); MS (APCI): m/z 535 [M+1]⁺; Anal. calcd for

C₂₉H₃₈N₆O₄(534.7): C 65.15, H 7.16, N 15.72, found: C 64.65, H 6.98,

N 16.20; R_f=0.40 [CH₃OH/25% NH₄OH/CHCl₃(14:2:84)]; HPLC: t_R=

4.77 min, purity: 92.8% [CH₃CN/H₂O/TFA (50:50:0.1%)].

N 16.51; R_f=0.54 [CH₃OH/25% NH₄OH/CHCl₃(16:2:82)]; HPLC: t_R=

4.51 min, purity: 96.6% [CH₃CN/H₂O/TFA (40:60:0.1%)].

l-pAad-l-His(1-benzyl-2-cyclopentyl)-l-ProNH₂(16e). Yield: 28%;

mp: 80–838C (dec.); ¹H NMR (CD₃OD): d=7.31 (m, 3H), 7.14 (m,

2H), 6.93 (s, 1H), 4.88 (s, 2H), 4.81 (m, 1H), 4.38 (m, 1H), 4.01 (m,

1H), 3.75 (m, 1H), 3.43 (m, 1H), 3.05 (m, 1H), 2.96 (m, 2H), 2.28–

1.60 (m, 18H); MS (APCI): m/z 535 [M+1]⁺; Anal. calcd for

C₂₉H₃₈N₆O₄(534.7): C 65.15, H 7.16, N 15.72, found: C 65.34, H 6.94,

N 16.07; R_f=0.58 [CH₃OH/25% NH₄OH/CHCl₃(16:2:82)]; HPLC: t_R=

5.39 min, purity: 97.8% [CH₃CN/H₂O/TFA (40:60:0.1%)].

l-pGlu-l-His[1-benzyl-2-(adamantan-1-yl)]-l-ProNH₂(10n). Yield:

13%; mp: 116–1188C (dec.); ¹H NMR (CD₃OD): d=7.29 (m, 3H),

7.04 (m, 2H), 6.77 (s, 1H), 5.41 (m, 2H), 4.80 (m, 1H), 4.39 (m, 1H),

4.15 (m, 1H), 3.78 (m, 1H), 3.57 (m, 1H), 2.95 (m, 2H), 2.44–1.28 (m,

23H); MS (APCI): m/z 587 [M+1]⁺; Anal. calcd for C₃₃H₄₂N₆O₄

(586.6): C 67.55, H 7.22, N 14.32, found: C 67.88, H 7.66, N 14.57;

R_f=0.60 [CH₃OH/25% NH₄OH/CHCl₃(14:2:84)]; HPLC: t_R=5.92 min,

purity: 92.8% [CH₃CN/H₂O/TFA (50:50:0.1%)].

l-pAad-l-His(1-benzyl-2-cyclohexyl)-l-ProNH₂(16 f). Yield: 23%;

mp: 88–908C (dec.); ¹H NMR (CD₃OD): d=7.31 (m, 3H), 7.16 (m,

2H), 6.92 (s, 1H), 5.14 (s, 2H), 4.81 (m, 1H), 4.38 (m, 1H), 4.01 (m,

1H), 3.78 (m, 1H), 3.45 (m, 1H), 2.96 (m, 2H), 2.82 (m, 1H, CH),

2.29–1.28 (m, 20H); MS (APCI): m/z 549 [M+1]⁺; Anal. calcd for

C₃₀H₄₀N₆O₄(548.7): C 65.67, H 7.35, N 15.32, found: C 65.98, H 7.23,

N 14.97; R_f=0.62 [CH₃OH/25% NH₄OH/CHCl₃(16:2:82)]; HPLC: t_R=

6.32 min, purity: 93.1% [CH₃CN/H₂O/TFA (40:60:0.1%)].

General method for the synthesis of l-pAad-l-His(1,2-di-

alkyl)-l-ProNH₂(16a–m)

The dipeptide salt (13a–m, 1 mmol) was neutralized by addition of

a solution of 7n NH₃in CH₃OH (10 mL) for 10 min at 08C. A solu-

tion of free peptide in DMF (4 mL) was cooled to 48C, and pAad-

OPfp (15, 0.81 mmol) was added, and the reaction mixture was

stirred at 48C for 36 h. The solvent was removed under reduced

pressure, and the resulting residue was purified by column chro-

matography over neutral alumina using CH₃OH/CHCl₃(2:8) as

eluent to afford tripeptides 16a–m.

l-pAad-l-His[1-benzyl-2-(adamantan-1-yl)]-l-ProNH₂(16g). Yield:

17%; mp: 96–988C (dec.); H NMR (CD₃OD): d=7.29 (m, 3H), 7.02

1

(m, 2H), 6.76 (s, 1H), 5.37 (m, 2H), 4.71 (m, 1H), 4.38 (m, 1H), 3.99

(m, 1H), 3.80 (m, 1H), 3.58 (m, 1H), 2.94 (m, 2H), 2.27 (m, 4H),

2.14–1.22 (m, 21H); MS (APCI): m/z 601 [M+1]⁺; Anal. calcd for

C₃₄H₄₄N₆O₄(600.3): C 67.98, H 7.38, N 13.99, found: C 68.25, H 7.18,

N 13.50; R_f=0.52 [CH₃OH/25% NH₄OH/CHCl₃(8:2:90)]; HPLC: t_R=

9.49 min, purity: 94.4% [CH₃CN/H₂O/TFA (40:60:0.1%)].

l-pAad-l-His(1-benzyl-2-propyl)-l-ProNH₂(16a). Yield: 26%; mp:

74–768C (dec.); ¹H NMR (CD₃OD): d=7.31 (m, 3H), 7.16 (m, 2H),

6.93 (s, 1H), 5.12 (s, 2H), 4.79 (m, 1H), 4.40 (m, 1H), 4.00 (m, 1H),

3.81 (m, 1H), 3.44 (m, 1H), 2.95 (m, 2H), 2.59 (t, 2H, J=7.5 Hz),

2.29–1.66 (m, 10H), 1.58 (m, 2H, CH₂), 0.89 (t, 3H, J=7.2 Hz); MS

(APCI): m/z 509 [M+1]⁺; Anal. calcd for C₂₇H₃₆N₆O₄(508.6): C 63.76,

H 7.13, N 16.52, found: C 64.07, H 7.31, N 16.19; R_f=0.48 [CH₃OH/

25% NH₄OH/CHCl₃(16:2:82)]; HPLC: t_R=4.71 min, purity: 99.8%

[CH₃CN/H₂O/TFA (40:60:0.1%)].

l-pAad-l-His(1-methyl-2-propyl)-l-ProNH₂(16h). Yield: 32%; mp:

64–668C (dec.); H NMR (CDCl₃): d=8.19 (bs, 1H), 6.63 (s, 1H), 4.77

1

(m, 1H), 4.50 (m, 1H), 4.02 (m, 1H), 3.67 (m, 1H), 3.52 (s, 3H), 3.21

(m, 1H), 2.97 (m, 2H), 2.53 (t, 2H, J=7.5 Hz), 2.42–1.72 (m, 10H),

1.58 (m, 2H, CH₂), 0.90 (t, 3H, J=7.2 Hz); MS (APCI): m/z 433

[M+1]⁺; Anal. calcd for C₂₁H₃₂N₆O₄(432.5): C 58.32, H 7.46, N 19.43,

found: C 58.64, H 7.57, N 19.11; R_f=0.55 [CH₃OH/25% NH₄OH/

CHCl₃(20:2:78)]; HPLC: t_R=4.93 min, purity: 94.9% [CH₃CN/H₂O/

TFA (20:80:0.1%)].

l-pAad-l-His(1-benzyl-2-isopropyl)-l-ProNH₂(16b). Yield: 13%;

mp: 68–708C (dec.); ¹H NMR (CD₃OD): d=7.31 (m, 3H), 7.14 (m,

2H), 6.92 (s, 1H), 5.15 (s, 2H), 4.82 (m, 1H), 4.38 (m, 1H), 4.01 (m,

1H), 3.79 (m, 1H), 3.45 (m, 1H), 3.04 (m, 1H), 2.97 (m, 2H), 2.29–

1.68 (m, 10H), 1.18 (m, 6H); MS (APCI): m/z 509 [M+1]⁺; Anal.

calcd for C₂₇H₃₆N₆O₄(508.6): C 63.76, H 7.13, N 16.52, found: C

l-pAad-l-His(1-methyl-2-isopropyl)-l-ProNH₂(16i). Yield: 38%;

mp: 70–718C (dec.); ¹H NMR (CD₃OD): d=7.16 (s, 1H), 4.96 (m,

1H), 4.45 (m, 1H), 4.05 (m, 1H), 3.81 (m, 1H), 3.77 (s, 3H), 3.56 (m,

1H), 3.37 (m, 1H), 3.11 (m, 2H), 2.28 (m, 4H), 2.09–1.72 (m, 6H),

1.38 (m, 6H); MS (APCI): m/z 433 [M+1]⁺; Anal. calcd for

C₂₁H₃₂N₆O₄(432.5): C 58.32, H 7.46, N 19.43, found: C 58.44, H 7.59,

N 19.58; R_f=0.55 [CH₃OH/25% NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=

4.85 min, purity: 95.5% [CH₃CN/H₂O/TFA (20:80:0.1%)].

64.07,

H 7.31, N 16.19; R_f=0.46 [CH₃OH/25% NH₄OH/CHCl₃

(16:2:82)]; HPLC: t_R=4.63 min, purity: 95.9% [CH₃CN/H₂O/TFA

(40:60:0.1%)].

l-pAad-l-His(1-benzyl-2-tert-butyl)-l-ProNH₂(16c). Yield: 19%;

mp: 77–808C (dec.); ¹H NMR (CD₃OD): d=7.29 (m, 3H), 7.07 (m,

2H), 6.81 (s, 1H), 5.36 (s, 2H), 4.81 (m, 1H), 4.39 (m, 1H), 4.00 (m,

1H), 3.81 (m, 1H), 3.58 (m, 1H), 2.96 (m, 2H), 2.30–1.70 (m, 10H),

1.37 (s, 9H); MS (APCI): m/z 523 [M+1]⁺; Anal. calcd for C₂₈H₃₈N₆O₄

(522.6): C 64.35, H 7.33, N 16.08, found: C 64.46, H 7.55, N 15.79;

R_f=0.42 [CH₃OH/25% NH₄OH/CHCl₃(16:2:82)]; HPLC: t_R=4.82 min,

purity: 98.9% [CH₃CN/H₂O/TFA (40:60:0.1%)].

l-pAad-l-His(1-methyl-2-tert-butyl)-l-Pro-NH₂(16j). Yield: 38%;

mp: 74–758C (dec.); ¹H NMR (CD₃OD): d=7.20 (s, 1H), 5.00 (m,

1H), 4.43 (m, 1H), 4.05 (m, 1H), 3.93 (s, 3H), 3.77 (m, 1H), 3.59 (m,

1H), 3.09 (m, 2H), 2.45–1.72 (m, 10H), 1.52 (s, 9H); MS (APCI): m/z

447 [M+1]⁺; Anal. calcd for C₂₂H₃₄N₆O₄(446.5): C 59.17, H 7.67, N

18.82, found: C 58.99, H 7.53, N 19.11; R_f=0.57 [CH₃OH/25%

NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=5.42 min, purity: 99.2%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

l-pAad-l-His(1-benzyl-2-cyclopropyl)-l-ProNH₂(16d). Yield: 24%;

mp: 72–748C (dec.); ¹H NMR (CD₃OD): d=7.27 (m, 3H), 7.11 (m,

2H), 6.94 (s, 1H), 5.22 (s, 2H), 4.77 (m, 1H), 4.37 (m, 1H), 3.99 (m,

1H), 3.75 (m, 1H), 3.43 (m, 1H), 2.92 (m, 2H), 2.28–1.67 (m, 11H),

0.88 (m, 4H); MS (APCI): m/z 507 [M+1]⁺; Anal. calcd for

C₂₇H₃₄N₆O₄(506.6): C 64.01, H 6.76, N 16.59, found: C 63.96, H 6.64,

l-pAad-l-His(1-methyl-2-cyclopentyl)-l-ProNH₂(16k). Yield: 27%;

mp: 77–798C (dec.); ¹H NMR (CD₃OD): d=6.83 (s, 1H), 4.82 (m,

1H), 4.38 (m, 1H), 4.02 (m, 1H), 3.81 (m, 1H), 3.59 (s, 3H), 3.55 (m,

1H), 3.17 (m, 1H), 2.93 (m, 2H), 2.27–1.28 (m, 18H); MS (APCI): m/z

459 [M+1]⁺; Anal. calcd for C₂₃H₃₄N₆O₄(458.7): C 60.24, H 7.47, N

18.33, found: C 60.22, H 7.42, N 18.31; R_f=0.60 [CH₃OH/25%

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R. Jain et al.

MED

NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=6.98 min, purity: 96.5%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

116.19, 136.01, 139.60, 168.86, 174.53, 177.07, 216.50; MS (APCI):

m/z 404 [M+1]⁺; Anal. calcd for C₂₀H₂₉N₅O₄(403.5): C 59.54, H 7.24,

N 17.36, found: C 59.83, H 7.49, N 17.39; R_f=0.55 [CH₃OH/25%

NH₄OH/CHCl₃(12:2:86)]; HPLC: t_R=4.95 min, purity: 99.6%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

l-pAad-l-His(1-methyl-2-cyclohexyl)-l-ProNH₂(16l): Yield: 22%;

mp: 82–848C (dec.); ¹H NMR (CD₃OD): d=6.77 (s, 1H), 4.80 (m,

1H), 4.36 (m, 1H), 4.02 (m, 1H), 3.81 (m, 1H), 3.57 (s, 3H), 3.53 (m,

1H), 2.92 (m, 2H), 2.73 (m, 1H), 2.30–1.26 (m, 20H); MS (APCI): m/z

473 [M+1]⁺; Anal. calcd for C₂₄H₃₆N₆O₄(472.7): C 61.00, H 7.68, N

17.78, found: C 61.15, H 7.98, N 17.47; R_f=0.62 [CH₃OH/25%

NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=8.95 min, purity: 97.8%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

(1R)-3-Ocp-l-His(1-isopropyl)-l-ProNH₂(21d). Yield: 20%; mp:

1

88–908C (dec.); H NMR (CD₃OD): d=7.64 (s, 1H), 7.11 (s, 1H), 4.80

(m, 1H), 4.40 (m, 1H), 3.77 (m, 1H), 3.36 (m, 1H), 3.04 (m, 1H), 2.96

(m, 3H), 2.35–1.95 (m, 10H), 1.46 (d, 6H, J=6.0 Hz); ¹³C NMR

(CD₃OD): d=24.33, 25.84, 26.46, 29.38, 33.09, 37.57, 40.16, 42.36,

46.81, 47.95, 51.08, 60.20, 112.02, 133.40, 137.63, 169.49, 174.84,

177.84, 177.51, 216.25; MS (APCI): m/z 404 [M+1]⁺; Anal. calcd for

C₂₀H₂₉N₅O₄(403.5): C 59.54, H 7.24, N 17.36, found: C 59.68, H 6.96,

N 17.74; R_f=0.53 [CH₃OH/25% NH₄OH/CHCl₃(12:2:86)]; HPLC: t_R=

4.79 min, purity: 97.6% [CH₃CN/H₂O/TFA (20:80:0.1%)].

l-pAad-l-His[1-methyl-2-(adamantan-1-yl)]-l-ProNH₂

(16m).

Yield: 15%; mp: 88–908C (dec.); ¹H NMR (CDCl₃): d=6.61 (s, 1H),

4.83 (m, 1H), 4.46 (m, 1H), 4.00 (m, 1H), 3.73 (s, 3H), 3.66 (m, 1H),

3.31 (m, 1H), 2.91 (m, 2H), 2.35 (m, 4H), 2.18–1.25 (m, 21H); MS

(APCI): m/z 525 [M+1]⁺; Anal. calcd for C₂₈H₄₀N₆O₄(524.6): C 64.10,

H 7.68, N 16.02, found: C 63.92, H 7.96, N 15.73; R_f=0.59 [CH₃OH/

25% NH₄OH/CHCl₃(20:2:78)]; HPLC: t_R=24.78 min, purity: 94.7%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

(1R)-3-Ocp-l-His(1-benzyl)-l-ProNH₂(21e). Yield: 18%; mp: 96–

1

988C (dec.); H NMR (CDCl₃): 7.71 (s, 1H), 7.35 (m, 5H), 7.05 (s, 1H),

5.19 (m, 2H), 4.80 (m, 1H), 4.44 (m, 1H), 3.79 (m, 1H), 3.35 (m, 1H),

3.07 (m, 1H), 2.96 (m, 2H), 2.35–1.71 (m, 10H); ¹³C NMR (CD₃OD):

d=23.87, 24.84, 29.38, 32.00, 37.30, 39.95, 42.05, 42.81, 50.10,

50.94, 60.59, 117.19, 127.13, 128.20, 128.99, 136.18, 137.63, 139.05,

169.27, 174.57, 177.59, 216.05; MS (APCI): m/z 452 [M+1]⁺; Anal.

calcd for C₂₄H₂₉N₅O₄(451.5): C 63.84, H 6.47, N 15.51, found: C

Synthesis of (1R)-3-Ocp-l-His(1-alkyl)-l-ProNH₂(21a–e)

The dipeptide salt (19a–e, 1 mmol) was neutralized with a solution

of 7n NH₃in CH₃OH (10 mL) for 10 at 08C min. HOBt (1.1 mmol)

was added to a solution of free dipeptide in anhydrous DMF

(10 mL). The reaction mixture was cooled to ꢀ108C, and DIC

(1.1 mmol) was added in one portion. The reaction mixture was

stirred for an additional 5 min at ꢀ108C. (1R)-3-Ocp-OH (20,

1 mmol) was then added to the reaction mixture, and stirring was

continued for an additional 36 h at 48C. The solvent was evaporat-

ed under reduced pressure, and the crude product was purified by

column chromatography on neutral alumina using CH₃OH/CHCl₃

(10:90) as eluent to afford tripeptides 21a–e.

64.06,

H 6.20, N 15.65; R_f=0.59 [CH₃OH/25% NH₄OH/CHCl₃

(12:2:86)]; HPLC: t_R=8.43 min, purity: 97.0% [CH₃CN/H₂O/TFA

(20:80:0.1%)].

General method for the synthesis of (1R)-3-Ocp-l-His(1,2-di-

alkyl)-l-ProNH₂(22a–l)

Protected dipeptide (12, 0.73 mmol) was treated with a solution of

40% TFA in CH₂Cl₂(15 mL) at 48C for 30 min. The salt was neutral-

ized with a solution of 7n NH₃in CH₃OH (10 mL) for 10 min at

08C. HOBt (1.1 mmol) was added to a solution of free dipeptides in

anhydrous DMF (10 mL). The reaction mixture was cooled to

ꢀ108C, and DIC (1.1 mmol) was added in one portion. The reaction

mixture was stirred for an additional 5 min at ꢀ108C. (1R)-3-Ocp-

OH (20, 1 mmol) was then added to the reaction mixture, and stir-

ring was continued for an additional 36 h at 48C. The solvent was

removed, and the crude product was purified by column chroma-

tography on neutral alumina using CH₃OH/CHCl₃(10:90) to afford

tripeptides 22a–l.

(1R)-3-Ocp-l-His(1-methyl)-l-ProNH₂(21a). Yield: 30%; mp: 101–

1038C (dec.); ¹H NMR (CD₃OD): d=7.50 (s, 1H), 6.94 (s, 1H), 4.79

(m, 1H), 4.39 (m, 1H), 3.72 (m, 1H), 3.67 (s, 3H), 3.43 (m, 1H), 3.05

(m, 1H), 2.96 (m, 2H), 2.35–1.91 (m, 10H); ¹³C NMR (CD₃OD): d=

24.57, 25.84, 29.38, 31.97, 33.02, 37.57, 40.16, 42.36, 46.81, 50.51,

60.20, 117.33, 137.31, 139.77, 169.40, 174.84, 177.59, 216.11; MS

(APCI): m/z 376 [M+1]⁺; Anal. calcd for C₁₈H₂₅N₅O₄(375.4): C 57.59,

H 6.71, N 18.65, found: C 57.97, H 6.45, N 18.51; R_f=0.44 [CH₃OH/

25% NH₄OH/CHCl₃(12:2:86)]; HPLC: t_R=3.98 min, purity: 99.5%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

(1R)-3-Ocp-l-His(1-benzyl-2-propyl)-l-ProNH₂(22a). Yield: 27%;

1

(1R)-3-Ocp-l-His(1-ethyl)-l-ProNH₂(21b). Yield: 49%; mp: 96–

988C (dec.); H NMR (CD₃OD): d=7.58 (s, 1H), 7.03 (s, 1H), 4.80 (m,

mp: 110–1128C (dec.); H NMR (CD₃OD): d=7.31 (m, 3H), 7.17 (m,

1

2H), 6.93 (s, 1H), 5.13 (s, 2H), 4.81 (m, 1H), 4.39 (m, 1H), 3.78 (m,

1H), 3.46 (m, 1H), 3.03 (m, 1H), 2.95 (m, 2H), 2.61 (t, 2H, J=

7.8 Hz), 2.34–1.93 (m, 10H), 1.57 (m, 2H), 0.89 (t, 3H, J=7.2 Hz);

¹³C NMR (CD₃OD): d=14.44, 21.69, 24.33, 25.84, 29.38, 29.88, 32.20,

37.57, 40.16, 42.36, 46.81, 48.60, 51.06, 60.20, 117.97, 127.15,

128.82, 128.20, 135.48, 135.65, 148.95, 169.28, 174.64, 177.59,

216.11; MS (APCI): m/z 494 [M+1]⁺; Anal. calcd for C₂₇H₃₅N₅O₄

(493.6): C 65.70, H 7.15, N 14.19, found: 65.94, H 7.29, N 14.28; R_f=

0.52 [CH₃OH/25% NH₄OH/CHCl₃(12:2:86)]; HPLC: t_R=5.14 min,

purity: 99.0% [CH₃CN/H₂O/TFA (40:60:0.1%)].

1H), 4.39 (m, 1H), 4.00 (q, 2H, J=7.2 Hz), 3.75 (m, 1H), 3.38 (m,

1H), 3.06 (m, 1H), 2.96 (m, 2H), 2.34–1.90 (m, 10H), 1.40 (t, 3H, J=

7.2 Hz); ¹³C NMR (CD₃OD): d=15.72, 24.10, 26.20, 29.70, 31.67,

38.10, 39.95, 41.34, 42.70, 46.60, 50.99, 60.45, 115.35, 135.84,

138.74, 169.27, 174.0, 177.10, 215.87; MS (APCI): m/z 390 [M+1]⁺;

Anal. calcd for C₁₉H₂₇N₅O₄(389.5): C 58.60, H 6.99, N 17.98, found:

C 58.91, H 7.07, N 17.61; R_f=0.48 [CH₃OH/25% NH₄OH/CHCl₃

(12:2:86)]; HPLC: t_R=4.30 min, purity: 97.4% [CH₃CN/H₂O/TFA

(20:80:0.1%)].

(1R)-3-Ocp-l-His(1-propyl)-l-ProNH₂(21c). Yield: 31%; mp: 90–

928C (dec.); H NMR (CD₃OD): d=7.56 (s, 1H), 7.01 (s, 1H), 4.78 (m,

1H), 4.40 (m, 1H), 3.92 (t, 2H, J=6.6 Hz), 3.78 (m, 1H), 3.40 (m,

1H), 3.06 (m, 1H), 2.96 (m, 2H), 2.35–1.95 (m, 10H), 1.79 (m, 2H),

0.89 (t, 3H, J=6.9 Hz); ¹³C NMR (CD₃OD): d=11.05, 23.64, 24.08,

25.94, 29.08, 32.10, 37.12, 40.44, 42.97, 46.50, 48.24, 51.01, 60.20,

(1R)-3-Ocp-l-His(1-benzyl-2-isopropyl)-l-ProNH₂(22b). Yield:

24%; mp: 120–1228C (dec.); ¹H NMR (CD₃OD): d=7.30 (m, 3H),

7.16 (m, 2H), 6.90 (s, 1H), 5.15 (s, 2H), 4.83 (m, 1H), 4.38 (m, 1H),

3.81 (m, 1H), 3.51 (m, 1H), 3.05 (m, 2H), 2.95 (m, 2H), 2.34–1.93 (m,

10H), 1.17 (d, 6H, 2ꢁJ=6.9 Hz); ¹³C NMR (CD₃OD): d=22.20, 24.17,

25.80, 28.50, 29.31, 33.19, 37.54, 40.10, 42.48, 46.98, 49.35, 51.13,

1

540

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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ChemMedChem 2011, 6, 531 – 543

Thyrotropin-Releasing Hormone Analogues

60.60, 116.20, 127.06, 128.43, 128.50, 134.14, 135.05, 153.87, 169.50,

174.37, 177.55, 215.13; MS (APCI): m/z 494 [M+1]⁺; Anal. calcd for

C₂₇H₃₅N₅O₄(493.6): C 65.70, H 7.15, N 14.19, found: 65.79, H 7.38, N

14.23; R_f=0.50 [CH₃OH/25% NH₄OH/CHCl₃(10:2:88)]; HPLC: t_R=

4.95 min, purity: 99.4% [CH₃CN/H₂O/TFA (40:60:0.1%)].

N 12.29; R_f=0.62 [CH₃OH/25% NH₄OH/CHCl₃(10:2:88)]; HPLC: t_R=

11.07 min, purity: 98.3% [CH₃CN/H₂O/TFA (40:60:0.1%)].

(1R)-3-Ocp-l-His(1-methyl-2-propyl)-l-ProNH₂(22h). Yield: 28%;

1

mp: 100–1038C (dec.); H NMR (CD₃OD): d=6.81 (s, 1H), 4.77 (m,

1H), 4.39 (m, 1H), 3.78 (m, 1H), 3.57 (s, 3H), 3.45 (m, 1H), 3.08 (m,

1H), 2.96 (m, 2H), 2.64 (t, 2H, J=7.5 Hz), 2.35–1.94 (m, 10H), 1.67

(m, 2H), 0.96 (t, 3H, J=7.2 Hz); MS (APCI): m/z 418 [M+1]⁺; Anal.

calcd for C₂₁H₃₁N₅O₄(417.5): C 60.41, H 7.48, N 16.77, found: C

(1R)-3-Ocp-l-His(1-benzyl-2-tert-butyl)-l-ProNH₂(22c). Yield:

10%; mp: 126–1288C (dec.); ¹H NMR (CD₃OD): d=7.30 (m, 3H),

7.08 (m, 2H), 6.90 (s, 1H), 5.36 (m, 2H), 4.81 (m, 1H), 4.39 (m, 1H),

3.79 (m, 1H), 3.56 (m, 1H), 3.03 (m, 1H), 2.94 (m, 2H), 2.35–1.95 (m,

10H), 1.38 (s, 9H); ¹³C NMR (CD₃OD): d=24.15, 25.89, 28.28, 29.34,

32.05, 32.86, 37.57, 40.10, 43.36, 46.70, 50.34, 51.20, 60.16, 115.90,

126.96, 128.13, 128.76, 135.00, 135.83, 154.98, 169.49, 174.52,

177.51, 216.15; MS (APCI): m/z 508 [M+1]⁺; Anal. calcd for

C₂₈H₃₇N₅O₄(507.6): C 66.25, H 7.35, N 13.80, found: 66.36, H 7.04, N

14.17; R_f=0.56 [CH₃OH/25% NH₄OH/CHCl₃(10:2:88)]; HPLC: t_R=

5.26 min, purity: 95.5% [CH₃CN/H₂O/TFA (40:60:0.1%)].

60.52,

H 7.61, N 17.10; R_f=0.43 [CH₃OH/25% NH₄OH/CHCl₃

(12:2:86)]; HPLC: t_R=5.46 min, purity: 98.4% [CH₃CN/H₂O/TFA

(20:80:0.1%)].

(1R)-3-Ocp-l-His(1-methyl-2-isopropyl)-l-ProNH₂(22i). Yield:

31%; mp: 96–988C (dec.); ¹H NMR (CD₃OD): d=6.80 (s, 1H), 4.80

(m, 1H), 4.39 (m, 1H), 3.79 (m, 1H), 3.59 (s, 3H), 3.50 (m, 1H), 3.11

(m, 2H), 2.93 (m, 2H), 2.36–1.93 (m, 10H), 1.28 (m, 6H); MS (APCI):

m/z 418 [M+1]⁺; Anal. calcd for C₂₁H₃₁N₅O₄(417.5): C 60.41, H 7.48,

N 16.77, found: C 60.25, H 7.37, N 16.61; R_f=0.42 [CH₃OH/25%

NH₄OH/CHCl₃(12:2:86)]; HPLC: t_R=5.17 min, purity: 98.3%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

(1R)-3-Ocp-l-His(1-benzyl-2-cyclopropyl)-l-ProNH₂(22d). Yield:

29%; mp: 116–1188C (dec.); ¹H NMR (CD₃OD): d=7.32 (m, 3H),

7.21 (m, 2H), 6.83 (s, 1H), 5.23 (s, 2H), 4.77 (m, 1H), 4.36 (m, 1H),

3.76 (m, 1H), 3.44 (m, 1H), 3.00 (m, 1H), 2.92 (m, 2H), 2.40–1.92 (m,

10H), 1.84 (m, 1H), 0.88 (m, 4H); ¹³C NMR (CD₃OD): d=8.52, 8.87,

24.13, 25.80, 29.30, 32.10, 37.26, 40.19, 42.40, 46.81, 49.57, 51.11,

60.17, 115.79, 127.34, 128.20, 128.82, 134.79, 136.59, 152.02, 169.54,

174.64, 177.77, 216.30; MS (APCI): m/z 492 [M+1]⁺; Anal. calcd for

C₂₇H₃₃N₅O₄(491.6): C 65.97, H 6.77, N 14.25, found: C 66.38, H 7.16,

N 13.85; R_f=0.53 [CH₃OH/25% NH₄OH/CHCl₃(10:2:88)]; HPLC: t_R=

4.72 min, purity: 96.0% [CH₃CN/H₂O/TFA (40:60:0.1%)].

(1R)-3-Ocp-l-His(1-methyl-2-tert-butyl)-l-ProNH₂(22j). Yield:

1

35%; mp: 104–1068C (dec.); H NMR (CD₃OD): d=6.78 (s, 1H), 4.79

(m, 1H), 4.37 (m, 1H), 3.78 (m, 1H), 3.73 (s, 3H), 3.53 (m, 1H), 3.07

(m, 1H), 2.89 (m, 2H), 2.36–1.89 (m, 10H), 1.40 (s, 9H, 3ꢁCH₃); MS

(APCI): m/z 432 [M+1]⁺; Anal. calcd for C₂₂H₃₃N₅O₄(431.5): C 61.23,

H 7.71, N 16.23; C 60.99, H 8.09, N 15.97; R_f=0.46 [CH₃OH/25%

NH₄OH/CHCl₃(12:2:86)]; HPLC: t_R=5.88 min, purity: 99.7%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

(1R)-3-Ocp-l-His(1-benzyl-2-cyclopentyl)-l-ProNH₂(22e): Yield:

23%; mp: 122–1248C (dec.); ¹H NMR (CD₃OD): d=7.31 (m, 3H),

7.15 (m, 2H), 6.91 (s, 1H), 5.14 (s, 2H), 4.71 (m, 1H), 4.36 (m, 1H),

3.81 (m, 1H), 3.50 (m, 1H), 3.06 (m, 2H), 2.96 (m, 2H), 2.34–1.60 (m,

18H); ¹³C NMR (CD₃OD): d=24.78, 25.84, 29.38, 31.82, 37.50, 39.15,

40.16, 42.36, 46.71, 48.88, 51.17, 60.32, 116.44, 127.13, 128.43,

128.65, 134.41, 135.17, 150.33, 169.50, 174.64, 177.58, 214.13; MS

(APCI): m/z 520 [M+1]⁺; Anal. calcd for C₂₉H₃₇N₅O₄(519.6): C 67.03,

H 7.18, N 13.48, found: C 67.50, H 7.47, N 13.03; R_f=0.57 [CH₃OH/

25% NH₄OH/CHCl₃(10:2:88)]; HPLC: t_R=5.98 min, purity: 93.0%

[CH₃CN/H₂O/TFA (40:60:0.1%)].

(1R)-3-Ocp-l-His(1-methyl-2-cyclohexyl)-l-ProNH₂(22k). Yield:

19%; mp: 110–1128C (dec.); H NMR (CD₃OD): d=6.78 (s, 1H), 4.80

1

(m, 1H), 4.38 (m, 1H), 3.79 (m, 1H), 3.57 (s, 3H), 3.47 (m, 1H), 3.08

(m, 1H), 2.90 (m, 2H), 2.74 (m, 1H), 2.35–1.28 (m, 20H); MS (APCI):

m/z 458 [M+1]⁺; Anal. calcd for C₂₄H₃₅N₅O₄(457.6): C 63.00, H 7.71,

N 15.31, found: C 63.08, H 7.78, N 15.36; R_f=0.54 [CH₃OH/25%

NH₄OH/CHCl₃(12:2:86)]; HPLC: t_R=10.45 min, purity: 96.6%

[CH₃CN/H₂O/TFA (20:80:0.1%)].

(1R)-3-Ocp-l-His[1-methyl-2-(adamantan-1-yl)]-l-ProNH₂

(22l).

Yield: 25%; mp: 118–1208C (dec.); ¹H NMR (CD₃OD): d=6.75 (s,

1H), 4.79 (m, 1H), 4.37 (m, 1H), 3.79 (m, 1H), 3.77 (s, 3H), 3.54 (m,

1H), 3.08 (m, 1H), 2.89 (m, 2H), 2.34 (m, 2H), 2.29–1.81 (m, 23H);

MS (APCI): m/z 510 [M+1]⁺; Anal. calcd for C₂₈H₃₉N₅O₄(509.6): C

65.99, H 7.71, N 13.74, found: C 66.28, H 8.05, N 13.35; R_f=0.59

[CH₃OH/25% NH₄OH/CHCl₃(12:2:86)]; HPLC: t_R=26.14 min, purity:

95.1% [CH₃CN/H₂O/TFA (20:80:0.1%)].

(1R)-3-Ocp-l-His(1-benzyl-2-cyclohexyl)-l-ProNH₂(22 f). Yield:

24%; mp: 130–1328C (dec.); ¹H NMR (CD₃OD): d=7.31 (m, 3H),

7.16 (m, 2H), 6.90 (s, 1H), 5.14 (s, 2H), 4.85 (m, 1H), 4.37 (m, 1H),

3.80 (m, 1H), 3.47 (m, 1H), 3.02 (m, 1H), 2.94 (m, 2H), 2.70 (m, 1H),

2.34–1.28 (m, 20H); ¹³C NMR (CD₃OD): d=24.30, 25.76, 26.13,

27.03, 29.13, 32.01, 32.23, 35.14, 37.57, 39.96, 42.12, 46.81, 49.08,

51.04, 60.03, 116.36, 127.13, 128.20, 128.82, 134.57, 135.29, 150.08,

169.34, 174.64, 177.59, 216.12; MS (APCI): m/z 534 [M+1]⁺; Anal.

calcd for C₃₀H₃₉N₅O₄(533.7): C 67.52, H 7.37, N 13.12, found: C

Receptor binding assays

67.64,

H 7.50, N 13.38; R_f=0.58 [CH₃OH/25% NH₄OH/CHCl₃

(10:2:88)]; HPLC: t_R=7.27 min, purity: 99.3% [CH₃CN/H₂O/TFA

(40:60:0.1%)].

Receptor binding studies were carried out according to a proce-

dure described previously.^[36]Briefly, HEK293EM cells stably express-

ing either TRH-R1 or TRH-R2 were grown in Dulbecco’s modified

Eagle’s medium containing 10% fetal bovine serum (FBS) and

200 mgmL^ꢀ1hygromycin. For equilibrium binding experiments,

cells were seeded into 24-well plates (1.5ꢁ10⁵cells per well). After

48 h, cells were incubated at 378C for 1 h with [³H][N(1)-Me-

His]TRH (MeTRH, 2 nm) in Hanks’ balanced salt solution, pH 7.4,

and various doses of TRH analogues. Apparent inhibitory constants

(K_i) were derived from curves fitted by nonlinear regression analysis

and drawn with the Prism version 3 software package (GraphPad

Software, Inc.) using the formula K_i=(IC₅₀)/{1+([L]/K_d)}, in which

(1R)-3-Ocp-l-His[1-benzyl-2-(adamantan-1-yl)]-l-ProNH₂

(22g).

Yield: 18%; mp: 136–1388C (dec.); ¹H NMR (CD₃OD): d=7.38 (m,

3H), 7.15 (m, 2H), 7.04 (s, 1H), 5.56 (s, 2H), 4.97 (m, 1H), 4.42 (m,

1H), 3.72 (m, 1H), 3.59 (m, 1H), 3.06 (m, 2H), 3.00 (m, 1H), 2.33–

1.28 (m, 25H); ¹³C NMR (CD₃OD): d=24.34, 25.84, 29.31, 29.38,

32.16, 35.99, 37.44, 40.16, 40.96, 42.36, 46.65, 50.35, 51.26, 59.12,

115.87, 127.05, 128.76, 135.04, 136.55, 151.17, 169.49, 174.12,

177.14, 215.80; MS (APCI): m/z 586 [M+1]⁺; Anal. calcd for

C₃₄H₄₃N₅O₄(585.7): C 69.72, H 7.40, N 11.96, found: C 69.38, H 7.69,

ChemMedChem 2011, 6, 531 – 543

ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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R. Jain et al.

MED

IC₅₀is the concentration of unlabeled analogue that half-competes

and K_dis the equilibrium dissociation constant for [³H][N(1)-Me-

His]TRH.

trazole (PTZ, 65 mgkg^ꢀ1i.p.) was used to induce clonic seizures, re-

flecting a generalized seizure condition in humans. TRH/TRH ana-

logues (10 mmolkg^ꢀ1i.v.) were injected into mice (n=6–8 per

group) via the tail vein, 10 min prior to PTZ administration, where-

as the control group was administered saline. The animals were

observed for 10 min after PTZ challenge. Onset of, and latency to,

generalized clonic convulsions with falling was recorded.

Luciferase activity assays

On the day prior to transfection, cells stably expressing either TRH-

R1 or TRH-R2 were seeded into 24-well plates (1.5ꢁ10⁵cells per

well). After 16 h, the medium was aspirated, and the cells (~50%

confluent) were co-transfected with plasmid DNA encoding CREB

and the CREB-activated luciferase gene (PathDetect CREB trans-Re-

The electrical test employed was the maximal electric shock (MES)

seizure test. The MES-induced seizure model was used to assess

the potential of drugs against partial seizures. Seizures were in-

duced in mice by applying a current of 45 mA through a corneal

electrode for 0.2 s as described elsewhere with slight modifica-

tion.^[52]Mice (n=6–8 per group) were treated with saline or TRH

(10 mmolkg^ꢀ1) or TRH analogues (10 mmolkg^ꢀ1i.v.). Ten minutes

later, seizures were induced by MES, and the mice were observed

for duration of tonic hind limb extension in mice.

porting System^TM

, Stratagene) using the calcium phosphate

method. On the second day, 6 h before the assay, medium contain-

ing 10% FBS was changed to medium containing 1% FBS, and var-

ious concentrations of TRH and TRH analogues were added to the

medium. Luciferase activity was measured 24 h after transfection.

Cells were washed with phosphate-buffered saline and lysed with

0.2 mL lysis buffer (25 mm Gly-Gly, pH 7.8, 15 mm MgSO₄, 4 mm

EGTA, 1 mm dithiothreitol, and 1% Triton X-100). Cell lysates

(0.025 mL) were combined automatically with 0.125 mL reaction

buffer (25 mm Gly-Gly, pH 7.8, 15 mm MgSO₄, 4 mm EGTA, 1 mm di-

thiothreitol, 15 mm KH₂PO₄, and 2 mm ATP) and 0.025 mL luciferin

(0.4 mm) in reaction buffer, and the luminescence was measured

for 10 s in a TR717 Microplate luminometer (Tropix, Bedford, MA,

USA). The luciferase activity levels detected by this assay reflect the

activation of signaling by TRH analogues.

The Supporting Information contains detailed experimental proce-

dures and spectral data for intermediates 12a–m, 13a–m, 18a–e,

and 19a–e.

Acknowledgements

This work is supported by the Council of Scientific and Industrial

Research, New Delhi (Grant No. 01(1861)/03/EMR-II). V.M. thanks

the Department of Science and Technology, New Delhi (Grant No.

SR/FTP/CS-79/2005), for the award of a young scientist fellow-

ship. C.L.M. thanks the University Grant Commission for the

award of a senior research fellowship.

Analeptic activity

Male Swiss albino mice (20–30 g) were procured from the Central

Animal Facility (CAF), NIPER, S.A.S. Nagar. Animals were housed in

a room at 22ꢁ28C with 12 h light/dark cycles and were allowed

free access to pellet food and water. All procedures used in this

study were approved by the Institutional Animal Ethics Committee

(IAEC), NIPER (experimental protocol approval numbers IAEC/08/43

and IAEC/09/01) and were conducted in accordance with the

guidelines of the Committee for the Purpose of Control and Super-

vision of Experiments on Animals (CPCSEA), Government of India.

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Received: November 10, 2010

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Article Doi

Synthesis, Receptor Binding, and CNS Pharmacological Studies of New Thyrotropin-Releasing Hormone (TRH) Analogues

DOI: 10.1002/cmdc.201000481

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