Metabolism-based brain-targeting system for a thyrotropin-releasing hormone analogue

Article abstract of DOI:10.1021/jm980526i

Gln-Leu-Pro-Gly, a progenitor sequence for the thyrotropin-releasing hormone (TRH) analogue [Leu²]TRH (pGlu-Leu-Pro-NH²), was covalently and bioreversibly modified on its N- and C-termini (by a 1,4-dihydrotrigonellyl and a cholesteryl group, respectively) to create lipoidal brain-targeting systems for the TRH analogue. The mechanism of targeting and the recovery of the parent peptide at the target site involve several enzymatic steps, including the oxidation of the 1,4-dihydropyridine moiety. Due to the lipid insolubility of the peptide pyridinium conjugate obtained after this reaction, one of the rudimentary steps of brain targeting (i.e., trapping in the central nervous system) can be accomplished. Our design also included spacer amino acid(s) inserted between the N-terminal residue of the progenitor sequence and the dihydrotrigonellyl group to facilitate the posttargeting removal of the attached modification. The release of the TRH analogue in the brain is orchestrated by a sequential metabolism utilizing esterase/lipase, peptidyl glycine α-amidating monooxygenase (PAM), peptidase cleavage, and glutaminyl cyclase. In addition to in vitro experiments to prove the designed mechanism of action, the efficacy of brain targeting for [Leu²]TRH administered in the form of chemical-targeting systems containing the embedded progenitor sequence was monitored by the antagonistic effect of the peptide on the barbiturate-induced anesthesia (measure of the activational effect on cholinergic neurons) in mice, and considerable improvement was achieved over the efficacy of the parent peptide upon using this paradigm.

Full text of DOI:10.1021/jm980526i

J . Med. Chem. 1999, 42, 4563-4571
4563
Meta bolism -Ba sed Br a in -Ta r getin g System for a Th yr otr op in -Relea sin g
Hor m on e An a logu e†
Laszlo Prokai,* Katalin Prokai-Tatrai, Xudong Ouyang, Ho-Seung Kim, Whei-Mei Wu, Alevtina Zharikova, and
Nicholas Bodor
Center for Drug Discovery, College of Pharmacy, University of Florida, Gainesville, Florida 32610-0497
Received September 18, 1998
Gln-Leu-Pro-Gly, a progenitor sequence for the thyrotropin-releasing hormone (TRH) analogue
[Leu2]TRH (pGlu-Leu-Pro-NH2), was covalently and bioreversibly modified on its N- and
C-termini (by a 1,4-dihydrotrigonellyl and a cholesteryl group, respectively) to create lipoidal
brain-targeting systems for the TRH analogue. The mechanism of targeting and the recovery
of the parent peptide at the target site involve several enzymatic steps, including the oxidation
of the 1,4-dihydropyridine moiety. Due to the lipid insolublity of the peptide pyridinium
conjugate obtained after this reaction, one of the rudimentary steps of brain targeting (i.e.,
trapping in the central nervous system) can be accomplished. Our design also included spacer
amino acid(s) inserted between the N-terminal residue of the progenitor sequence and the
dihydrotrigonellyl group to facilitate the posttargeting removal of the attached modification.
The release of the TRH analogue in the brain is orchestrated by a sequential metabolism
utilizing esterase/lipase, peptidyl glycine R-amidating monooxygenase (PAM), peptidase
cleavage, and glutaminyl cyclase. In addition to in vitro experiments to prove the designed
mechanism of action, the efficacy of brain targeting for [Leu2]TRH administered in the form of
chemical-targeting systems containing the embedded progenitor sequence was monitored by
the antagonistic effect of the peptide on the barbiturate-induced anesthesia (measure of the
activational effect on cholinergic neurons) in mice, and considerable improvement was achieved
over the efficacy of the parent peptide upon using this paradigm.
In tr od u ction
Carrier-based strategies (3) also sustain several limita-
tions (e.g., inadequate transport capacity, high cost, etc.)
in brain delivery of small peptides.1 A prodrug or related
approach (2) may be considered primarily. Recently, an
opioid peptide (a Leu-enkephalin analogue) has been
successfully transported into the CNS4 by using a
specific prodrug (chemical-targeting system) approach
based on retrometabolic design.5 In this method, a
redox-targeting moiety (1,4-dihydrotrigonellyl, T) is
conjugated to the N-terminus of the peptide via a spacer
amino acid and a lipophilic ester is anchored to the
C-terminus. The nonpeptidic, lipophilic groups furnish
lipid solubility to the molecule and also protect the
embedded peptide from peptidases; thus, penetration
through the BBB to the CNS is facilitated. Sequential
metabolism, starting with the enzymatic oxidation of
the dihydropyridine in the brain, affords retention
(“lock-in”) of the peptide conjugate and, then, the release
of the pharmacologically active neuropeptide analogue
at the site of action. The principles of this method can
be adapted to other neuropeptides with free amino- and
carboxyl-termini.6
The majority of hydrophilic peptides have poor access
to the central nervous system (CNS); they do not cross
the blood-brain barrier (BBB) due to their poor lipid
solubility and due to the absence of specific transport
or delivery systems present in the membrane.1 Admin-
istration of only large doses of peptides produces neu-
ropharmacological effects. Their metabolic instability
also implies that highly active neuropeptide-degrading
enzymes,2 such as the capillary-bound aminopeptidases,
endopeptidases, angiotensin-converting enzyme (ACE),
etc., represent an additional “enzymatic BBB”. There-
fore, the use of a targeting approach is highly justified,
when centrally active peptides are considered as poten-
tial neuropharmaceuticals.
Three strategies have been used to achieve drug
targeting: (1) development of site-specific, pharmaco-
logically active agents; (2) preparation of pharmacologi-
cally inactive agents that are activated preferentially
at the site of action; and (3) use of biological carrier
systems that selectively direct drugs to the specific site
of the body.3 When targeting peptides into the CNS, the
first approach focuses only on (mostly elusive) receptor
specificity and avoids implementing measures to over-
come the BBB and, therefore, is of limited usefulness.
TRH, pGlu-His-Pro-NH2, is the first hypothalamic
releasing factor characterized, establishing the funda-
mental proof for the existence of a neuroendocrine
regulation of pituitary functions by hypothalamic neu-
ronal structures.7,8 A variety of behavioral effects are
induced by peripheral and central application of TRH,
such as increased locomotor activity, “wet dog” shakes,
and forepaw tremor.9,10 The most interesting and best-
known CNS effect of this neuropeptide is the analeptic
† Abbreviations: DCC, 1,3-dicyclohexylcarbodiimide; DMAP, 4-(di-
methylamino)pyridine; HOBt, 1-hydroxybenzotriazole; DCM, dichlo-
romethane; DMF, N,N-dimethylformamide; Fmoc, fluorenylmethyl-
oxycarbonyl; Boc, tert-butyloxycarbonyl; MeOH, methanol; TFA, tri-
fluoroacetic acid; PG, propylene glycol; DIEA, N,N-diisopropylethyl-
amine; TEA, triethylamine; DMSO, dimethyl sulfoxide; Nic, nicotinoyl;
T+, trigonellyl or N-methylnicotinoyl; T, 1,4-dihydrotrigonellyl.
10.1021/jm980526i CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/14/1999

4564 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Prokai et al.
Sch em e 1. Retrometabolic Design for CNS Targeting of [Leu2]TRHa
a
Where T is a 1,4-dihydrotrigonellyl-type targeting group, T+ is trigonellyl, Sp represents spacer amino acid residue(s), and Ch o is
cholesteryl.
action manifested by the reduction of barbiturate nar-
His-Pro-NH2. During the posttranslational processing
of precursors of many secretory proteins in the endo-
plasmic reticulum, Golgi, or secretory vesicle, “unmask-
ing” of an N-terminal glutamine is actually a signal
causing its enzymatic cyclization to pyroglutamyl by
glutaminyl cyclase.31
cosis or haloperidol-induced catalepsy.11,12 TRH and its
analogues can also increase extracellular acetylcholine
(ACh) levels, accelerate ACh turnover, improve memory
and learning, and reverse the reduction in high-affinity
choline uptake induced by lesions of the medial septal
cholinergic neurons.13-15 Therefore, these peptides have
been indicated to be beneficial for treating motor neuron
diseases,16 spinal cord trauma,17 and Alzheimer’s dis-
ease.18,19 Various analogues of TRH have been synthe-
sized to separate the endocrine effect (i.e., TSH-
releasing activity) from the CNS effects.20-23 Several
compounds have been described having enhanced CNS
activity, but decreased or absent TSH-releasing action,
which is a desired feature concerning potential treat-
ment of CNS disorders (analogues where [His2] is
replaced by an aliphatic amino acid residue, such as by
leucine, isoleucine, or norvaline,23 are characteristic
representatives of agents meeting this requirement).
However, TRH and its analogues have poor access to
the CNS.24
The aim of the present study is to report the synthesis
and evaluation of chemical CNS-targeting systems for
a centrally active TRH analogue, [Leu2]TRH. Our design
is based on the hypothesis that CNS delivery of a
progenitor sequence embedded in an appropriate chemi-
cal-enzymatic-targeting system will eventually lead to
the formation of the target peptide via specific enzymes
inherently present at the site of action. We focused our
effort to evaluate the approach by examining the oc-
currence and relative rates of sequential metabolic
processes presumed to be involved in the formation of
the specific TRH analogue in the brain. We also assessed
the improvement, compared to the unmodified peptide
and partially modified compounds, based on a CNS-
specific effect of the target molecule.11,23
TRH analogues modified only at the His2 residue23
typify peptides, where no free N-terminal amino and
C-terminal carboxy groups are available for apparent
covalent modification. These peptides, therefore, convey
problems to the design of a targeting system due to the
presence of the amino-terminal pGlu residue and of the
prolinamide at the carboxy-terminus. The solutions to
these problems have been derived from considering the
biosynthesis of TRH, which involves posttranslational
cleavage of a large precursor polyprotein25 synthesized
on ribosomes. This precursor, consisting of 123 amino
acid residues, contains multiple sequences of Gln-His-
Pro-Gly.26 The TRH progenitor sequence is flanked by
dibasic residues that are typical sites of processing by
hormone secretase enzymes in polyproteins.27,28 Peptidyl
glycine R-amidating monooxygenase (PAM) converts the
C-terminal glycyl residue to an amide.29,30 The final
peptide is designed to develop from a precursor, Gln-
Resu lts
Design . We have postulated that TRH analogues
modified at the second amino acid residue can be
obtained from a Gln-Xaa-Pro-Gly progenitor sequence
(where Xaa is an unspecified amino acid residue).
Because pGlu-Leu-Pro-NH2 was found to exert a 2.5-
fold increase in CNS activity23 and also showed de-
creased TSH-releasing properties compared to TRH, we
have synthesized and evaluated chemical-targeting
systems based on a retrometabolic design for this CNS-
active TRH analogue.32
In the approach selected for brain targeting (Scheme
1), a 1,4-dihydrotrigonellyl targeting group (T) is at-
tached to the N-terminal Gln via spacer amino acid(s)
(Sp ). The 1,4-dihydrotrigonellyl undergoes an enzymati-
cally mediated oxidation (T f T+) resulting in a
hydrophilic, membrane-impermeable trigonellyl (T+)

Brain-Targeting System for a TRH Analogue
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4565
Ch a r t 1. CDSs of [Leu2]TRH
F igu r e 1. Comparison of barbiturate-induced sleeping times
in mice after the administration of the vehicle control, [Leu2]-
TRH (18, in vehicle), and 13a -f. Ten min after iv injection of
the compound (15 µmol/kg of body weight), pentobarbital (60
mg/kg, ip) was injected. Sleeping was recorded as the time
elapsed from the onset of the loss of the righting reflex until
the reflex was regained. The statistical significance of differ-
ences between groups was determined using analysis of
variance (ANOVA) followed by post hoc Fisher’s PLSD test
for multiple comparisons: *p < 0.05 vs vehicle control, **p <
0.05 vs [Leu2]TRH.
salt whose efflux from the brain is greatly reduced; thus,
it is retained in the CNS (“lock-in”). The lipophilic
cholesteryl (Ch o) moiety on the C-terminus is removed
by lipases/esterases further enhancing the lock-in.33
Then, the peptide conjugate with the liberated Gly on
the C-terminus produces the corresponding proline
amide29,30 followed by a cleavage that removes the T+-
Sp moiety releasing Gln-Leu-Pro-NH2, the immediate
precursor for pGlu-Leu-Pro-NH2. The role of Sp in this
design is to facilitate or control this cleavage via
appropriate enzymes, dipeptidyl dipeptidases34 (EC
3.4.14.2 and/or EC 3.4.14.5)34 or proline oligopeptidase
(POP, formerly known as postproline cleavage enzyme,
EC 3.4.21.26),35,36 that have specific cleaving sites
(primarily after Pro) in a peptide sequence. (Theoreti-
cally, POP may also cleave after Pro3 of the embedded
progenitor sequence in T+-(Sp )-Gln-Leu-Pro-Gly-OH.
However, since N-terminal elongation of the sequence
results in decreased specific rate constants,37 this reac-
tion is not expected to be competitive with the amidation
catalyzed by PAM.) Although dipeptidyl peptidase and
POP cleave primarily after Pro in the proper position,25-27
the Pro residue may be substituted with Ala.34,35
However, this replacement is accompanied by a de-
creased affinity of the substrate to the respective
enzyme. To show that the expected production of Gln-
Leu-Pro-NH2 and, after formation of [Leu2]TRH from
this immediate precursor, the pharmacological effect in
the CNS are associated with the given enzymatic
reaction, chemical-targeting systems containing Ala as
single- and double-residue spacers were also synthesized
as controls for pharmacological evaluation.
An a lep tic Activity. We used pharmacological re-
sponses11,23 to assess the enhanced brain-targeting of
this CNS-active peptide. The antagonism on the barbi-
turate-induced anesthesia should indicate the extent of
the activation of cholinergic neurons15 and, thus, the
successful central delivery. The results shown in Figure
1 indicate that, compared to the vehicle control (54.9 (
3.8 min), [Leu2]TRH (18) only decreased the sleeping
time (45.5 ( 3.8 min, a 17 ( 7% change) slightly, while
a significant (p < 0.05) decrease was observed after the
administration of the chemical-targeting systems (13a -
f). Compared to 18, peptide conjugates with the 1,4-
dihydrotrigonellyl (T) attached to a Pro residue showed
the most significant enhancement, with p < 0.05, of
analeptic effect in both the dipeptidyl dipeptidase- and
the POP-based releases of the TRH analogue. Com-
pound 13d with Pro, 13e with Pro-Pro, and 13f with
Pro-Ala as the spacer portion of the chemical-targeting
systems have resulted in a decrease of sleeping time to
29.2 ( 3.5 min (47 ( 6% change compared to the vehicle
control), 26.8 ( 4.0 min (55 ( 7%), and 24.3 ( 2.3 min
(56 ( 4%), respectively.
Syn th esis. The brain-targeting systems (13a-f, Chart
1) were obtained via stepwise elongation of the peptide
chain from the direction of the C-terminus utilizing Boc
chemistry in solution phase. The synthetic procedure
consisted of a series of coupling/deprotection steps.
Briefly, the first step involved the preparation of the
cholesteryl ester of the C-terminal Gly (1). The inter-
mediate peptide esters (2-11) were synthesized by
DCC/HOBt activation. In the final coupling nicotinic
acid was used. After each step, column chromatographic
purification was applied. The Nic-terminated peptide
cholesteryl esters (11a -f) were alkylated on the pyri-
dine ring with Me2SO4, and the obtained pyridinium
salts (12a -f) were reduced with sodium dithionite38 to
the corresponding peptide brain-targeting systems (13a-
f) for [Leu2]TRH. Other peptide conjugates important
in this study were obtained by routine solid-phase
peptide synthesis (SPPS) protocols.
We have also studied the analeptic effect of the
partially derivatized [Leu2]TRH analogues to demon-
strate, again, the need for a bulky, highly lipophilic
moiety (Ch o) together with the 1,4-dihydrotrigonellyl
(T) unit for successful brain-targeting of the parent
peptide, as shown in Figure 2. Other lipophilic com-
pounds such as Boc-Gln-Leu-Pro-Gly-OCho (4, one of
the synthetic intermediates containing the full progeni-
tor sequence), as well as T+-Pro-Pro-Gln-Leu-Pro-Gly-
OCho (12e) and T-Pro-Pro-Gln-Leu-Pro-Gly(-OH) (15),
did not show significant decrease in sleeping time under
the same experimental conditions that we used for
testing the analeptic activity of 13a -f.
In Vitr o Sta bility a n d Meta bolism . To prove that
the release of the parent peptide in the brain after the
systemic administration of 13a -f was according to the
designed sequential metabolism (Scheme 1), a series of
in vitro experiments were performed by using the

4566 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Prokai et al.
F igu r e 2. Comparison of barbiturate-induced sleeping times
in mice after the administration of the vehicle control, 18, 4,
12e, 13e, and 15 under the same experimental conditions as
given in Figure 1.
appropriate synthetic peptide conjugates as substrates.
Because of the limited weight of organs and tissue that
can be obtained from mice, brain, liver, and blood were
obtained from rats for these studies. The analeptic effect
of 13a and 13b was similar in rats32 and in mice
(present study); therefore, we considered the mechanism
of action in the two rodent species similar in principle.
By analogy of brain targeting of a [Leu5]enkephalin
analogue,4 our studies focused on the sequential meta-
bolic conversion of the intermediate T+-(Sp )-Gln-Leu-
Pro-Gly-OH obtained after the oxidation of T and the
subsequent rapid removal of the C-terminal ester func-
tion. One of the critical steps in the sequential metabo-
lism is the prerequisite for a rapid conversion of the
(Pro-)Gly carboxy-terminus to prolinamide in the brain.
In vitro studies on the synthetic T+-Ala-Gln-Leu-Pro-
Gly-OH (14a , m/z 605) as a model compound were used
to confirm this hypothesis. Electrospray ionization (ESI)
mass spectrometric (MS) analyses have revealed that
the conversion of this peptide conjugate to T+-Ala-Gln-
Leu-Pro-NH2 (16a , m/z 547) is only significant in brain
tissue.39 In blood and liver, the major peptide-metabo-
lizing tissues in the rest of the body, degradation occurs
primarily by removal of the C-terminal Gly that yields
T+-Ala-Gln-Leu-Pro-OH (m/z 548). The latter could only
provide further inactive metabolites. The metabolism
of 14a also is slower in the liver than in the brain, and
the compound is relatively stable in plasma (less than
10% of degradation in 2 h). Thus, the systemic formation
of the parent peptide (18) is practically prevented.
Consequently, this CDS also has mechanistic aspects
that are characteristic to enzymatic activation prefer-
entially at the target site, because PAM processing of
the intermediate (formed postdelivery by the loss of
cholesteryl from the C-terminal portion of the peptide
conjugate) is significant only in the brain.
F igu r e 3. (a) ESI mass spectrum demonstrating the forma-
tion of the metabolites T+-Pro-Pro-OH (T+-PP, m/z 332) and
Gln-Leu-Pro-NH2 (17, m/z 356; 10 times magnification in the
mass range indicated) from T+-Pro-Pro-Gln-Leu-Pro-NH2 (16e,
m/z 669) in rat brain homogenate (incubation: 37 °C, 30 min).
(b) MS/MS (product ions) of m/z 356 showing characteristic
fragment ions of Gln-Leu-Pro-NH2 (according to the nomen-
clature introduced by Roepstorff and Fohlman45), which are
identical to those of the synthetic peptide 17.
(MS/MS) confirmation of the structure of the key
compounds) on the in vitro metabolism of T+-Pro-Pro-
Gln-Leu-Pro-NH2 (16e), where POP is the apparent
processing enzyme, provided unequivocal evidence to
the specific peptidase cleavage leading to the release of
17 (Figure 3). The occurrence of this enzymatic reaction
was verified by incubating the peptide conjugate both
in rat brain homogenate and in pH 7.4 HEPES buffer
containing 25 µg/mL recombinant porcine brain POP
(J M-105 strain). On the basis of the mass spectrometric
analysis and the kinetic profiles obtained for the me-
tabolism of 16e in brain homogenate and for the POP-
catalyzed degradation,40 respectively, the “designed-in”
cleavage of the substrate and the concomitant formation
of both hydrolysis products (T+-Pro-Pro-OH and Gln-
Leu-Pro-NH2) were found to occur without significant
side reactions such as the C-terminal deamidation of
16e by POP. We observed that, after its formation,
deamidation of Gln-Leu-Pro-NH2 by POP to produce
Gln-Leu-Pro-OH did occur, but at a much slower rate
The subsequent release of Gln-Leu-Pro-NH2 (17), the
immediate precursor of the TRH analogue, involves a
“designed-in” peptidase cleavage. The half-lives of T+-
(Sp )-Gln-Leu-Pro-Gly-OH (14) intermediates in brain
homogenate at pH 7.40 and 37 °C were in the 20-40
min range. Therefore, the T+-(Sp )-Gln-Leu-Pro-NH2
(16) intermediates, which have half-lives ranging from
about 1 to 4 h, should be considered the “depot” forms
in the process of sequential metabolism in the brain.
ESI-MS studies (including tandem mass spectrometric

Brain-Targeting System for a TRH Analogue
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4567
than the intended primary cleavage. These observations
were consistent with prior studies about the substrate
specificity of POP.37 Conversion of N-terminal Gln to
pGlu by glutaminyl cyclase in the brain, the final step
in the sequential metabolism shown in Scheme 1, has
been well-documented for peptides containing an N-
terminal Gln.31,41,42 We also observed by using an HPLC/
UV assay that 17 (Gln-Leu-Pro-NH2) was processed very
rapidly (t1/2 of 0.8 min at 37 °C in 20% brain homoge-
nate) and with high degree of conversion (>70%, based
on determining the respective areas-under-the-curves
in the kinetic profiles) to 18 ([Leu2]TRH) in vitro.
However, the protonated pGlu-Leu-Pro-NH2 ([18 + H]+,
m/z 339) was not detected in the ESI mass spectrum of
the sample obtained after the incubation of 16e in the
brain homogenate (Figure 3), because ionization of this
compound via ESI was hampered by the lack of basic
nitrogen necessary for the detection of the compound
with an adequate sensitivity by ESI-MS. Evidence for
the sequential formation of 18 from 16e was obtained
by an alternative mass spectrometric assay that utilized
atmospheric pressure chemical ionization (APCI) to
generate protonated molecules and showed high sensi-
tivity in detecting the analyte (18).43 For consecutive
first-order reactions that describe the process shown
below,
F igu r e 4. Kinetics of the conversion of T+-Pro-Pro-Gln-Leu-
Pro-NH2 (16e) to [Leu2]TRH (18) in rat brain homogenate at
37 °C. The concentrations of 16e and 18 measured in brain
homogenate by LC-APCI-MS/MS are shown by the closed
squares (9) and the open triangles (∆), respectively. The
predicted concentration-time profile of 17 and 18 for an ideal
consecutive process (without side reactions) is given by the
dashed and dotted curves, respectively.
peptides; a lipophilic prodrug without a targeting moiety
can efflux from the CNS, and a 1,4-dihydrotrigonellyl-
conjugated peptide is not sufficiently lipophilic and/or
not camouflaged against neuropeptide-degrading en-
zymes without the bulky lipid-soluble moiety to enable
brain delivery by the passive transport through the
BBB. The major goal of the present paper has been to
show that a progenitor sequence is a suitable basis for
the rational design of brain-targeting systems for pep-
tides that are not amenable to straightforward chemical
modifications. In vitro experiments indicated that our
model peptide pGlu-Leu-Pro-NH2 (18) was, indeed,
formed and released in the brain according to a predict-
able metabolic pathway (Scheme 1) from its progenitor
sequence “inlayed” in an appropriate chemical-targeting
system. The bioactivation ultimately leading to the
regeneration of [Leu2]TRH in the brain was also affected
predictably (i.e., Pro is preferred in the Sp )44 by the
specific substrate properties in the metabolic step
utilized to remove the modifying moiety conjugated to
the target peptide sequence.
In conclusion, CNS delivery of peptides based on
covalent conjugation to specific carrier or targeting
functions may utilize appropriate progenitors, when
coupling of the mature peptide to such functions appears
to be cumbersome. Peptides with C-terminal amide and
N-terminal pGlu may rely on a Gly-extended sequence
and Gln as natural precursors, respectively, within the
framework of a rational design approach, as demon-
strated here through the example of chemical-targeting
systems for pGlu-Leu-Pro-NH2, a centrally active TRH
analogue.
the expected concentrations of 17 and 18 in the brain
homogenate versus time of incubation without consider-
ing side reactions are as follows:
c(17) ) c0‚k1/(k2 - k1)‚[exp(-k1t) - exp(-k2t)]
c(18) ) c0‚[k1‚k2/(k2 - k1)/(k3 - k1)‚exp(-k1t) + k1‚k2/
(k3 - k2)/(k1 - k2)‚exp(-k2t) + k1‚k2/(k2 - k3)/
(k1 - k3)‚exp(-k3t)]
where c0 is the initial concentration of 16e (30 µM) and
k1 (0.012 min-1), k2 (0.9 min-1), and k3 (0.01 min-1) are
the respective first-order rate constants determined
separately for the reaction steps indicated. The mea-
sured and predicted kinetic profiles for the sequential
metabolism of 16e leading to the formation of pGlu-Leu-
Pro-NH2 (18) are summarized in Figure 4. In agreement
with the ESI mass spectrum shown in Figure 3a, the
concentration of the intermediate Gln-Leu-Pro-NH2 (17)
should, indeed, remain low during the incubation based
on the calculated c(17) values. The concentration of 18
determined in the brain homogenate by LC-APCI-MS/
MS at the given times of incubation and comparison to
the calculated c(18) values indicated about 50% overall
conversion of 16e to the pharmacologically active TRH
analogue.
Exp er im en ta l Section
In str u m en ts a n d Ma ter ia ls. All chemicals used were
reagent grade or peptide synthesis grade. Solvents were
obtained from Fisher Scientific (Atlanta, GA). Resins and
amino acids were purchased from Bachem BioSci (Torrance,
CA). Cholesterol was purchased from Eastman Kodak Co.
(Rochester, NY). Melting points were determined on a Fisher-
J ohns melting point apparatus and are uncorrected. Thin-layer
chromatography (TLC) was done on silica gel-coated (Kieselgel
60 F254, 0.2-mm thickness) glass or aluminum plates (EM
Discu ssion
Our studies on analeptic activities of the compounds
synthesized for evaluation have demonstrated that the
chemical-targeting systems (13a -f) produced significant
pharmacological response. Comparison with related
prodrug forms of the TRH analogue selected has, again,
confirmed the rationale of a brain-targeting design for

4568 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Prokai et al.
Science, Gibbstown, NJ ) using the following solvent systems:
(a) CHCl3/MeOH (98:2); (b) CHCl3/MeOH (90:10); (c) CHCl3/
MeOH (88:12). Column chromatography was performed using
Davisil grade 643 silica gel (for compounds 1-11) or neutral
alumina (for compound 12). UV spectra were recorded in
MeOH on Cary 210 and Cary 3E UV-visible spectrophotom-
eters (Varian, Walnut Creek, CA). Elemental analyses were
performed by Atlantic Microlab, Inc. (Norcross, GA) and the
Department of Chemistry, University of Florida (Gainesville,
FL). Organic solutions that had been previously extracted with
aqueous solution were dried over anhydrous sodium sulfate.
A Coupler 1000 (DuPont-VEGA, Wilmington, DE) and Synthor
2000 (Peptide International, Louisville, KY) instruments were
applied for solid-phase peptide synthesis (SPPS) using stan-
dard Fmoc chemistry for obtaining T-(Sp )-Gln-Leu-Pro-NH2
(16), Gln-Leu-Pro-NH2 (17), and pGlu-Leu-Pro-NH2 (18).
Preparative RP-HPLC purification was performed on a system
consisting of a ThermoSeparation/SpectraPhysics (Fremont,
CA) SpectraSERIES P200 binary gradient pump, a Rheodyne
(Cotati, CA) model 7125 injector valve equipped with a 5-mL
loop, and a Spectra 100 UV/VIS detector set at 216 nm. The
preparative column (25-mm × 100-mm cartridge with Guard-
Pak insert from Waters Chrom) was packed with 15-µm Delta-
Pak C18. The mobile phase was mixed from 0.1% (v/v) TFA in
H2O and 0.08% (v/v) of TFA in CH3CN at a 5.0 mL/min flow
rate. Molecular weight of the synthetic compounds was
confirmed by ESI mass spectrometry on a Vestec 200 ES
instrument (Perspective Biosystem/Vestec, Houston, TX) using
MeOH or MeOH/CHCl3 as a solvent (to promote cation
attachment as a mechanism of ionization for lipophilic peptide
derivatives, the spraying solvent contained ca. 10 -4 M sodium
iodide). All other samples were sprayed from a methanolic
solution containing 2% (v/v) acetic acid.
Boc-Ala -Gln -Leu -P r o-Gly-OCh o (5): CCSG DCM/MeOH
(92:8, v/v), yield 87%; TLC Rf (b) 0.34; mp 110-112 °C; MS
m/z 976 [M + Na]+. Anal. (C53H88O9N6‚H2O) C, H, N.
Boc-Ala -Ala -Gln -Leu -P r o-Gly-OCh o (6): CCSG DCM/
MeOH (92:8, v/v), yield 87%; TLC Rf (b) 0.25; mp 183-185 °C;
MS m/z 1047 [M + Na]+. Anal. (C56H93O10N7‚H2O) C, H, N.
Boc-P r o-Ala -Gln -Leu -P r o-Gly-OCh o (7): CCSG DCM/
MeOH (88:12, v/v), yield 83%; TLC Rf (c) 0.41; mp 135-137
°C; MS m/z 1073 [M + Na]+. Anal. (C58H95O10N7‚2H2O) C, H,
N.
Boc-P r o-Gln -Leu -P r o-Gly-OCh o (8): CCSG DCM/MeOH
(92:8, v/v), yield 75%; TLC Rf (b) 0.35; mp 128-130 °C; MS
m/z 1002 [M + Na]+. Anal. (C58H95O10N7‚H2O) C, H, N.
Boc-P r o-P r o-Gln -Leu -P r o-Gly-OCh o (9): CCSG DCM/
MeOH (90:10, v/v), yield 81%; TLC Rf (b) 0.47; mp 137-139
°C; MS m/z 1099 [M + Na]+. Anal. (C60H97O10N7‚0.5 H2O) C,
H, N.
Boc-Ala -P r o-Gln -Leu -P r o-Gly-OCh o (10): CCSG DCM/
MeOH (90:10, v/v), yield 63%; TLC Rf (b) 0.42; mp 135-137
°C; MS m/z 1073 [M + Na]+. Anal. (C58H95O10N7‚H2O) C, H,
N.
Nicot in oyl-Ala -Gln -Leu -P r o-Gly-OCh o (11a ): CCSG
DCM/MeOH (90:10, v/v), yield 49%; TLC Rf (c) 0.34; mp 160-
162 °C dec; MS m/z 981 [M + Na]+. Anal. (C54H83O8N10‚2 H2O)
C, H, N.
Nicotin oyl-Ala -Ala -Gln -Leu -P r o-Gly-OCh o (11b): CCSG
DCM/MeOH (88:12, v/v), yield 65%; TLC Rf (c) 0.42; mp 169-
171 °C dec; MS m/z 1052 [M + Na]+. Anal. (C57H88O9N8) C, H,
N.
Nicotin oyl-P r o-Ala -Gln -Leu -P r o-Gly-OCh o (11c): CCSG
DCM/MeOH (88:12, v/v), yield 65%; TLC Rf (c) 0.33; mp 156-
158 °C; MS m/z 1078 [M + Na]+. Anal. (C59H90O9N8‚H2O) C,
H, N.
Nicot in oyl-P r o-Gln -Leu -P r o-Gly-OCh o (11d ): CCSG
DCM/MeOH (88:12, v/v), yield 71%; TLC Rf (c) 0.36; mp 140-
142 °C; MS m/z 1007 [M + Na]+. Anal. (C56H85O8N7‚1.5H2O)
C, H, N.
Nicotin oyl-P r o-P r o-Gln -Leu -P r o-Gly-OCh o (11e): CCSG
DCM/MeOH (86:14, v/v), yield 94%; TLC Rf (c) 0.37; mp 150-
152 °C; MS m/z 1104 [M + Na]+. Anal. (C61H92O9N8‚H2O) C,
H, N.
Syn th esis. Boc-Gly-OCh o (1). A solution of Boc-glycine
(24.71 g, 14.11 mmol), DMAP (24.01 g, 196.53 mmol), and DCC
(43.73 g, 21.16 mmol) in DCM (165 mL) was cooled to 0 °C
with an ice bath. Cholesterol (28 g, 72.41 mmol) dissolved in
DCM (100 mL) was added and the mixture was stirred at 0
°C for 30 min, then at room temperature for 24 h. The
precipitate (DCU) was filtered off, and the filtrate was washed
with citric acid (5%, 5 × 100 mL), saturated sodium bicarbon-
ate (5 × 100 mL), and water (5 × 100 mL). The volatiles were
removed in vacuo. The crude product was purified by column
chromatography on silica gel (CCSG) with DCM/MeOH (99:1,
v/v) eluent affording 29.6 g (yield 75%) of white solid: TLC Rf
(a) 0.61; mp 115-116 °C; MS m/z 567 [M + Na]+. Anal.
(C34H57O4N‚0.5H2O) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of P ep tid e
Ch olester yl Ester s (Com p ou n d s 2-11). The Boc-protected
peptide cholesteryl ester, dissolved in DCM, was deprotected
with TFA (60 min at room temperature). The volatiles were
removed in vacuo. The residue was dissolved in DCM and
neutralized with DIEA. A solution of Boc-protected amino acid
or, in the final step, nicotinic acid (1.5 equiv, 1.5:1 molar ratio
to the cholesteryl ester), HOBt (1.7 equiv), and DCC (2.8
equiv), dissolved in DCM, was added and the mixture was
stirred at room temperature for 24 h. The precipitate (DCU)
obtained was separated and the filtrate was diluted with DCM
and washed with 5% citric acid solution (4×), saturated
aqueous sodium bicarbonate (4×), and water (4×). The organic
layer was separated and the solvent was removed in vacuo.
The residue was purified by column chromatography on silica
gel (CCSG).
Nicotin oyl-Ala -P r o-Gln -Leu -P r o-Gly-OCh o (11f): CCSG
DCM/MeOH (83:17, v/v), yield 68%; TLC Rf (c) 0.51; mp 146-
147 °C; MS m/z 1078 [M + Na]+. Anal. (C59H90O9N8‚1.5H2O)
C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of T+-(Sp )-Gln -
Leu -P r o-Gly-OCh o 12a -f. Compounds 11a -f (1 equiv) were
alkylated in DCM with approximately 15 equiv of dimethyl
sulfate at room temperature until the reaction was completed.
Then the solvent was removed in vacuo and the solid residue
obtained was washed with ether. The crude products were
recrystallized from chloroform and ether. The products crystal-
lized with 0.5-1 equiv of alkylating agent.
Tr igon ellyl-Ala -Gln -Leu -P r o-Gly-OCh o (12a ): yield 98%;
mp 172-174 °C; MS m/z 973 (Q+); UVλmax 265 nm.
Tr igon ellyl-Ala -Ala -Gln -Leu -P r o-Gly-OCh o (12b): yield
98%; mp 173-175 °C; MS m/z 1044 (Q+); UVλmax 265 nm.
Tr igon ellyl-P r o-Ala -Gln -Leu -P r o-Gly-OCh o (12c): yield
98%; mp 176-178 °C; MS m/z 1070 (Q+); UVλmax 265 nm.
Tr igon ellyl-P r o-Gln -Leu -P r o-Gly-OCh o (12d): yield 99%;
mp 194-196 °C; MS m/z 999 (Q+); UVλmax 268 nm.
Tr igon ellyl-P r o-P r o-Gln -Leu -P r o-Gly-OCh o (12e): yield
92%; mp 186-188 °C; MS m/z 1096 (Q+); UVλmax 264 nm.
Tr igon ellyl-Ala -P r o-Gln -Leu -P r o-Gly-OCh o (12f): yield
98%; mp 181-183 °C; MS m/z 1070 (Q+); UVλmax 264 nm.
Gen er a l P r oced u r e for th e Sod iu m Dith ion ite Red u c-
tion (13a -f). Under continuous nitrogen atmosphere the
pyridinium salts 12 (1 equiv) were dissolved in 50% (v/v)
aqueous MeOH; then the mixture of sodium bicarbonate (12
equiv) and sodium dithionite (10 equiv) was added under ice
cooling. After 15 min the cooling was removed and the stirring
was continued at room temperature for 40 min under nitrogen
atmosphere. Then the reaction mixture was extracted several
times with cold, degassed DCM. The combined organics were
Boc-P r o-Gly-OCh o (2): CCSG DCM/MeOH (99:1, v/v),
yield 63%; TLC Rf (a) 0.3; mp 173-175 °C; MS m/z 664 [M +
Na]+. Anal. (C39H64O5N2) C, H, N.
Boc-Leu -P r o-Gly-OCh o (3): CCSG DCM/MeOH (98:2, v/v),
yield 75%; TLC Rf (b) 0.65; mp 59-61 °C; MS m/z 777 [M +
Na]+. Anal. (C45H75O6N3‚H2O) C, H, N.
Boc-Gln -Leu -P r o-Gly-OCh o (4): CCSG DCM/MeOH (93:
7, v/v), yield 76%; TLC Rf (b) 0.4; mp 105-107 °C; MS m/z
905 [M + Na]+. Anal. (C50H83O8N5‚H2O) C, H, N.

Brain-Targeting System for a TRH Analogue
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4569
washed several times with cold, degassed water, and then the
solvent was removed in vacuo. The crude products were
purified by column chromatography on neutral alumina with
DCM/MeOH (97:3) (v/v).
1,4-Dih ydr otr igon ellyl-Ala-Gln -Leu -P r o-Gly-OCh o (13a):
yield 35%; TLC Rf (c) 0.42; mp 138-140 °C dec; UVλmax 348
nm. Anal. (C55H87O8N7‚2H2O) C, H, N.
1,4-D ih y d r o t r ig o n e lly l-Ala -Ala -G ln -L e u -P r o -G ly -
OCh o (13b): yield 34%; TLC Rf (c) 0.46; mp 150-152 °C dec;
UVλmax 348 nm. Anal. (C58H92O9N8‚2.5H2O) C, H, N.
1,4-D ih y d r o t r ig o n e lly l-P r o -Ala -G ln -L e u -P r o -G ly -
OCh o (13c): yield 25%; TLC Rf (c) 0.43; mp 145-147 °C dec;
UVλmax 348 nm. Anal. (C60H94O9N8‚3H2O) C, H, N.
1,4-Dih ydr otr igon ellyl-P r o-Gln -Leu -P r o-Gly-OCh o (13d):
yield 27%; TLC Rf (c) 0.4; mp 142-144 °C dec; UVλmax 348 nm.
Anal. (C57H89O8N7‚2.5H2O) C, H, N.
1,4-D ih y d r o t r ig o n e lly l-P r o -P r o -G ln -L e u -P r o -G ly -
OCh o (13e): yield 24%; TLC Rf (c) 0.4; mp 140-142 °C dec;
UVλmax 348 nm. Anal. (C62H96O9N8‚3.5H2O) C, H, N.
1,4-D ih y d r o t r ig o n e lly l-Ala -P r o -G ln -L e u -P r o -G ly -
OCh o (13f): yield 21%; TLC Rf (c) 0.45; mp 163-165 °C dec;
UVλmax 348 nm. Anal. (C60H94O9N8‚3H2O) C, H, N.
Tr igon ellyl-(Sp )-Gln -Leu -P r o-Gly-OH 14a-f. Componds
12a -f (200 mg) in methanesulfonic acid (2.8 mL) containing
anisole (0.3 mL) were kept at 0 °C for 10 min and at room
temperature for 30 min. Then 400 mL of diethyl ether was
added. The precipitated amorphous solids were separated,
washed with diethyl ether, and dried in vacuo. The solids
(identified by MS) were obtained with approximately 95%
yield; UVλmax 264 nm.
1,4-Dih yd r ot r igon ellyl-P r o-P r o-Gln -Leu -P r o-Gly-OH
(15). Compound 14e (100 mg, 0.14 mmol) was dissolved in
degassed distilled water (25 mL) at 0 °C; then the mixture of
sodium bicarbonate (236.0 mg, 2.8 mmol) and sodium dithion-
ite (407.4 mg, 2.3 mmol) was added under argon followed by
the addition of DCM (20 mL). The mixture was stirred at 0 °C
for 20 min and then at room temperature for 40 min.
Methylene chloride (30 mL) and 1 g of sodium chloride were
added. The layers were separated and the organic layer was
washed several times with deaerated water, then evaporated
to dryness resulting in 40 mg (yield 40%) of fine yellowish
powder: MS m/z 753 [M + Na]+; UVλmax 348 nm; peptide
content (spectrophotometric titration with methanolic 0.1 M
AgNO3) > 90%.
Tr igon ellyl-(Sp)-Gln -Leu -P r o-NH2 (16): prepared by SPPS
utilizing standard Fmoc protocol; after purification (RP-HPLC)
the white solids obtained were identified by ESI-MS and APCI-
MS.
Gln -Leu -P r o-NH2 (17) tr iflu or oa ceta te: semisolid; MS
m/z 356 [M + H]+. Anal. (C18H30O6N5F 3‚1.5H2O) C, H, N.
p Glu -Leu -P r o-NH2 (18): yellowish semisolid; MS m/z 339
[M + H]+. Anal. (C16H27O4N4‚H2O) C, H, N.
An a lep tic Activity. The effect on pentobarbital-induce
sleeping times was studied in Swiss Webster mice (Harlan
Sprague-Dawley). All animals experiments were conducted
in accordance with the guidelines set forth in the Declaration
of Helsinki and the Guiding Principles in the Care and Use of
Animals (DHEW Publication, NIH 80-23). Ten to sixteen Swiss
Webster mice (30 ( 2 g) were used in each group. Test
compounds were dissolved in a vehicle consisting of PG and
DMSO (2:1) (v/v). The vehicle alone (1.5 mL/kg of body weight)
was administered to the animals in the control group. Equimo-
lar doses of test compounds including [Leu2]TRH itself (18;
15 µmol/kg of body weight) were injected through the tail vein.
After 10 min, each animal received an intraperitoneal (ip)
injection of sodium pentobarbital solution at a dose of 60 mg/
kg of body weight. The sleeping time was recorded as the time
elapsed from the onset of loss of the righting reflex until the
reflex was regained. The significance of differences between
groups was determined using analysis of variance (ANOVA)
followed by Fisher’s protected least significant difference
(PLSD) test for multiple comparisons.
phate buffer, plasma, and brain and liver homogenates.
Approximately 50 nmol of the compound (dissolved in water)
was added to 1 mL of rat plasma or brain or liver homogenate
(20%, w/w, in pH 7.4 Tris buffer), respectively, and the mixture
was incubated at 37 °C in a temperature-controlled, shaking
water bath. For the experiment involving recombinant porcine
brain POP (J M-105 strain), the enzyme (25 µg/mL) was
dissolved in pH 7.4 HEPES buffer, and the solution was
preincubated at 37 °C for 5 min before the addition of the
compound studied. Aliquots (100 µL) were removed after 5,
15, 30, 45, 60, and 90 min of incubation, respectively, and
transferred into 1.5-mL plastic centrifuge tubes containing 200
µL of ice-cold 1 M aqueous acetic acid. After centrifugation at
12500g for 15 min, the supernatant was removed and analyzed
by microbore HPLC. Analyses were done on a system consist-
ing of a ThermoSeparation/SpectraPhysics (Fremont, CA)
SpectraSERIES P200 binary gradient solvent delivery system,
a Rheodyne (Cotati, CA) model 7125 injector valve equipped
with a 5-µL sample loop, a Spectroflow 757 variable wave-
length UV/VIS detector (Kratos Analytical, Manchester, U.K.)
operated at 216 nm, and a Hewlett-Packard model HP 3395
computing integrator (Palo Alto, CA). A 30-cm × 1.0-mm i.d.
Supelcosil LC-18 (5-µm) reversed-phase column (Supelco,
Bellefonte, PA) was used at a flow rate of 50 µL/min that was
maintained by using a dynamic split (before the injection
valve) via a balance column (25-mm × 4.6-mm i.d.) connected
in parallel with the microbore column. The mobile phase was
mixed from 0.1% (v/v) TFA in water and 0.08% (v/v) TFA in
acetonitrile, and gradient elution was done from 5% to 95% of
organic modifier that changed in a linear profile at a rate of
1%/min. HPLC calibration curves for determining concentra-
tions were obtained by adding known amount of peptide into
aliquots of brain homogenate transferred into ice-cold 1 M
acetic acid solution and analyzing the supernatant after
centrifugation. Concentration-time profiles were analyzed by
exponential fitting, assuming a pseudo-first-order degradation.
Half-lives (t1/2) were calculated from the rate constants (k) as
0.693/k. Supernatants obtained from samples removed from
the incubation mixtures were desalted on reversed-phase
cartridges (1-mL Supelclean LC-18, Supelco, Bellefonte, PA)
for ESI mass spectrometry. After treating the cartridge by
washing with methanol (2 mL) followed by 3% (v/v) aqueous
acetic acid (2 mL), the sample was adsorbed. Poorly retained
sample components were removed by washing with 3% aque-
ous acetic acid solution (2 mL), and the analytes were obtained
by water/methanol/acetic acid (49/49/2, v/v, 400 µL) as an
eluent. The sample solution was supplied for analysis at 3-5
µL/min by syringe pumps fitted with a 100- or 250-µL gastight
syringe (Hamilton, Nevada) and connected to the ESI source
of a mass spectrometer via fused silica tubing (100-µm i.d.).
ESI mass spectra were recorded on
a Vestec/PerSeptive
Biosystems (Houston, TX) model 200 ES instrument controlled
by a Vector/Two data system (Teknivent, St. Louis, MO) and/
or on an LCQ (Finnigan MAT, San J ose, CA) quadrupole ion
trap controlled by a Microsoft Windows NT-based system
(Navigator 1.1, Finnigan MAT). The mass spectra were
acquired in the 100-1000 mass-to-charge (m/z) range. On the
LCQ, narrow mass-range scans (ZoomScan; covering an m/z
range of 10 u) were also recorded for the ESI ions of interest
in order to achieve the resolution of the isotope peaks. APCI
mass spectra were acquired on the LCQ. LC-MS and LC-
MS/MS analyses were performed by using a 5-cm × 4.0-mm
i.d. C18 reversed-phase cartridge (SGE International, Ring-
wood, Australia). The mobile phase was 25% (v/v) acetonitrile
and 2 % (v/v) acetic acid in deionized water, the flow rate was
maintained at 1.0 mL/min by a Spectroflow 400 (Kratos
Analytical) isocratic solvent delivery system, and 5 µL of the
desalted sample solution was injected for analysis.
Ack n ow led gm en t. This research has been partially
supported by grants from the National Institutes of
Health (AG10485, NS30616, RR12023, DA10543, and
MH59360). Graduate Research Assistantships funded
by Genentec Corp. (to X.O. through N.B.) and by the
In Vitr o Sta bility a n d Meta bolism Stu d ies. Stability
studies of selected compounds have been performed in phos-

4570 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22
Prokai et al.
(17) Faden, A. I.; Vink, R.; McIntosh, T. K. Thyrotropin-releasing
hormone and central nervous system trauma. Ann. N. Y. Acad.
Sci. 1989, 553, 380-384.
(18) Sunderland, A.; Mellow, A.; Cohen, R.; Lawlor, B.; J ill, J .;
Newhouse, P.; Cohen, M.; Murphy, D. Acute effects of high-dose
thyrotropin releasing hormone infusions in Alzheimer’s disease.
Psychopharmacology 1989, 98, 403-407.
Division of Sponsored Research at the University of
Florida (to H.-S.K. through L.P.) are also acknowledged.
The recombinant porcine brain POP was generously
provided by Dr. Laszlo Polgar, Institute of Enzymology,
Hungarian Academy of Sciences, Budapest.
(19) Lampe, T.; Norris, J .; Risse, S.; Owens-Williams, E.; Keenan,
T. Basic mechanisms, diagnosis and therapeutic strategies. In
Alzheimer’s Disease; Iqbal, K., McLachlan, D. R. C., Winbald,
B., Wisniewski, H. M., Eds.; J ohn Wiley and Sons, Ltd.: New
York, 1991; p 643.
(20) Yamamoto, M.; Shimizu, M. Effects of a new TRH analogue, YM-
14673 on the central nervous system. Naunyn-Schmiedebergs
Arch. Pharmacol. 1987, 336, 561-565.
(21) Friedrichs, E.; Schwertner, E.; Herrling, S.; Gunzler, W.-A.;
Ottig, F.; Flohe, L. In Structure and activity of natural peptides;
Voelter, W., Weitzel, G., Eds.; Walter de Gruyter: Berlin,
Germany, 1981; p 461.
Su p p or tin g In for m a tion Ava ila ble: Mass spectrometric,
LC-MS, and kinetic analyses that show the involvement of
PAM, peptidase cleavage, and glutaminyl cyclase in the
designed sequential metabolic process leading to the formation
of [Leu2]TRH in rat brain in vitro according to Scheme 1. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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us to conclude that the product with the carboxamide-terminus

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J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 22 4571
(16a ) was in large excess compared to the one with the carboxy-
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