Novel l-prolyl-l-leucylglycinamide (PLG) tripeptidomimetics based on a 2-azanorbornane scaffold as positive allosteric modulators of the D2R

Article abstract of DOI:10.1039/c6ob02248k

An efficient and straightforward orthogonal methodology was successfully developed to achieve constrained l-prolyl-l-leucylglycinamide (PLG) analogues starting from two proline mimetics based on a 2-azanorbornane scaffold. A preliminary dopamine D₂ receptor radiolabeled binding assay with [³H]-N-propylnorapomorphine shows that enantiopurity of PLG peptidomimetics based on 2-azanorbornane is a requirement to achieve statistically significant positive modulators of the D₂ receptor. This is the first documented active peptidomimetic of PLG whose bioactivity is not correlated with the C-terminal carboxamide pharmacophore and which cannot adopt the hypothesized type II β-turn conformation.

Full text of DOI:10.1039/c6ob02248k

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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Novel L-prolyl-L-leucylglycinamide (PLG) Tripeptidomimetics based on 2-
DOI: 10.1039/C6OB02248K
azanorbornane Scaffold as Positive Allosteric Modulators of D2R
Ivo E. Sampaio-Dias†a, Carlos A. D. Sousab, Xerardo García-Merac, Joana Ferreira da Costad, Olga Caamañoc,
José E. Rodríguez-Borges†a
a. UCIBIO / REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007
Porto, Portugal.
b. LAQV / REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto,
Portugal.
c.
Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, E-15782 Santiago de
Compostela, Spain.
d. Instituto de Química, Universidade Federal da Bahia, Campus de Ondina, Salvador-BA, 40170-290 Brazil.
† Corresponding authors e-mails: idias@fc.up.pt (Ivo E. Sampaio-Dias); e-mail: jrborges@fc.up.pt (José E. Rodríguez-Borges)
Replacement of L-prolyl residue in PLG sequence by enantiopure (1R,3S,4S)-2-azanorbornane scaffold
afforded an active peptidomimetic compatible with suppression of C-terminal carboxamide pharmacophore.
Abstract
An efficient and straightforward orthogonal methodology was successfully developed to achieve
constrained L-prolyl-L-leucylglycinamide (PLG) analogues starting from two proline mimetics based
on 2-azanorbornane scaffold. Preliminar dopamine D2 receptor radiolabeled binding assay with [3H]-
N-propylnorapomorphine shown that enantiopurity of PLG peptidomimetics based on 2-
azanorbornane is a requirement to achieve statistically significant positive modulators of D2 receptor.
This is the first documented active peptidomimetic of PLG that bioactivity is not correlated to C-
terminal carboxamide pharmacophore and which cannot adopt the hypothesized type II -turn
conformation.
Keywords
PLG; Allosteric Modulation; Constrained Proline Mimetics; 2-azanorbornane; Peptidomimetics.

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DOI: 10.1039/C6OB02248K
Introduction
L
-prolyl- -leucylglycinamide (PLG, Fig. 1) is a short length peptide suitable as a lead
L
molecule for the development of novel compounds capable of crossing the blood-brain
barrier.1 Pharmacological studies demonstrate that its biological functions rely on the
modulation of dopaminergic neuronal transmission in the nigrostriatal pathway2,3 and
therefore it has been linked to Parkinson's Disease and tardive dyskinesia, where it has shown
therapeutic activity.4
The accurate mechanism by which PLG modulates dopaminergic receptors remains
unknown since the precise allosteric site at which PLG exhibits modulation in dopamine
receptors it is not known.3 This provides an interesting field of research to the development of
new potential pharmaceuticals.
Although studies point out that modifications at -Pro residue of PLG has a significant
L
influence in the activity of PLG analogues,5 there are few examples reporting PLG proline
mimetics.6,7
Fig. 1
L
-prolyl- -leucylglycinamide (PLG) and a new class of analogues based on 2-azanorbornane scaffold.
L
In previous publications our research group described the synthesis of 3,5-
disubstituted proline derivatives and its effects on allosteric modulation of the dopamine
receptor,1 endorsing the importance of proline mimetics to achieve active analogues of PLG.
Constrained amino acids, such as rigid pseudoprolines, can provide important
information about the bioactive conformation of prolyl-containing peptides,6 by stabilizing
certain peptide secondary structure elements.7
The bicyclic system exo-2-azanorbornane-3-carboxylic acid (Ψ
P) is a constrained
proline mimetic that has been used in peptide synthesis,7,8 which can be regarded as a trans-
β,δ-substituted proline analogue,8 in a permanent Cγ-endo envelope conformation (Fig. 2).

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DOI: 10.1039/C6OB02248K
Fig. 2 Proline envelope conformers present in many peptides and proteins: (
2-azanorbornane scaffold as proline mimetic in permanent Cγ-endo conformation and (
proline mimetic, Ψ
A
) Cγ-exo and (
B) Cγ-endo conformations. (C)
D
) a possible starting material for
P
.
The rationale behind this project is therefore developing constrained analogues of PLG
in order to assess the importance of restricting the conformational space of proline’s envelope
conformers in PLG and its effects in modulation of D2 receptor by replacing it for a non-
proteinogenic constrained proline mimetic based in 2-azanorbornane scaffold in a permanent
Cγ-endo mimetic such as
1 (Fig. 1).
To meet this goal, it is imperative to develop a robust methodology for the
development of the desired peptidomimetics.
Discussion
Organic Chemistry: optimization of the synthetic route
C-termini dipeptides were prepared using the convenient protected amino acids 2a-c
and 3a-c in presence of Hünig’s base and TBTU9 as coupling reagent in dry CH2Cl2,
affording the dipeptides 4a-c in good to excellent yields (88-98%, scheme 1).
It was found (TLC) that the reactions were completed in about 1h and that longer
reaction times (> 24h) led to the formation of side products that made purification of
dipeptides 4a-c more laborious. These short reaction periods required for peptide coupling
using TBTU are in agreement with previous work by Balalaie et al. using different carboxylic
acids and primary amines at rt.10
Acidolytic removal of tert-butoxycarbonyl (Boc) protecting group from dipeptides 4a-
c
was performed with 2,2,2-trifluoroacetic acid (TFA) in dry CH2Cl2 for about 1h. Azeotropic
elimination of TFA with CH2Cl2 afforded the corresponding deprotected dipeptides 5a-c as
trifluoracetate salts with practically quantitative yields (98-99%, Scheme 1).

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DOI: 10.1039/C6OB02248K
Scheme 1 Synthesis of dipeptides 5a-c. Reagents and conditions: i) TBTU, DIEA, CH2Cl2, rt; ii) TFA, CH2Cl2, 0˚C, 1h.
With these C-termini dipeptides in hands we studied the most straightforward way to
achieve peptidomimetics 1a-c (Scheme 2) from 2-azanorbornane scaffolds as constrained
prolines. Following a diversity-oriented synthesis approach, three main families of analogues
in their C-terminal ester form (precursors of C-terminal amides) were designed, namely
ΨPVA
series,
acids,
(
1a), ΨPLG
-leucine (L) and glycine (G) residues were replaced by their structure-related amino
-valine (V) and
-alanine (A), respectively; in ΨPLG series the sequence is
( (
1b) and ΨPGL 1c). With regard to PLG native sequence, in ΨPVA
L
L
L
maintained and in ΨPGL the sequence of C-terminus dipeptide made of two hydrophobic
amino acids were inverted. This allows assessment of the robustness of methodologies while
introducing chemical diversity, as shown in Scheme 2.
The simplest and most direct route to achieve peptidomimetics 1a-c is expected to be
method
by hetero-Diels-Alder reaction between the protonated methyl 2-(benzylimino)acetate
(prepared in situ) and cyclopentadiene (CPD) as early reported.11 Bicyclic unit (
)- is easily
obtained by treatment of ( )- with LiOH in a mixture of THF/H2O at room temperature (rt),
followed by work-up with dropwise addition of H2SO4 1M at 0˚C (91%).
A represented in scheme 2. Thus, starting with cycloadduct (±)-6 – which is obtained
±
7
±
6
Then, (
±
)-
7
was coupled with dipeptides 5a-c using the same peptide condensation
)- is a racemate, N-benzylated tripeptides 8a-c 8a'-c' were obtained as
protocol. Since (
±
7
/
mixtures of diastereoisomers in reasonable yields (66-76%).
The saturation of the olefin and hydrogenolysis (N-debenzylation) were carried out by
catalytic hydrogenation of the tripeptides 8a-c 8a'-c', using Pd(OH)2/C and H2 (1 atm) at rt,
affording the respective N-deprotected tripeptides 1a-c 1a'-c' in good yields (70-83%). It was
/
/
verified (TLC) that the saturation of olefin was achieved quite rapidly (about 1-3h) followed
by the N-debenzylation which was carried out between 4-8 days. Despite the fair results,
hydrogenolysis reactions were very long-standing and sometimes incomplete, ensuing in
troublesome chromatographic purifications.

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DOI: 10.1039/C6OB02248K
In order to circumvent these difficulties, we tested the adoption of method
B (scheme
2). Boc-protected cycloadduct (
±
)-
9
(exo) is obtained from aza-Diels-Alder reaction in water
between CPD and the iminium ion generated in situ from ethyl glyoxylate and aqueous
NH4Cl, as previously described,12,13 and posteriorly N-Boc protected.
Hydrogenation of (
±
)-
9
was carried using triethylsilane (TES) as a source of H2 in situ
)-10 in a short reaction time (10
in presence of Pd/C, affording the saturated cycloadducts (
±
min) with quantitative yield. This result is in agreement with the saturation of olefins
described by Mandal and McMurray using TES.14
The corresponding carboxylic acid was achieved by alkaline hydrolysis of (
±)-10
under the same conditions mentioned before, obtaining ( )-11 in excellent yield (94%). Here,
±
special attention was taken during the acidification work-up procedure to prevent acidolytic
removal of N-Boc protective group.
Coupling of (±)-11 with dipeptide 5b was performed in the same conditions of peptide
coupling mentioned before using TBTU, resulting in tripeptides 12b/12b' as diastereomeric
mixtures in very good yield (90%).
Deprotection of N-Boc tripeptides was conducted by the same acidolysis conditions
mentioned above, affording tripeptides 1b/1b' in practically quantitative yield (98%),
avoiding the need of chromatographic purifications. It’s worth to mention that all N-Boc
1
protected intermediates exhibit rotamers in both H- and 13C-NMR spectra (CDCl3), which is
known and well described for N-Boc protected compounds derivatives of 2-azanorbornane.15
Although the latter approach (method
more practical, cleaner and also offers better overall yields) it has two major drawbacks: 1)
starting material ( )- is of difficult obtainment since its free amine precursor is acquired as
B) offers clear advantages over method A (it is
±
9
a minor adduct from the referred aza-Diels-Alder reaction (exo adduct obtained with 18%
yield);12,13 2) only racemates are obtained by using this method.
Enantiopurity may be a crucial issue, since in PLG the natural proline residue has the
specific (S)-configuration. Thus, for a properly evaluation of the influence of 2-
azanorbornane scaffold as
), pure (1R,3S,4S)-2-azanorbornane should be used since chromatographic attempts to
separate the mixtures of diastereoisomers mentioned before were unfruitful.
L
-proline mimetic in it permanent Cγ-endo conformation (Fig. 2,
C

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In order to benefit from the better of the two mentioned methodologies, we adopted
DOI: 10.1039/C6OB02248K
method
C
(Scheme 2). Starting from adduct (1S,3S,4R)-13, which may be accomplished by a
similar process of compound (
±
)-9 from ethyl 2-((R)-(1-phenylethyl)imino)acetate and
CPD.7 Enantiomerically pure proline mimetic (1R,3S,4S)-14 was efficiently achieved by
catalytic hydrogenation followed by addition of conc. HCl in EtOH/Et2O until it precipitated
as colourless crystals, which were then submitted to N-deprotection by catalytic
hydrogenolysis. Subsequent N-Boc protection by treatment with Boc2O in the presence of
Hünig’s base yielded enantiomerically pure (1R,3S,4S)-10 (97%) which was converted in
pure carboxylic acid (1R,3S,4S)-11 (89%) by following the same alkaline hydrolysis
procedure for (±)-11.
From there on, compounds (1R,3S,4S)-1a and (1R,3S,4S)-1b were obtained as
diastereomericaly pure peptidomimetic by following the same protocol applied before in
method B.
Scheme 2 Synthesis of constrained bicyclic PLG peptidomimetics 1a-c based on orthogonal methodologies: starting with
proline analogues ( )- ), ( )- ) and (1S,3S,4R)-13 ). Reagents and conditions: i) LiOH, MeOH/H2O, rt; ii) TBTU,
±
6
(
A
±
9
(
B
(C
DIEA, CH2Cl2, rt; iii) H2 (1 atm), cat. 20% Pd(OH)2/C, rt; iv) TES, MeOH, Pd/C 10% w/w; v) LiOH, EtOH/H2O, rt; vi)
TFA, CH2Cl2, 0˚C, 1h; vii) H2 (50 psi), Pd/C 10% in abs. EtOH, 2h at rt followed by conc. HCl in EtOH/Et2O; viii) H2 (90
psi), Pd/C 10% in abs. EtOH, 4d at rt; ix) Boc2O, DIEA, CH2Cl2, 2h. Yields were determined after purification by flash
chromatography except for (±)-7, (±)-11, 11,
14 and 1(a,b) . TFA.

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DOI: 10.1039/C6OB02248K
Pharmacology: photoaffinity-labeling binding assays
There are studies showing that the replacement of
L
-Pro by its enantiomer -Pro in the
D
PLG sequence revealed activity comparable to that of PLG.16 Conversely, a similar
epimerization at the bicyclic L-Pro analogue yields a diastereoisomer, whose integration as proline
mimetic in PLG may influence the pharmacological outcome.
Therefore, compounds 1a-c/1a'-c' (mixture of diastereoisomers) and enantiopure
tripeptides (1R,3S,4S)-1a and (1R,3S,4S)-1b were tested for their ability to potentiate the
maximum binding of radiolabelled agonist N-propylnorapomorphine ([3H]-NPA) to cloned
human dopamine D2S receptors as described in literature17 (Fig. 3 and Fig. S41 of SI). These
compounds were tested at eight different concentrations in the range between 1 pM and 10

M.
The binding assays revealed absence of modulatory effect by the mixture of
diastereoisomers 1a-c
1a'-c' in increasing [3H]-NPA binding (data not shown). On the other
/
hand, the tested enantiopure tripeptides (1R,3S,4S)-1a and (1R,3S,4S)-1b exhibited a bell-
shaped dose response curve that is typical of the allosteric modulation of the dopamine
receptor by PLG and its analogues, compatible with previous findings from in vivo and
clinical experiments using PLG.17 This data evidences that these compounds are acting as
allosteric modulators of dopamine D2S receptors by increasing the binding of the
radiolabelled agonist [3H]-NPA to this preparation.
A statistically significant enhancement (P < 0.05) of the [3H]-NPA response was
observed for peptidomimetic (1R,3S,4S)-1a at 10 pM and 100 pM (the same concentrations
for PLG). The maximum effect for (1R,3S,4S)-1a was 15 ± 6% at 10 pM (Fig. 3) against 22 ±
9% for PLG at the same concentration. The response by (1R,3S,4S)-1b was not statistically
significant due to its large variability (P > 0.05).
The effect observed with PLG in our work is different to that previously reported by
Verma et al.,17 showing to increase [3H]-NPA binding at lower concentrations than those
previously reported. This difference can be explained by the different host cell where human
D2S receptors were expressed as it has been previously reported that allosteric modulators are
sensitive to environmental changes which may condition the different active conformations
elicited by the endogenous agonists on GPCRs.18
Earlier studies have shown C-terminal carboxamide is a key pharmacophore in PLG
allosteric activity.19,20 In our series, the tested compounds display C-terminal methyl esters
and the results obtained for the derivative (1R,3S,4S)-1a is considered most interesting since

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it shows that absence of carboxamide pharmacophore can be accepted in 2-azanorbornane
DOI: 10.1039/C6OB02248K
based PLG peptidomimetics.
The acceptance of this replacement by PLG binding site adds interesting information
to the discussion about the bioactive conformation of PLG, which was postulated in several
studies to be a type II 
-turn.5,19,20
To our best knowledge this represents the first documented class of compounds
exhibiting modulatory effects without the C-terminal carboxamide pharmacophore.19,20
Without the C-terminal carboxamide group it is impossible for peptidomimetic (1R,3S,4S)-1a
to adopt type II -turn conformation, so it is speculated an extended bioconformation for this
derivative. This observation also supports the hypothesis of the existence of a second
bioactive conformation for PLG based in its extended form.5,21
(1 R ,3 S ,4 S )-1 a
P L G
40
20
0
40
20
0
*
*
*
*
10 -1 2 10 -1 1 10 -1 0 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4
10 -1 2 10 -1 1 10 -1 0 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4
-20
-20
[ ] / M
[ ] / M
Fig. 3 Modulation of [3H]-NPA binding of PLG and (1R,3S,4S)-1a exerted by the different compounds at eight different
concentrations. Points represent the mean ± standard deviation (vertical bars) of three independent experiments carried out
with duplicate points. *P < 0.05 (ANOVA test; post-hoc Dunnet T3 test).
It is important to denote that this scenario is not against or infringe the hypothesis that
bioactive conformation of PLG is a type-II β-turn conformation or the role of carboxamide
pharmacophore, but more studies are needed to fully understand the chemical requirements
towards positive modulation of D2 receptors in order to develop new potential
pharmaceuticals to neurological disorders related to dopaminergic pathways.
Since the diastereoisomeric mixtures of 1a/1a' and 1b/1b' presented no modulatory
effect at all, but their pure enantiomers showed bell-shaped profiles similar to the PLG,
particularly (1R,3S,4S)-1a demonstrating very interesting results, only enantiopure 2-
azanorbornanes are useful to be submitted to identical binding assays. Since enantiomeric
pure compounds are often very expensive, the use of optimized, well established and
effective protocols is mandatory.

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DOI: 10.1039/C6OB02248K
Conclusion
An efficient and straightforward methodology was developed to furnish
diastereomerically pure constrained PLG analogues derived from enantiomerically pure 2-
azanorbornane scaffold. In opposite to diastereoisomeric mixture 1a 1a' – obtained from
/
racemic 2-azanorbornane scaffold – peptidomimetic (1R,3S,4S)-1a revealed a statistically
significant activity in the binding [3H]-NPA to D2 receptor displaying a bell-shaped profile
similar of PLG, proving that enantiopurity of 2-azanorbornane is crucial to achieve
significantly active modulators of dopamine receptor.
This provides an important insight for the rational design of novel PLG analogues in
order to achieve active peptidomimetics, opening a new field of investigation seeking for
potential pharmaceuticals. Further studies on the structure-activity relationship of optically
pure peptidomimetics of PLG of type
will be reported in due course.
1 as dopamine receptor modulators are in progress and
Acknowledgements
This study was funded by Fundação para a Ciência e Tecnologia (FCT) through national funds and
co-financed by FEDER, under the Partnership Agreement PT2020 with references
UID/QUI/50006/2013–POCI/01/0145/FERDER/007265
and
UID/MULTI/04378/2013–
POCI/01/0145/FERDER/007728. IESD and CADS thank FCT for the grants, SFRH/BD/93632/2013
and SFRH/BPD/80100/2011, respectively.
Conflict of interest
The authors declare that they have no conflict of interests.
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