Synthesis and biological evaluation of a novel pyroglutamyl-modified TRH analogue

Article abstract of DOI:10.1016/S0014-827X(02)01232-6

The TRH analogue 3, incorporating the (S)-isothiazolidine-1,1-dioxide-3-carboxylic acid (1) moiety in place of the native L-pyroglutamic acid (pGlu) residue, has been synthesized and fully characterized by ¹H and ¹³C NMR. The effects

Full text of DOI:10.1016/S0014-827X(02)01232-6

Il Farmaco 57 (2002) 479–486
www.elsevier.com/locate/farmac
Synthesis and biological evaluation of a novel
pyroglutamyl-modified TRH analogue
b
L. Brunetti a, I. Cacciatore a, A. Di Stefano a, S. Dupre` , A. Giorgi b, G. Luisi a,
B. Michelotto a, G. Orlando a, F. Pinnen a,c,*, L. Recinella a, P. Sozio a, A. Spirito b
a Dipartimento di Scienze del Farmaco, Uni6ersita` degli Studi ‘G. D’Annunzio’, Via dei Vestini 31, I-66013 Chieti, Italy
b Dipartimento di Scienze Biochimiche, Uni6ersita` degli Studi ‘La Sapienza’, P.le Aldo Moro 5, I-00185 Rome, Italy
c Centro di Studio per la Chimica del Farmaco del CNR, Uni6ersita` degli Studi ‘La Sapienza’, Rome, Italy
Received 13 December 2001; accepted 17 February 2002
Abstract
The TRH analogue 3, incorporating the (S)-isothiazolidine-1,1-dioxide-3-carboxylic acid (1) moiety in place of the native
1
L
-pyroglutamic acid (pGlu) residue, has been synthesized and fully characterized by H and 13C NMR. The effects of replacing
pGlu with its sulphonamido counterpart on biological activity have been investigated. This peptide, which is significantly
stabilized towards hydrolysis by pyroglutamyl peptidase type I (PP I, EC 3.4.19.3), has shown to maintain in vitro prolactin-re-
´
leasing activity. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Pyroglutamic acid; Pyroglutamyl peptidases; Sulphonamidopeptides; Synaptosomes; Thyrotropin-releasing hormone
unique class of enzymes, the pyroglutamyl peptidases
1. Introduction
(PPs) [5]; interestingly, two out of the three distinct PP
forms identified to date in mammalian tissues show a
substrate specificity restricted to the endocrine and
Distinctive biological activities of many peptides,
such as their ability to recognize and bind to receptors
and their relative stability towards inactivation by pep-
tidases, are governed by specific structural modifica-
tions at the amino- and carboxy-terminal positions. The
pyroglutamic acid (pGlu) residue is a frequent struc-
tural determinant in hormones and neuropeptides,
where it is postulated to originate at the N-terminus
from the post-translational cyclization of amino termi-
nal glutamyl or glutaminyl residues; relevant physiolo-
gical functions for this amino acid are suggested by the
existence of enzymatic systems involved in its formation
[1–4]. The pyroglutamate residue is hydrolytically re-
moved from pyroglutamyl peptides and proteins by a
CNS-active thyrotropin-releasing hormone (TRH:
L-
pyroglutamyl-L–histidyl–
TRH-like peptides.
L-prolinamide, Fig. 1), and to
Much interest is focused on these peptidases, since
they are thought to play a role in regulating TRH
availability to its receptors, as well as the extent and
duration of its neuroendocrine activities. So far, a
number of pyroglutamyl-modified TRH analogues have
been synthesized in order to overcome susceptibility to
PP degradation and consequently to prolong intrinsic
activity [6–10]. It is worth noting that several of these
Abbre6iations: Abbreviations follow the recommendations of the
IUPAC-IUB Commission on Biochemical Nomenclature as given in
Eur. J. Biochem. 138 (1984) 9–37. Additional abbreviations:; Boc,
tert-butyloxycarbonyl; DCC, dicyclohexylcarbodiimide; DMF, N,N%-
dimethylformamide; DMSO-d6, hexadeuterated dimethyl sulfoxide;
EDTA, ethylenediamine tetraacetic acid; MeOH, methanol; TEA,
triethylamine; Z, benzyloxycarbonyl.
* Corresponding author.
E-mail address: fpinnen@unich.it (F. Pinnen).
Fig. 1. TRH.
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(02)01232-6

480
L. Brunetti et al. / Il Farmaco 57 (2002) 479–486
analogues were shown to possess enhanced CNS effects
as a consequence of increased bioavailability [8,11].
In the course of our studies on the modification of
bioactive peptides [12–15], we became interested in
sulphonamidopeptides [16–18]. These pseudopeptides,
characterized by an SO2NH versus CONH replacement,
contain a peptide bond surrogate which appears very
attractive for more than one reason: stability toward
enzymatic hydrolysis, H-bonding capacity, high polar
character, and tetrahedral structure suitable to design
transition state analogue protease inhibitors. In a pre-
liminary communication, we reported the synthesis and
some chemical properties of (S)-isothiazolidine-1,1-
dioxide-3-carboxylic acid (1), the sulphonamido ana-
logue of pyroglutamic acid [19].
Scheme 1. Synthesis of TRH analogue 3. Reagents and conditions: (a)
HꢀHisꢀProꢀNH2, isobutyl chloroformate, TEA, DMF, −10 °C, 20
min; 0 °C, 3 h; then r.t., 20 h; (b) H2, PdꢀC 10%, MeOH, r.t., 2 h.
As an extension of our research in this field and
taking into account the biological significance of the
TRH/PP system, we were prompted to design a pyro-
glutamyl-modified TRH analogue containing the new
g-sultam 1, with a view to achieving stabilization of the
PP sensitive pGluꢀHis peptide bond as well as possible
dissociation of the CNS from the hormonal activities.
Here we report data on the synthesis, characterization
and biological activity of the novel TRH analogue 3.
N-benzyloxycarbonyl derivative for the subsequent
coupling step. Direct condensation of N-unprotected 1
with HꢀHisꢀProꢀNH2 was found in fact to be unconve-
nient, owing to its low solubility in the solvents usually
employed in coupling reactions, and in accordance with
the well-known tendency of the SO2NH group to un-
dergo intra- and inter-molecular acylation reactions
[21]. Coupling of (S)-2-benzyloxycarbonyl-isothiazo-
lidine-1,1-dioxide-3-carboxylic acid with HꢀHisꢀProꢀ
NH2 was found to be difficult, due to the reactivity of
the imidazo basic group. A variety of conditions were
tried in order to overcome the problem and finally the
precursor tripeptide was obtained in acceptable yields
by using mixed anhydride activation. The resulting
(S,S,S)-N-(2-benzyloxycarbonyl-isothiazolidine-1,1-di-
oxide-3-carbonyl)-histidyl-prolinamide (2) was then
converted by catalytic hydrogenolysis with 10% PdꢀC
into the desired TRH analogue 3 without any side
reactions and in good yields (Scheme 1). It should be
noted here that, in accordance with the known low
stability of sultams of type 1 under acidolytic condi-
tions [22], a complex reaction mixture was obtained
when HBr in acetic acid was used for the N-deprotec-
tion of 2. The final peptide was purified by gel-perme-
ation chromatography and fully characterized by NMR
spectroscopy.
2. Chemistry
In the synthesis of the TRH analogue 3, still main-
taining the central histidine residue of the parent pep-
tide, we coped with some problems connected with the
reactivities of both the g-sultam and the imidazole
rings. The TRH analogue 3 was synthesized using
solution procedures by stepwise elongation of the pep-
tide chain in the C-to-N direction as outlined in Scheme
1. Dipeptide amide HꢀHisꢀProꢀNH2 [20] was prepared
by conventional DCC condensation of BocꢀHisꢀOH
with HꢀProꢀNH2 and subsequent acidolytic removal of
the Boc protecting group. The modified amino acid
(S)-isothiazolidine-1,1-dioxide-3-carboxylic acid (1) was
synthesized according to the methods reported in our
previous paper [19] and opportunely converted into its
A correlation of the 1H and 13C NMR chemical shifts
of the new sulphonamido analogue 3 with those of
native TRH in DMSO-d6 solution [23,24] is reported in
Table 1. An analysis of spectral data indicates that 3
presents great conformational similarities to TRH. In
particular, a trans/cis isomerism about the HisꢀPro
peptide bond is observed for 3. Measurement of the cis

L. Brunetti et al. / Il Farmaco 57 (2002) 479–486
481
content, made from relative peak intensities of Cb and
Cg atoms in DMSO-d6 solution at room temperature
[25], reveals a cis-configurated population of about
10–15%, comparable to that of TRH in the same
solvent. The Pro Cb and Cg atoms of 3 show prevailing
resonances (l 29.97 and 25.02 ppm, respectively) typical
of trans XaaꢀPro bonds, which are accompanied by
resonances at 31.83 and 22.89 l ppm, typical of cis
bonds [26]. Furthermore, as in the case of TRH, several
resonances accompanied by satellite peaks, reflecting
the cis content of the prolyl residue, are present in the
1H NMR spectrum of compound 3 [23,25,27].
Table 1) suggest for analogue 3 a preferentially stag-
gered conformation of the histidine side chain with its
imidazole ring folded on the proline moiety [25].
A further significant spectral feature of the isothiazo-
1
lidine ring is represented by the H and 13C resonances
at the Cg position. In agreement with the presence of
the SO2 group, both the 1H and 13C NMR signals
appear downfield shifted (l 3.12, 3.22 and 46.09, res-
pectively) [18,21,29] as compared with the correspon-
ding pGlu resonances in TRH (l 2.46 and 26.57,
respectively; see Table 1).
A characteristic spectral feature of 3 is connected
with the preferred conformation of the histidyl residue.
The observed NaH–CaH coupling constant for his-
tidine in DMSO-d6 solution (7.5 Hz) is within the
7.2–8.0 Hz range which is reported for the same cou-
pling in TRH [25,28] and in accordance with a prefe-
rentially extended backbone conformation. Further-
more, the values of His CaH–CbH vicinal coupling
constants (6.6 Hz) as well as the anisotropic effect
observed on proline CdH2 and CONH2 atoms (see
3. Results and discussion
Our first interest was the investigation of the effects
of the pGlu replacement with its sulphonamido coun-
terpart on enzymatic stability towards PP I (EC
3.4.19.3). Metabolic stability was monitored by diffe-
rential MALDI–TOF MS analysis [30] of TRH ana-
logue 3 and TRH following PP I treatment [31]. While
TRH was easily hydrolysed as expected and as
Table 1
NMR data a for TRH and TRH analogue 3
Residue
TRH
3
lH (ppm) [23]
lC (ppm) [24]
lH (ppm)
lC (ppm)
pGlu or (S)-isothiazolidine-1,1-dioxide-3-carboxylic acid
Ca
Cb
4.44
2.23
2.46
2.46
56.83
30.65
3.96
1.98
2.12
3.12
3.22
55.24
27.07
Cg
26.57
46.09
CO (peptide)
CO
173.56
181.03
170.49 b
NH
8.19
7.34
His
Ca
Cb
4.98
3.23
53.67
32.63
4.61
2.86
2.91
8.08
7.62
51.55
30.03
Im C2
Im C4
Im C5
Im NH
CO
7.88
7.30
136.50
n.o.
n.o.
135.60
117.49
133.32
n.o.
8.58
11.90
7.92
171.46
170.01 b
NH
Pro
Ca
Cb
4.59
2.23
2.46
2.23
61.59
30.83
n.o.
23.45
25.85
47.69
48.29
175.54
4.16
2.15
2.56
1.65
2.02
3.36
3.54
60.73
29.97
31.83
22.89
25.02
47.42
n.o.
Cg
Cd
3.61
3.89
CO
174.51
NH2
7.30
8.51
6.94
7.00
a In DMSO at 23 °C.
b Assignments may be interchanged.

482
L. Brunetti et al. / Il Farmaco 57 (2002) 479–486
evidenced by the disappearance after 1 h of its molecu-
lar ion (m/z 363), the analogue 3 was found stable to
enzymatic hydrolysis, the molecular ion (m/z 399) re-
maining present and unchanged during the same experi-
ment. This result shows that peptide 3 is significantly
stabilized towards hydrolysis by PP I as compared to
TRH and may represent a degradation-resistant TRH
analogue with prolonged in vivo activity.
evaluate here the activity of compound 3 on dopamine
release in comparison with TRH, by using rat hypotha-
lamic synaptosomal fractions. It was found that in
contrast with native TRH, which is able to inhibit
depolarization-induced dopamine release, the TRH
analogue 3 has lost this activity (Fig. 3).
In the present pharmacological studies a comparative
evaluation of the hormonal effects of TRH and its
sulphonamido analogue 3 has been initially considered.
TRH is known to induce the secretion of the pituitary
lactogenic hormone prolactin (PRL), through hypophy-
seal lactotropic cell TRH receptor activation. We report
here the effects of 3 and TRH on PRL secretion in vitro
by using anterior pituitary cell cultures from adult male
Wistar rats. It is found that the TRH analogue 3 is able
to stimulate PRL secretion in a dose-dependent man-
ner, although with lower potency than TRH (Fig. 2). In
particular, the doubling of the control values is ob-
tained with 87.0 nM TRH analogue 3 and 1.2 nM
TRH, respectively. Thus, upon modification of the
pyroglutamyl moiety, the PRL-releasing activity is re-
tained, albeit to a lesser extent, suggesting that the
endocrine TRH receptor binding properties in com-
pound 3 are maintained.
4. Experimental
4.1. Peptide synthesis
Column chromatography was carried out on silica
gel 60 (230–400 mesh, Merck). TLC was performed on
precoated silica gel 60 F254 Merck plates, developed
with the following solvent systems: (A) CHCl3–MeOH
(85:15); (B) CHCl3–MeOH (3:1). Optical rotations
were taken at 20 °C with a Schmidt-Haensch Polar-
tronic D polarimeter. H and 13C NMR spectra of the
1
new compounds were determined in DMSO-d6 solution
on a Varian VXR 300 MHz instrument (l expressed in
ppm). Elemental analyses for the new compounds are
within the 90.4% of the theoretical values. Compound
1 and its N-benzyloxycarbonyl derivative were synthe-
sized as described in Ref. [19].
There is a growing evidence that CNS-activating
pharmacological effects of TRH are mediated through
various neurotransmitters, most prominently cate-
cholamines and acetylcholine [11,32–35]. In a prelimi-
nary study we found that TRH is able to inhibit
hypothalamic dopamine release from male rat neuronal
endings (synaptosomes) in vitro [36]. This suggests a
possible TRH involvement either in central mechanisms
controlling the anorectic behaviour or in the modula-
tion of PRL release via an indirect pathway; the hypo-
thalamic dopamine is in fact the main putative PRL
inhibiting factor (PIF). It seemed then interesting to
4.1.1. (S,S,S)-N-(2-Benzyloxycarbonyl-isothiazolidine-
1,1-dioxide-3-carbonyl)-histidyl-prolinamide (2)
To a solution of (S)-2-benzyloxycarbonyl-isothiazo-
lidine-1,1-dioxide-3-carboxylic acid (2.8 g, 9.4 mmol) in
DMF (40 ml) TEA (1.0 g, 10.3 mmol) and isobutyl
chloroformate (1.4 g, 10.3 mmol) were added dropwise
alternately at −10 °C. After 20 min stirring, a solution
of 2HCl·HꢀHisꢀProꢀNH2 [20] (4.6 g, 14.2 mmol) in
DMF (40 ml) and TEA (4.3 g, 42.6 mmol) were added,
and the mixture stirred at 0 °C for 3 h and at room
temperature (r.t.) for 20 h. The reaction mixture was
Fig. 2. Effect of TRH (a) and TRH analogue 3 (b) (1 nM–1 mM) on PRL release from anterior pituitary cell cultures. Trypsin dispersed cells were
plated into 24-well dishes (300,000 cells/well). After 2–3 days in culture, the cells were washed twice with 1 ml DMEM and release experiments
were performed by 1 h incubations with graded concentrations of TRH or TRH analogue 3 in DMEM. Each column represents the mean9SEM
of 12–15 wells; ANOVA PB0.0001, *PB0.001 vs. control.

L. Brunetti et al. / Il Farmaco 57 (2002) 479–486
483
Fig. 3. Areas under the time–response curves (AUCs) relative to the effects of TRH (a) and TRH analogue 3 (b) (10 nM–1 mM) on
depolarization-induced dopamine release. After a 30 min equilibration perfusion with buffer alone, a 23 min perfusion with graded concentrations
of the peptides was started, where in the final 3 min, K+ concentrations in the perfusion buffer were elevated to 15 mM (after removal of
equimolar concentrations of Na+). Then, the Krebs–Ringer buffer perfusion was restarted for 9 min to record a return to basal. Controls
followed the same protocol, but without peptides in perfusion buffer. Each column represents the mean9SEM of 3–5 experiments performed in
triplicate; ANOVA, PB0.0001; *PB0.001 vs. controls.
filtered, the solvent removed in vacuo and the residue
chromatographed on silica gel using CHCl3–MeOH
(85:15) as eluant, to give N-protected tripeptide 2 as an
oil in 50% yield. Anal. (C23H28N6O7S) C, H, N, S; Rf
4.2. Biological studies
PP I (EC 3.4.19.3) from calf liver was purchased
from Sigma. Differential mass spectrum analyses were
obtained with a Voyeger-DE (PE Biosystems) instru-
ment. Some aliquots of analogue 3 and TRH (12 mmol)
were treated with 7.7 U of PP I in 1 ml of 50 mM
potassium phosphate buffer, pH 8.0, containing 5 mM
mercaptoethanol and 5 mM EDTA. At different times,
10 ml aliquots were dissolved with an appropriate
MALDI matrix in MeCN solution containing 0.1%
trifluoroacetic acid.
1
(A) 0.3, Rf (B) 0.4; [h]D= −55° (c=1, MeOH). H
NMR: l 1.7–1.9 (m, 3H, Pro g-CH2 and b-CHB), 2.0
(m, 1H, Pro b-CHA), 2.25 (m, 1H, SO2ꢀCH2ꢀCHB),
2.55 (m, 1H, SO2ꢀCH2ꢀCHA), 2.7–2.9 (m, 2H, His
b-CH2), 3.1 (m, 1H, Pro d-CHB), 3.25–3.65 (m, 3H,
SO2ꢀCH2 and Pro d-CHA), 4.2 (m, 1H, Pro a-CH), 4.55
(m, 1H, His a-CH), 4.7 (m, 1H, SO2ꢀN(Z)ꢀCH), 5.2
(m, 2H, CH2O), 6.8 (s, 1H, Im C4), 7.0 (s, 1H, NH),
7.3–7.4 (m, 5H, aromatics), 7.55 (s, 1H, Im C2), 8.15 (s,
1H, NH), 8.6 (d, J=6.46 Hz, 1H, His NH), 11.8 (s,
1H, Im NH). 13C NMR: l 22.76 (Pro Cg), 24.32
(SO2ꢀCH2ꢀCH2), 29.30 (Pro and His Cb), 46.72
(SO2ꢀCH2), 47.18 (Pro Cd), 51.65 (Pro Ca), 57.35
(SO2ꢀN(Z)ꢀCH), 60.03 (His Ca), 67.63 (CH2O), 117.50
(Im C4), 127.47, 128.13, and 128.42 (Ph), 133.81 (Im
C5), 134.99 (Ph), 135.39 (Im C2), 150.05 (OCO), 168.43,
169.71, and 173.94 (3×CO).
4.3. Pharmacological studies
4.3.1. Materials and methods
4.3.1.1. Anterior pituitary cell cultures. Cell dispersion
was performed as previously described [37], under ste-
rile conditions and the media were supplemented with
penicillin (35 mg/ml) and streptomycin (50 mg/ml). The
anterior pituitaries were minced with a blade into small
fragments and sequentially incubated in Dulbecco’s
modified eagle’s medium (DMEM) at 37 °C in a Dub-
noff shaking bath, with 0.5% trypsin (type XII S) for 15
min, 0.02% deoxyribonuclease I for 1 min, 0.1% soy-
bean trypsin inhibitor for 4 min, 2 and 1 mM EDTA
(in Ca2+/Mg2+-free Earle’s basic salt solution, EBSS)
for 4 and 15 min, respectively. The remaining tissue
fragments were mechanically dispersed into single cells
by gentle suction and extrusion through a narrow tip
Pasteur pipette; the cell suspension was then filtered
through a 100 mm nylon mesh and centrifuged at
4.1.2. (S,S,S)-N-(isothiazolidine-1,1-dioxide-3-
carbonyl)-histidyl-prolinamide (3)
Peptide 2 (2.2 g, 4.1 mmol) was hydrogenated in
MeOH (100 ml) in the presence of 10% Pd on activated
charcoal (0.2 g). After 2 h, the catalyst was filtered off
and the filtrate evaporated under reduced pressure. The
crude product was purified by column chromatography
on Sephadex LH-20 using H2O–MeOH (2:1) as eluant
to yield deprotected tripeptide 3 as a foam (1.5 g, 92%).
Anal. (C15H22N6O5S) C, H, N, S; Rf (B) 0.2, [h]D=
−41° (c=1, MeOH).

484
L. Brunetti et al. / Il Farmaco 57 (2002) 479–486
1000×g for 10 min through a layer of 3% bovine
serum albumin (BSA) in DMEM. Finally, cells were
resuspended in DMEM–0.3% BSA, checked for viabil-
ity by Trypan blue exclusion test, counted by a haemo-
cytometer (ca. 2 million cells/pituitary), plated into
24-well dishes (300,000 cells/well), and incubated with
DMEM supplemented with 10% newborn calf serum,
at 37 °C in ambient air/CO2 95/5%. After 2–3 days in
culture, the cells were washed twice with 1 ml DMEM
and release experiments were performed by 1 h incuba-
tions with graded concentrations of TRH or TRH
analogue 3 in DMEM (supplemented with 0.1% BSA,
0.006% ascorbic acid and 40 IU/ml aprotinin); release
aliquots were stored at −20 °C until assay. Chemicals
and cell culture reagents were purchased from Sigma,
except DMEM and serum from Gibco.
PRL radioimmunoassay was performed as previously
described [37]. PRL was iodinated with Na125I by the
chloramine-T method. Rat PRL antibody and standard
were provided by NIDDK, National Hormone and
Pituitary Program, USA. Hundred microlitres of rat
PRL standard or samples were incubated at 5 °C with
the PRL antibody at a final dilution of 1:12,500 in
assay buffer for 24 h; thereafter, 3000 cpm 125PRL were
added to each tube and incubated for 48 h. Finally,
anti-rabbit goat serum (1:200) and 0.4% polyethyleneg-
lycol were added to separate bound and free fractions.
After 24 h tubes were centrifuged for 30 min at 4000×
g, supernatants were aspirated, and pellets counted in a
g-counter. The sensitivity of the assay was 50 pg/tube.
turn to basal). To evaluate the effects of the peptides on
neurotransmitter release during depolarization [3 min
perfusion with K+(15 mM), after removal of equimolar
concentrations of Na+ in perfusion buffer], TRH or
TRH analogue 3 were added, after the equilibration
period, both 20 min prior (pre-stimulus) and 3 min
during K+(15 mM) perfusion (stimulus). Then, Krebs–
Ringer buffer perfusion was restarted for 9 min to
record a return to basal. Finally, b-emission from per-
fusate fractions, corresponding to [3H]dopamine re-
lease, was detected by liquid scintillation scanning. All
chemicals were purchased from Carlo Erba, Italy, ex-
cept [7,8-3H]dopamine (1 mCi/ml) from Amersham
Pharmacia Biotech, Italy.
4.3.2. Analysis of data
PRL release has been evaluated as group means9
standard error of mean (SEM). Each group represents
the mean9SEM of 12–15 wells.
Dopamine release has been calculated as either the
means9SEM of the percentage of [3H]dopamine re-
covered in each fraction respect to total (fractions+
filter) or the means9SEM of the area under the
time–response curve (AUC); each group represents the
mean9SEM of 3–5 experiments performed in tripli-
cate. Treatment and control group data from experi-
ments run on a single pool of hypothalamic tissue.
Treatment and control group means have been com-
pared by the analysis of variance (ANOVA), followed
by the Student–Newman–Keul test (GRAPH
PAD
PRISM 2.00 Software).
4.3.1.2. Hypothalamic synaptosomes. Hypothalamic
synaptosomes were prepared according to Ref. [38].
Briefly, male Wistar rats (200–250 g) were sacrificed by
decapitation, the hypothalami quickly dissected, ho-
mogenized in 0.32 M saccharose and centrifuged, first
at 1000×g for 5 min, and then at 12,000×g for 20
min, to isolate neuronal endings from cell nuclei and
glia. Then, the synaptosome suspension was incubated,
at 37 °C, under O2/CO2 95/5%, pH 7.2–7.4, in Krebs–
Ringer buffer (mM/l: 125 NaCl, 3 KCl, 1.2 MgSO4, 1.2
CaCl2, 5 NaH2PO4, 10 Tris–HCl, 10 glucose, 1, ascor-
bic acid), supplemented with 0.05 mM fluoxetine, a
selective serotonin reuptake inhibitor, with 0.05 mM
[3H]dopamine, for 15 min, to make synaptosomes up-
take [3H]dopamine, substituting for the endogenous
dopamine pool. Then, synaptosomes were layered onto
0.8 mM Millipore filters, placed into 37 °C water-jacke-
ted superfusion chambers (18 different chambers for
each experiment), and perfused with the above buffer
(0.6 ml/min). After 30 min, to allow stable release
(equilibration period), perfusate was collected in 2 min
fractions, and after the first 3–4 fractions (basal re-
lease) graded concentration of TRH or TRH analogue
3 were added to the perfusion buffer for 10 min (stimu-
lus), followed by 10 min with Krebs buffer alone (re-
5. Conclusions
In summary, we have found that the replacement of
the pGlu residue with its sulphonamido counterpart,
the (S)-isothiazolidine-1,1-dioxide-3-carboxylic acid, at
the N-terminus of TRH stabilizes the scissile XaaꢀHis
amide bond to hydrolysis by the pyroglutamyl pepti-
dases, which represents the main mechanism responsi-
ble for the peptide in vivo inactivation. This result is
valuable for the design of metabolically stabilized TRH
and TRH-like peptide analogues to be considered for
clinical trials.
In the pharmacological investigations, analogue 3
was found to retain, albeit with a weaker potency, the
hormonal activity of TRH, as measured by the pro-
lactin-releasing effect. When the analogue was tested
for its ability to inhibit dopamine release, through
activation of hypothalamic TRH receptors, it was
found devoid of activity. These differences are in line
with emerging data supporting the hypothesis that the
endocrine and CNS effects of TRH may be mediated
through different TRH receptor subtypes: the high-
affinity, pituitary-like binding sites, and the low-affinity
binding sites in hypothalamus and cortex [39,40].

L. Brunetti et al. / Il Farmaco 57 (2002) 479–486
485
[9] C. Mapelli, L.F. Elrod, F.L. Switzer, C.H. Stammer, Conforma-
tional properties of 2,3-methanopyroglutamic acid in peptides:
NMR and X-ray diffraction studies, Biopolymers 28 (1989)
123–128.
[10] B. Koksch, D. Ullmann, H.-D. Jakubke, N. Sewald, K. Burger,
Structure and biological activity of trifluoromethyl substituted
GnRH and TRH analogues, Peptides (1994) 323–324.
[11] I. Fukuchi, T. Asahi, K. Kawashima, Y. Kawashima, M. Yama-
mura, Y. Matsuoka, K. Kinoshita, Effects of taltirelin hydrate
(TA-0910), a novel thyrotropin-releasing hormone analog, on in
vivo dopamine release and turnover in rat brain, Arzneim.-
Forsch./Drug Res. 48 (1998) 353–359.
[12] A. Calcagni, M. Kajtar-Peredy, G. Lucente, G. Luisi, F. Pinnen,
L. Radics, D. Rossi, Nine-membered cyclodepsitripeptides con-
taining the retroisomeric sequence of ergot peptides, Int. J. Pept.
Protein Res. 42 (1993) 84–92.
[13] A. Calcagni, S. Dupre`, G. Lucente, G. Luisi, F. Pinnen, D.
Rossi, A. Spirito, Synthesis and activity of the glutathione
Although the pyroglutamic acid moiety is not consid-
ered to be involved in the stabilization of the putative
bioactive conformation of TRH [23], its cyclic structure
and conformation are important in maintaining the
right spatial location of the lactam thus stabilizing the
hydrogen bond formation in the receptor interaction
[41]. By taking into account the conformational anal-
ogy between 3 and the native molecule at the level of
the HisꢀProꢀNH2 fragment, the change in the hor-
monal potency as well as the lack of the CNS activity
should be ascribed to alterations in receptor interac-
tions connected with the specific properties of the
SO2NH group. In this context, an unfavourable electro-
static potential of sulphonamides [42] and/or the pres-
ence of the more sterically demanding tetrahedral
structure of the SO2 group as compared with the planar
amide carbonyl should play an important role.
analogue g-(
L
-(g-oxaglutamyl)-L-cysteinyl-glycine, Arch. Pharm.
Pharm. Med. Chem. 329 (1996) 498–502.
[14] A. Calcagni, G. Lucente, G. Luisi, F. Pinnen, D. Rossi, E.
Gavuzzo, Approaches to pseudopeptidic ergopeptines. Part 3.
Consequences of the incorporation of an a-azaphenylalanine
residue into the ergotamine oxa-cyclolic system, J. Chem. Soc.,
Perkin Trans 1 (1997) 2223–2227.
Acknowledgements
[15] A. Calcagni, G. Lucente, G. Luisi, F. Pinnen, D. Rossi, Novel
glutathione analogues containing the dithiol and disulfide form
of the CysꢀCys dyad, Amino acids 17 (1999) 257–265.
[16] G. Luisi, A. Calcagni, F. Pinnen, C(SO2ꢀNH) Transition state
isosteres of peptides. Synthesis of the glutathione disulfide ana-
logue [g-GluꢀC(SO2ꢀNH)ꢀCysꢀGly]2, Tetrahedron Lett. 34
(1993) 2391–2392.
Financial support from Ministero dell’Universita` e
della Ricerca Scientifica e Tecnologica (MURST, 60%)
(Italy) is gratefully acknowledged. The authors wish to
thank Professor G. Lucente for his suggestions and
helpful discussions.
[17] A. Calcagni, D. Rossi, M. Paglialunga Paradisi, G. Lucente, G.
Luisi, E. Gavuzzo, F. Mazza, G. Pochetti, M. Paci, Peptides
containing the sulfonamide junction. Synthesis, structure and
conformation of Z-TauꢀProꢀPheꢀNHiPr, Biopolymers 41 (1997)
555–567.
References
[18] A. Calcagni, P.G. Ciattini, A. Di Stefano, S. Dupre`, G. Luisi, F.
Pinnen, D. Rossi, A. Spirito, c(SO2NH) Transition state
isosteres of peptides. Synthesis and bioactivity of sulfonamido
pseudopeptides related to carnosine, Farmaco 54 (1999) 673–
677.
[19] G. Luisi, F. Pinnen, Synthesis and properties of (S)-isothiazo-
lidine-1,1-dioxide-3-carboxylic acid, a new g-sultam analogue of
pyroglutamic acid, Arch. Pharm. 326 (1993) 139–141.
[20] M. Bienert, Gy. Ko¨ller, H. Niedrich, Synthese von Thyreoliberin
(TRF), Pharmazie 32 (1977) 397–398.
[1] P.G. Board, K.A. Moore, J.E. Smith, Purification and properties
of gamma-glutamylcyclotransferase from human erythrocytes,
Biochem. J. 173 (1978) 427–431.
[2] W.H. Fischer, J. Spiess, Identification of a mammalian glu-
taminyl cyclase converting glutaminyl into pyroglutamyl pep-
tides, Proc. Natl. Acad. Sci. USA 84 (1987) 3628–3632.
[3] W.H. Busby Jr., G.E. Quackenbush, J. Humm, W.W. Young-
blood, J.S. Kizer, An enzyme(s) that converts glutaminyl-pep-
tides into pyroglutamyl-peptides. Presence in pituitary, brain,
adrenal medulla, and lymphocytes, J. Biol. Chem. 262 (1987)
8532–8536.
[4] L. Prokai, X. Ouyang, K. Prokai-Tatrai, J.W. Simpkins, N.
Bodor, Synthesis and behavioral evaluation of a chemical brain-
targeting system for a thyrotropin-releasing hormone analogue,
Eur. J. Med. Chem. 33 (1998) 879–886.
[21] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, F. Pinnen, G.
Pochetti, D. Rossi, Structure and conformation of peptides
containing the sulfonamide junction. II. Synthesis and conforma-
tion of cyclo[ꢀMeTauꢀPheꢀ
34 (1989) 471–479.
D
Proꢀ], Int. J. Peptide Protein Res.
[22] W.F. Erman, H.C. Kretschmar, Syntheses and facile cleavage of
five-membered ring sultams, J. Org. Chem. 26 (1961) 4841–4850.
[23] M. Montagut, B. Lemanceau, A.-M. Bellocq, Conformational
analysis of thyrotropin releasing factor by proton magnetic
resonance spectroscopy, Biopolymers 13 (1974) 2615–2629.
[24] R. Deslauriers, C. Garrigou-Lagrange, A.-M. Bellocq, I.C.P.
Smith, Carbon-13 nuclear magnetic resonance studies on thyro-
tropin-releasing factor and related peptides, FEBS Lett. 31
(1973) 59–66.
[25] B. Donzel, J. Rivier, M. Goodman, Conformational studies on
the hypothalamic thyrotropin releasing factor and related com-
pounds by 1H nuclear magnetic resonance spectroscopy, Bio-
polymers 13 (1974) 2631–2647.
[5] P.M. Cummins, B. O’Connor, Pyroglutamyl peptidase: an
overview of the three known enzymatic forms, Biochim. Bio-
phys. Acta 1429 (1998) 1–17.
[6] L. Flohe´, K. Bauer, E. Friderichs, W.A. Gu¨nzler, H.H. Hennies,
8
S. Herrling, F. Lagler, F. Otting, E. Schwertner, Biological
effects of degradation-stabilized TRH analogues, in: E.C.
Griffiths, G.W. Bennett (Eds.), Thyrotropin Releasing Hormone,
Raven Press, New York, 1983, pp. 327–340.
[7] R.F. Nutt, F.W. Holly, C. Homnick, R. Hirschmann, D.F.
Veber, B.H. Arison, Synthesis of thyrotropin-releasing hormone
analogues with selective central nervous system effects, J. Med.
Chem. 24 (1981) 692–698.
[8] G. Metcalf, Regulatory peptides as a source of new drugs—the
clinical prospects for analogues of TRH which are resistant to
metabolic degradation, Brain Res. 257 (1982) 389–408.
[26] H. Kessler, Conformation and biological activity of cyclic pep-
tides, Angew. Chem. Int. Ed. Engl. 21 (1982) 512–523.

486
L. Brunetti et al. / Il Farmaco 57 (2002) 479–486
[27] J. Feeney, G.R. Bedford, P.L. Wessels, 1H Nuclear magnetic
resonance studies of thyrotropin releasing factor (TRF), FEBS
Lett. 42 (1974) 347–351.
[28] S. Fermandjian, P. Pradelles, P. Fromageot, Proton NMR stu-
dies on thyrotropin releasing factor, FEBS Lett. 28 (1972) 156–
160.
[29] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, F. Pinnen, G.
Pochetti, D. Rossi, Structure and conformation of peptides
containing the sulphonamide junction. III. Synthesis, crystal and
molecular structure of a taurine containing peptidic oxa-cyclol,
Int. J. Peptide Protein Res. 37 (1991) 167–173.
[30] B.T. Chait, S.B.H. Kent, Weighing naked proteins: practical,
high-accuracy mass measurement of peptides and proteins,
Science 257 (1992) 1885–1894.
[31] A. Szewczuk, M. Mulczyk, Pyrrolidonyl peptidase in bacteria.
The enzyme from Bacillus subtilis, Eur. J. Biochem. 8 (1969)
63–67.
[32] H.H. Keller, G. Bartholini, A. Pletscher, Enhancement of cere-
bral noradrenaline turnover by thyrotropin-releasing hormone,
Nature 248 (1974) 528–529.
[33] G.G. Yarbrough, TRH potentiates excitatory actions of acetyl-
choline on cerebral cortical neurons, Nature 263 (1976) 523–524.
[34] A. Horita, M.A. Carino, H. Lai, Pharmacology of thyrotropin
releasing hormone, Annu. Rev. Pharmacol. Toxicol. 26 (1986)
311–332.
[35] A. Horita, An update on the CNS actions of TRH and its
analogs, Life Sci. 62 (1998) 1443–1448.
[36] L. Brunetti, G. Orlando, B. Michelotto, L. Recinella, M. Vacca,
Cocaine- and amphetamine-regulated transcript peptide-(55-102)
and thyrotropin releasing hormone inhibit hypothalamic do-
pamine release, Eur. J. Pharmacol. 409 (2000) 103–107.
[37] L. Brunetti, P. Preziosi, E. Ragazzoni, M. Vacca, Effects of
vindesine on hypothalamic–pituitary–adrenal axis, Toxicol.
Lett. 75 (1995) 69–73.
[38] L. Brunetti, B. Michelotto, G. Orlando, M. Vacca, Leptin
inhibits norepinephrine and dopamine release from rat hypotha-
lamic neuronal endings, Eur. J. Pharmacol. 372 (1999) 237–240.
[39] H. Asai, K. Kinoshita, M. Yamamura, Y. Matsuoka, Diversity
of thyrotropin-releasing hormone receptors in the pituitary and
discrete brain regions of rats, Jpn. J. Pharmacol. 79 (1999)
313–317.
[40] S. Vonhof, G.Z. Feuerstein, L.A. Cohen, V.M. Labroo,
Norvaline2-TRH: binding to TRH receptors in rat brain ho-
mogenates, Eur. J. Pharmacol. 180 (1990) 1–12.
[41] H.J. Goren, L.G. Bauce, W. Vale, Forces and structural limita-
tions of binding of thyrotrophin-releasing factor to the thyro-
trophin-releasing receptor: the pyroglutamic acid moiety, Mol.
Pharm. 13 (1977) 606–614.
[42] J.L. Radkiewicz, M.A. McAllister, E. Goldstein, K.N. Houk, A
theoretical investigation of phosphonamidates and sulfonamides
as protease transition state isosteres, J. Org. Chem. 63 (1998)
1419–1428.