Calcitonin gene-related peptide analogues with aza and indolizidinone amino acid residues reveal conformational requirements for antagonist activity at the human calcitonin gene-related peptide 1 receptor

Article abstract of DOI:10.1021/jm061343w

Calcitonin gene-related peptide antagonists have potential for the treatment and prevention of disease states such as non-insulin-dependent diabetes mellitus, migraine headache, pain, and inflammation. To gain insight into the spatial requirements for CGR

Full text of DOI:10.1021/jm061343w

J. Med. Chem. 2007, 50, 1401-1408
1401
Calcitonin Gene-Related Peptide Analogues with Aza and Indolizidinone Amino Acid Residues
Reveal Conformational Requirements for Antagonist Activity at the Human Calcitonin
Gene-Related Peptide 1 Receptor
Damien Boeglin,† Fadi F. Hamdan,‡ Rosa E. Melendez,† Je´roˆme Cluzeau,† Andre Laperriere,‡ Madeleine He´roux,‡
Michel Bouvier,*,‡ and William D. Lubell*,†
De´partements de Chimie et de Biochimie, UniVersite´ de Montre´al, C. P. 6128, Succursale Centre Ville, Montre´al, Que´bec, Canada H3C 3J7
ReceiVed NoVember 20, 2006
Calcitonin gene-related peptide antagonists have potential for the treatment and prevention of disease states
such as non-insulin-dependent diabetes mellitus, migraine headache, pain, and inflammation. To gain insight
into the spatial requirements for CGRP antagonism, three strategies were employed to restrict the conformation
of the potent undecapeptide antagonist, [D31,P34,F35]CGRP27-37. First, aza-amino acid scanning was performed,
and ten aza-peptide analogues were synthesized and examined for biological activity. Second, (3S,6S,9S)-
2-oxo-3-amino-indolizidin-2-one amino acid (I2aa) and (2S,6S,8S)-9-oxo-8-amino-indolizidin-9-one amino
acid (I9aa) both were introduced at positions 31-32, 32-33, 33-34, and 34-35, regions of the backbone
expected to adopt turns. Finally, the conformation of the backbone and side-chain of the C-terminal residue,
Phe35-Ala36-Phe37-NH2, was explored employing (2S,4R,6R,8S)-9-oxo-8-amino-4-phenyl-indolizidin-9-one
amino acid (4-Ph-I9aa) as a constrained phenylalanine mimic. The structure-activity relationships exhibited
by our 26 analogues illustrate conformational requirements important for designing CGRP antagonists and
highlight the importance of â-turns centered at Gly33-Pro34 for potency.
Introduction
The very potent vasodilatory effects of CGRP originally
suggested that CGRP receptors agonists could represent clini-
cally valuable compounds for the treatment of severe hyperten-
sion, cerebrovascular vasospasm, and Raynaud’s syndrome;
however, drawbacks including lack of efficacy and tachycardia
have limited their development.18-20 CGRP antagonists, how-
ever, remain attractive candidates for treating a variety of
indications, including, non-insulin-dependent diabetes mellitus,
migraine headache, pain, inflammation, and morphine-induced
analgesia. For example, a dipeptide analogue (Figure 1) has
recently entered into clinical trials for the treatment of mi-
graine.21,22
Calcitonin gene-related peptide (CGRP,a Figure 1) is a 37
amino acid peptide produced in two forms (R and â) by an
alternative splicing of calcitonin mRNA.1 Both R and â human
CGRP are present in the central and peripheral nervous systems,
and although they differ by three amino acids, they produce
similar biological activity.1-3 CGRP controls many distinct
physiological responses including actions on the cardiovascular
system and the central nervous system, reproductive organs,
skeletal muscles, calcium metabolism, insulin regulation, and
gastric secretion.4
Two pharmacologically distinct receptors have been proposed
to mediate CGRP actions.5-7 The best characterized CGRP1
receptor is composed of the calcitonin receptor-like receptor
(CRLR), a seven transmembrane domain protein, and the
receptor-activity-modifying proteins (RAMPs) that determine
the pharmacological selectivity of the receptor. Indeed, whereas
the CRLR/RAMP1 complex leads to the CGRP1 receptor,
association of CRLR with either RAMP2 or RAMP3 leads to
adrenomedulin (AM) receptors.8-10 Although less well charac-
terized, a third protein, the receptor component protein (RCP),
has been proposed to act as a chaperone protein required for
receptor CGRP function.11,12 A second pharmacologically
distinct CGRP receptor, CGRP2, has been reported to occur in
reproductive organs13-17and to be less sensitive to CGRP8-37
antagonism. To this date, the molecular composition of this
receptor remains unknown and its existence as a tractable distinct
entity remains controversial.
The conformation of CGRP has been shown by NMR and
CD spectroscopy to comprise an amino-terminal disulfide-
bonded loop containing residues 2-7 that nucleates an R-helical
region between residues 8 and 18 which unfolds, Via a turn
about residues 19-21, into a less ordered sequence that may
adopt turn conformations.23-28 Initial examination of CGRP by
NMR spectroscopy in 1:1 trifluoroethanol:water identified the
presence of a turn-like conformation about residues 19-21.25
Examination of CGRP in DMSO by NMR spectroscopy and
computational analysis provided further evidence to suggest a
γ-turn between the amide carbonyl of Ser19 and the amide NH
of Gly21.27 Spectroscopic analysis of the C-terminal [But-
Cys18]h-â-CGRP19-37 fragment in DMSO also revealed a turn
conformation in the Ser19-Gly21 region as well as indicated a
second type I â-turn in the region of Asn31-Val32-Gly33.28
Although this second turn was not observed in spectroscopic
studies of the parent CGRP peptide, a comparison with a
C-terminal fragment of CGRP and two different agonists, both
constrained by a disulfide ring, one comprised at the C- and
the other at the N-terminal fragment, indicated that the Val32-
Gly33-Ser34 sequence may adopt an inverse γ-turn.29,30
* Corresponding authors. E-mail: michel.bouvier@umontreal.ca. lubell@
chimie.umontreal.ca.
† De´partement de Chimie.
‡ De´partement de Biochimie.
a Abbreviations: CGRP, calcitonin gene-related peptide; CRLR, calci-
tonin receptor like receptor; RAMP, receptor activity modifying proteins;
AM, adremodulin receptor; BTD, bicyclic turn dipeptide; BTC, bis(trichloro-
methyl)carbonate; TIS, triisopropylsilane; HBTU, O-benzotriazole-N,N,N′,N′-
tetramethyluronium hexafluorophosphate; Iaa, indolizidinone amino acid.
The analysis of abridged CGRP analogues has shown that
N-terminal fragments usually activate the receptor31 and that
the C-terminal fragments act as antagonists of CGRP action.32-38
10.1021/jm061343w CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/24/2007

1402 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Boeglin et al.
Figure 1. Structures of hR,â-CGRP, peptidic and nonpeptidic CGRP antagonists.
Exploring further the conformational requirements for the
activity of peptide based CGRP antagonists, we have employed
three strategies for restraining the geometry of the potent
undecapeptide [D31,P34,F35]CGRP27-37. Initially, aza-amino acid
scanning of the C-terminal peptide, i.e., sequential replacement
of amino acid residues in the peptide sequence by their aza-
amino acid counterparts, was performed by the synthesis of five
aza-analogues of [D31,P34,F35]CGRP27-37 (antagonist 1) and five
aza-analogues of [D31,P34,F35]CGRP29-37 (antagonist 2). This
approach was taken to probe the existence, location, and
importance of turn structures in antagonists 1 and 2, because
aza-peptides, peptide analogues in which the R-carbon of one
or more of the amino acid residues is replaced with a nitrogen
atom, retain normal side-chain functions and exhibit propensity
for adopting the central positions of a â-turn conformation.44-47
Moreover, aza-peptides of peptides 1 and 2 were examined
because of their potential to increase resistance to proteases and
provide mimics with longer duration of action relative to the
natural peptide.48-53
Figure 2. Representative indolizidinone amino acids.
In particular, CGRP27-37 has been shown to be the shortest
C-terminal peptide with a natural sequence having significant
antagonist activity. Extensive structure-activity relationship
studies of this peptide led to the discovery of [D31,P34,F35]-
CGRP27-37, an antagonist with nanomolar affinity at the
hCGRP1 receptor, representing a 100-fold increased affinity
compared to the unmodified CGRP segment.39-41 Additionally,
removal of its first two N-terminal residues associated with the
substitution of three residues at position 31, 34, and 35 did not
dramatically alter receptor binding and has furnished [D31,P34,F35]-
In the second phase of our study, indolizidin-2- and -9-one
N-(Fmoc)amino acids (Fmoc-I2aa and Fmoc-I9aa) were em-
ployed to induce restricted turn conformations in the backbone
at positions 31-32, 32-33, 33-34, and 34-35, to characterize
the geometry responsible for activity. X-ray structural analyses
of N-(Boc)amino indolizidin-2- and -9-one methyl esters (Boc-
I2aa-OMe and Boc-I9aa-OMe)54 have indicated that the central
dihedral angles constrained within the heterocycle were similar
to the ideal values for the i + 1 and i + 2 residues of a type II′
â-turn. By NMR spectroscopy and computational analysis of
model peptides, I2aa has been shown to adopt the i/i + 1
positions as well as the i + 1/i + 2 positions of a â-turn and
the i/i + 1 positions of a γ-turn.55,56 Similar preferences for
both the i/i + 1 positions and the i + 1/i + 2 positions of â-turn
conformations have been previously observed when the thiain-
dolizidinone amino acid (BTD, Figure 3) was introduced
respectively into cyclic hexapeptide mimics of tendamistat57 and
into the antibiotic cyclic peptide gramicidin S.58 The synthesis
of peptides respectively possessing I2aa and I9aa residues was
intended to identify the region and backbone dihedral angle
geometry of an active turn.
CGRP29-37, an antagonist displaying only a 5-fold decrease of
42
binding affinity compared with CGRP27-37
.
Examination of
[D31,P34,F35]CGRP27-37 and related analogues by CD spectros-
copy revealed that increases in ordered structure correlated with
high binding affinity and suggested that replacement of the
original Ser34 by proline reinforced a turn already present in
the unmodified segment.42 Studies of analogues of CGRP27-37
indicated that Thr30, Val32, and Phe37 also play critical roles for
antagonist potency on hCGRP1, and computational and NMR
spectroscopic studies suggested the participation of Thr30 in a
turn conformation.42 Two alternative turn conformations have
also been proposed to be important for antagonist activity of
C-terminus fragments: a possible γ-turn around Pro34 in
[D31,P34,F35]CGRP27-37 and a left-handed helical turn from V32
to A36 in [D31,A34,F35]CGRP27-37. More recently, the introduc-
tion of the turn inducing thiaindolizidinone amino acid (BTD,
Figure 2) at positions 19, 20 and 33, 34 of the antagonist hR-
CGRP8-37 produced constrained analogues that exhibited similar
antagonist activity (pA2 ) 6.0 to 6.2) as the parent peptide (pA2
(CGRP27-37) ) 6.0).17,43 This observation has led to a suggestion
that the active conformer possesses two â-bends at positions
18-21 and 32-35.
In the final strategy for constraining peptides 1 and 2, the
importance of the backbone and side-chain geometry of the
aromatic residues in the C-terminal peptide was explored by
the use of (2S,4R,6R,8S)-4-phenyl-indolizidin-9-one N-(Fmoc)-
amino acid (Fmoc-4-Ph-I9aa) as a constrained Phe derivative.

Calcitonin Gene-Related Peptide Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1403
Figure 3. Positioning of BTD in peptide turns.
Figure 4. Dose-response curve of R-CGRP-promoted cAMP produc-
tion. HEK293 stably expressing CRLR were challenged with increasing
concentrations of R-CGRP, and cAMP levels were measured as
described in the Experimental Section. Data are means ( SEM of three
independent experiments. pEC50 is calculated from nonlinear regression
fitting using GraphPad Prism 4.
Introduction of 4-Ph-I9aa at positions 34-35 and 36-37 was
used to explore the possibility of a turn region with a specifically
oriented aromatic ring for the Pro-Phe and Ala-Phe regions. In
summary, this application of aza-peptides and peptides con-
strained with different indolizidinone amino acids was focused
on defining the significance of turn geometry for peptide activity
and furnishing leads for the design of more effective mimics.
peptides were treated with neat TFA for 45 min to cleave the
resin and to deprotect amino acid side chains. The resin was
washed twice with TFA, and the TFA layers were combined
and treated with excess Et2O to precipitate peptides. The
peptides were removed by centrifugation, and the solution was
evaporated to an oil, that was precipitated again with ether. The
combined white solids were dried under vacuum, dissolved in
50% water/50% acetonitrile, and purified on semipreparative
HPLC/MSQ using an Alltech C18 column (25 cm × 22 mm),
a 20 mL/min flow rate, and a gradient of water:acetonitrile
containing 0.1% of TFA. Purification furnished peptides in 23%
average overall yields.
Biological Examinations. Inhibition of Agonist-Promoted
cAMP Production by CGRP(27-37) Analogues. To assess
the ability of various CGRP27-37 peptide analogues to inhibit
agonist-promoted cAMP production by the CGRP1 receptor, we
used a HEK293 cell line that stably expresses human CRLR.70
As shown in Figure 4, treatment of these cells with R-CGRP
resulted in a dose-dependent accumulation of cAMP with an
EC50 of ∼0.2 nM, which is similar to what was previously
reported for the CGRP1 receptor.69 This indicated that the
CRLR-HEK293 cells used herein contain sufficient endogenous
RAMP-1 and RCP proteins for the generation of a functional
CGRP1 receptor. Therefore, this cell line was further used to
test the effect of peptide analogues based on the CGRP1 receptor
antagonist [D31,P34,F35]CGRP27-37 on R-CGRP-promoted cAMP
production by CGRP1. The inhibition potency and efficacy of
individual peptide analogues are summarized in Table 2. The
potency is expressed as pIC50 whereas the efficacy is presented
as % inhibition of the cAMP production promoted by 0.5 nM
R-CGRP.
Results
Chemistry. Aza-peptides. Aza-Phe27 (8), aza-Pro34 (5), and
aza-Phe35 (6 and 11) peptide analogues were prepared as
previously reported by using N-(Boc)-aza1-dipeptide building
blocks. The respective N-Boc-Aza1-dipeptides were first syn-
thesized in solution and subsequently incorporated into aza-
peptides 26-29 by using a Boc protection strategy on oxime
resin.59,60 After cleavage of the peptide from the resin with
saturated NH3 in MeOH:DCM and removal of the side chain
protecting groups [Thr(OBn) and Asp(OBn)] by hydrogenolysis,
the Boc group of the final peptide was removed using 25% TFA
in DCM, and aza-peptides 5, 6, 8, and 11 were purified by RP-
HPLC to furnish product suitable for biological analysis in 10-
20% yields based on initial loading.
Aza-Asp31 (3 and 9) and aza-Ala36 (7 and 12) analogues were
synthesized on acid-labile Rink resin61 as previously reported
by using Fmoc-aza-amino acid chloride building blocks;62 aza-
Gly33 (4 and 10) was introduced by way of its 1,3,4-oxadiazol-
2(3H)-one.63 Standard Fmoc/tBu protocols with Fmoc amino
acids and HBTU as coupling agent were used to elongate the
resin-bound peptide.64 After acylation with the aza-amino acid
residue, the subsequent Fmoc amino acid was coupled by using
BTC as coupling agent,65 and peptide synthesis was continued.
Attempts failed to acylate the aza-Asp(OtBu) residue on the
resin-bound peptide using Fmoc-Thr(OtBu)-OH and a number
of activation methods, such that it was introduced as its
oxazolidine carboxylate analogue. After simultaneous cleavage
of the aza-peptide from the support and deprotection of side
chain protecting groups with a TFA/TIS/H2O (95/2.5/2.5, v/v/
v) solution, the aza-peptides were isolated by precipitation with
Et2O, dissolved in a 1:1 acetonitrile/H2O solution, and lyoph-
ilized to white foams that were directly analyzed by RP-HPLC
to assess purity. Samples requiring additional purification were
isolated by preparative RP-HPLC.
Deletion of amino acids 27 and 28 of [D31,P34,F35]CGRP27-37
did not affect its potency or efficacy for inhibiting cAMP
production, indicating that amino acids 27 and 28 may not be
essential for the antagonistic activity of the peptide. On the other
hand, aza-amino acid scanning of the shorter CGRP29-37 peptide
resulted in total loss of cAMP inhibition when the respective
residues (Asp31, Gly33,Phe35, and Ala36) were replaced by an
aza-amino acid, and a ∼6 fold reduction in potency in the case
of Pro.34 Aza-peptide analogues exhibited better potency in the
longer peptides. For example, replacement of Phe35 and Ala36
of [D31,P34,F35]CGRP27-37 resulted in only a 5 - 8-fold
reduction in potency instead of promoting complete loss of
activity in [D31,P34,F35]CGRP29-37. Aza-residues at Phe27 and
Asp31 did not significantly alter the potency nor efficacy relative
to the parent peptide. Moreover, replacement of Gly33 by its
aza-amino acid counterpart resulted in an approximately 10-
fold increase in potency.
Chemistry. Indolizidinone Peptides. The rigid dipeptide
surrogates I2aa, I9aa, and 4-Ph-I9aa were synthesized according
to literature procedures that delivered the Boc-protected ana-
logues.54,66,67 Subsequent N-deprotection and conversion to the
Fmoc-amino acid was achieved as described for I2aa in the
Experimental Section. Standard peptide chemistry using an
Fmoc-protection strategy and the respective indolizidinone
N-(Fmoc)amino acid (Fmoc-I2aa, Fmoc-I9aa, and Fmoc-4-Ph-
I9aa) was performed on BHA resin modified with a Rink linker
by way of an amino caproic acid spacer. After treatment with
piperidine to remove the terminal Fmoc protection, the final

1404 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Boeglin et al.
Table 1. Analytical Data for the Peptide, Aza-peptide, and Azabicycloalkane-Containing Peptide Analogues Tested in This Study
HPLC tR
,
HPLC tR
,
M + 1
mass spectral
purity (%)
at 214 nm
1
2
no.
peptide
structure
min (system 1) min (system 2) (calcd) analysis (M + 1)
1a
[D31,P34,F35]CGRP27-37
FVPTDVGPFAF-NH2
PTDVGPFAF-NH2
18.4
15.1
14.2
15.8
-
22.7
21.5
26.2
24.9
-
1195.6
949.5
950.5
950.5
950.5
1196.0
949.5
950.5
950.4
950.5
>98
>99
86
90
88
91
96
>99
86
95
92
95
>99
>98
91
60/30
86
2a
[D31,P34,F35]CGRP29-37
3b
[azaD31,P34,F35]CGRP29-37
[D31,azaG33,P34,F35]CGRP29-37
[D31,azaP34,F35]CGRP29-37
[D31,P34,azaF35]CG RP29-37
[D31,P34,F35,azaA36]CGRP29-37
[azaF27,D31,P34,F35]CGRP27-37
[azaD31,P34,F35]CGRP27-37
[D31,azaG33,P34,F35]CGRP27-37
[D31,P34,azaF35]CGRP27-37
[D31,P34,F35,azaA]CGRP27-37
[I2aa31-32,P34,F35]CGRP29-37
[I9aa31-32,P34,F35]CGRP29-37
[D31,I2aa34-35]CGRP29-3 7
[D31,I9aa34-35]CGRP29-3 7
[I2aa31-32,P34,F35]CGRP27-37
[I9aa31-32,P34,F35]CGRP27-37
[D31,I2aa32-33,P34,F35]CGRP 27-37
[D31,I9aa32-33,P34,F35]CGRP 27-37
[D31,I2aa33-34,F35]CGRP 27-37
[D31,I9aa33-34,F35]CGRP 27-37
[D31,I2aa34-35]CGRP27-3 7
[D31,I9aa34-35]CGRP27-3 7
PTazaDVGPFAF-NH2
PTDVazaGPFAF-NH2
PTDVGazaPFAF-NH2
PTDVGPazaFAF-NH2
PTDVGPFazaAF-NH2
azaFVPTDVGPFAF-NH2
FVPTazaDVGPFAF-NH2
FVPTDVazaGPFAF-NH2
FVPTDVGPazaFAF-NH2
FVPTDVGPFazaAF-NH2
PTI2aaGPFAF-NH2
4b
5c
6c
-
-
950.5
950.5
950.5
950.4
7b
16.3
-
16.3
16.1
-
17.5
14.5
15.0
8.2
25.2
-
27.8
26.8
-
8c
1196.6
1196.6
1196.6
1196.6
1196.6
915.5
915.5
885.4
885.4
1161.7
1161.7
1219.6
1219.6
1221.6
1221.6
1131.6
1131.6
961.5
1196.3
1196.6
1196.6
1196.6
1196.5
915.7
915.7
885.3
885.3
1161.2
1162.0
1219.6
1219.6
1221.7
1221.7
1131.8
1131.8
961.7
9b
10b
11c
12b
13a
14a
15a
16a
17a
18a
19b
20b
21b
22b
23a
24a
27.4
20.6
20.8
17.7
17.8/18.1
22.4
22.5
26.3
26.8
28.6
28.3
19.9
20.1
21.7
22.5
23.1
23.8
PTI9aaGPFAF-NH2
PTDVGI2aaAF-NH2
PTDVGI9aaAF-NH2
9.4
FVPTI2aaGPFAF-NH2
FVPTI9aaGPFAF-NH2
FVPTDI2aaPFAF-NH2
FVPTDI9aaPFAF-NH2
FVPTDVI2aaFAF-NH2
FVPTDVI9aaFAF-NH2
FVPTDVGI2aaAF-NH2
FVPTDVGI9aaAF-NH2
PTDVGXaaAF-NH2
17.8
18.6
16.3
16.9
18.2
17.4
12.6
13.6
16.6
17.0
20.1
21.0
87
,
>99
>99
>99
>99
91
86
25a,d [D31,Xaa34-35]CGRP29-3 7
>99
>99
>99
92
26a,d [D31,P34,F35,Xaa36-37]CGRP29-3 7
27a,d [D31,Xaa34-35]CGRP27-3 7
PTDVGPFXaa-NH2
FVPTDVGXaaAF-NH2
FVPTDVGPFXaa-NH2
987.5
1207.6
1233.6
987.7
1207.4
1233.7
28a,d [D31,P34,F35,Xaa36-37]CGRP27-3 7
Analytical HPLC analyses were performed on an TARGA column from Higgins Analytical, Inc (4.6 × 250 mm, 5 µm, C18) with a flow rate of 1.5
mL/min ausing water (0.1% TFA)/CH3CN (0.1% TFA) (system 1) eluents from 80/20 to 50/50 in 30 min and 50/50 to 10/90 in 5 min and water (0.1%
b
TFA)/MeOH (0.1% TFA) (system 2) from 80/20 to 20/80 in 20 min, 20/80 to 10/90 in 5 min, using a 40 min linear gradient from water (0.1% TFA) to
CH3CN (0.1% TFA) (system 1) or MeOH (0.1% TFA) (system 2). c See ref 59 for analytical datas. d Xaa ) 4-Ph-I9aa. e Two signals (2:1 ratio) corresponding
to the same mass were observed, which may correspond to either a conformational or a configurational mixture.
Table 2. Antagonist Activities of [D31,P34,F35]-CGRP27-37 (1) and [D31,P34,F35]-CGRP29-39 (2) and Their Constrained Analogues on hRCGRP-Induced
cAMP Accumulation in HEK293 Cells
% inhibitionb
nc
b
no.
peptides
structure
pIC50
1
2
3
4
5
6
7
8
[D31,P34,F35]CGRP27-37
FVPTDVGPFAF-NH2
PTDVGPFAF-NH2
6.15 ( 0.18
6.11 ( 0.18
N.A.a
N.A.
5.35 ( 0.16
N.A.
68 ( 4
81 ( 7
N.A.
N.A.
68 ( 8
N.A.
4
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
5
3
3
3
3
3
2
3
[D31,P34,F35]CGRP29-37
[azaD31,P34,F35]CGRP29-37
[D31,azaG33,P34,F35]CGRP29-37
[D31,azaP34,F35]CGRP29-37
[D31,P34,azaF35]CGRP29-37
[D31,P34,F35,azaA36]CGRP29-37
[azaF27,D31,P34,F35]CGRP27-37
[azaD31,P34,F35]CGRP27-37
[D31,azaG33,P34,F35]CGRP27-37
[D31,P34,azaF35]CGRP27-37
[D31,P34,F35,azaA36]CGRP 27-37
[I2aa31-32,P34,F35]CGRP29-37
[I9aa31-32,P34,F35]CGRP29-37
[D31,I2aa34-35]CGRP29-37
PTazaDVGPFAF-NH2
PTDVazaGPFAF-NH2
PTDVGazaPFAF-NH2
PTDVGPazaFAF-NH2
PTDVGPFazaAF-NH2
azaFVPTDVGPFAF-NH2
FVPTazaDVGPFAF-NH2
FVPTDVazaGPFAF-NH2
FVPTDVGPazaFAF-NH2
FVPTDVGPFazaAF-NH2
PTI2aaGPFAF-NH2
N.A.
N.A.
6.23 ( 0.34
6.42 ( 0.30
7.17 ( 0.17
5.24 ( 0.26
5.45 ( 0.38
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
6.97 ( 0.17
N.A.
N.A.
N.A.
69 ( 25
59 ( 3
70 ( 6
69 ( 5
51 ( 5
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
70 ( 6
N.A.
N.A.
N.A.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
PTI9aaGPFAF-NH2
PTDVGI2aaAF-NH2
[D31,I9aa34-35]CGR P29-37
[I2aa31-32,P34,F35]CGRP27-37
[I9aa31-32,P34,F35]CGRP27-37
[D31,I2aa32-33,P34,F35]CGRP27-37
[D31,I9aa32-33,P34,F35]CGRP27-37
[D31,I2aa33-34,F35]CGRP27-37
[D31,I9aa33-34,F35]CGRP27-37
[D31,I2aa34-35]CGRP27-37
PTDVGI9aaAF-NH2
FVPTI2aaGPFAF-NH2
FVPTI9aaGPFAF-NH2
FVPTDI2aaPFAF-NH2
FVPTDI9aaPFAF-NH2
FVPTDVI2aaFAF-NH2
FVPTDVI9aaFAF-NH2
FVPTDVGI2aaAF-NH2
FVPTDVGI9aa AF-NH2
PTDVGXaaAF-NH2
,
[D31,I9aa34-35]CGRP27-37
[D31,Xaa34-35]CGRP29-37
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
[D31,P34,F35,Xaa36-37]CGRP29-37
[D31,Xaa34-35]CGRP27-37
PTDVGPFXaa-NH2
FVPTDVGXaaAF-NH2
FVPTDVGPFXaa-NH2
[D31,P34,F35,Xaa36-37]CGRP27-37
a
N.A., not applicable: no inhibition was detected with peptide concentrations up to 100 µM. b Data are represented as mean ( SEM from at least three
independent experiments. c Number of experiments.
The difference between the short and long CGRP analogue
was particularly spectacular when considering the replacement
of Gly33 that led to a complete loss of potency in [D31,P34,F35]-
CGRP29-37 (4) but promoted a 10-fold increase in potency when
introduced into [D31,P34,F35]CGRP27-37 (10). This indicates that
the short and long analogues do not share identical binding

Calcitonin Gene-Related Peptide Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1405
Table 3. Backbone Torsion Angles Observed in Various Ideal â-Turn
and γ-Turn Types, in Dipeptide Surrogates I2aa and I9aa, and in
Aza-amino Acid-Induced Turns
aza- and indolizidinone amino acids (Table 3), we can speculate
that a type II′ beta-turn with Gly33-Pro34 at the i + 1 and i + 2
positions may likely be the active antagonist conformation in
the parent peptide.
amino acid or
dipeptide surrogate
æi+1
ψi+1
æi+2
ψi+2
In contrast to the enhanced activity observed in I2aa-analogue
21, use of I9aa amino acid as a Gly33-Pro34 surrogate in
[D31,P34,F35]CGRP27-37 (1) did not produce a compound with
significant activity. Although such sensitivity to the change in
heterocycle may suggest that a precise set of backbone torsion
angles in the turn is a prerequisite for the biologically active
conformer of [D31,P34,F35]CGRP27-37 (1), different steric effects
of the two ring systems may also have differentially affected
binding. Similarly, although the introduction of I2aa at positions
other than 33-34 and the use of other indolizidinone amino
acids (I9aa and 4-Ph-I9aa) were not well tolerated by the CGRP1
receptor, these results do not necessarily refute the presence of
turns at these positions in the receptor-bound conformation of
the parent peptide, because the steric bulk of the Iaa residues
as well as the removal of key side chains may have interfered
with binding in such cases. Interestingly, the aza-amino acid
scan did produce other active compounds (aza-peptides 5, 8, 9,
11, and 12) that displayed either equal or moderately lower
potency than the parent peptide. This indicates that the
introduced constraints did not prevent the analogues from
adopting active conformers that may result from the presence
and orientation of the amino acid side chains. Finally, the
striking difference of the activities of the aza-Gly33 analogues
of the longer and shorter peptide antagonists 1 and 2 may
indicate that the two peptides bind the receptor in a different
manner and as a consequence information gained from the
scanning of the shorter peptide cannot be extrapolated to obtain
information on the binding modalities of the longer peptides.
As mentioned, spectroscopic analyses of related C-terminal
analogues have suggested a turn geometry about the residues
from positions 31 to 36, including a type I â-turn in the region
of Asn31-Val32-Gly33,28 a possible γ-turn around Pro34 and a
left-handed helical turn from V32 to A36.42,71 Consistent with
this notion, insertion of the thiaindolizidinone BTD at positions
33, 34 of the antagonist hR-CGRP8-37 did not affect antagonist
activity, indicative of the presence of a type II′ turn centered at
this position.17,43 Recently, computational analysis, NMR spec-
troscopy, and the insertion of turn-inducing amino acid sur-
rogates into [D31,P34,F35]-CGRP27-37 have suggested that Pro29
and Thr30 exist in a type I â-turn conformation and that a
hydrophobic residue at position 29 enhances receptor affin-
ity.42,71 The presence of multiple turn conformations has,
-
-
-
-
-
-
ideal â I turn
ideal â I turn
ideal â II turn
ideal â II turn
ideal â III turn
ideal â III turn
ideal â IV turn
ideal â V turn
ideal â V turn
ideal â VIa turn
-60
60
-60
60 -120
-60
60
-61
-80
80
-30
30
120
-90
90
80
-80
-60 -30
60
-53
-80
80 -80
-90
-60
0
0
0
0
-30
30
10
30
17
80
-
-
-
80
-80
120
120
-30 -120
-64
69
-176
-141
-23 -102
-60
0
0
ideal â VIb turn -120
-
ideal â VIII turn
classic γ turn
inverse γ turn
â II type turna
â II type turna
â I type turnb
â II type turnb
â II type turnb
-60
75
120
-
-
-
-
-78
-34
-
-79
-
-
I2aa (i + 1/I + 2)
I9aa (I + 1/i + 2)
Aza-aa (i + 1)
-
-
-
-74
12
12
21
59 -153
-90
71
Aza-aa (i + 2)
-60
130
a Data obtained from X-ray analysis of Boc-I2aa-OMe and Boc-I9aa-
OMe, see ref 51. b Data obtained from computational analysis of Ac-
AzaGly/Ala-L-Ala-NHMe and Ac-L-Ala-AzaGly/Ala-NHMe, see ref 74.
modes with the receptor and suggests that the two peptides need
to be considered independently for the rational design of more
potent/efficacious ligands.
In the second part of this study, the effects of substituting
indolizidin-2- and -9-one N-(Fmoc)-amino acids (Fmoc-I2aa and
Fmoc-I9aa) at positions 31-32, 32-33, 33-34, and 34-35 of
CGRP27-37 and CGRP29-37 were investigated. For both
CGRP27-37 and CGRP29-37, the introduction of I9aa at positions
31-32, 32-33, 33-34, and 34-35 resulted in a failure to
inhibit R-CGRP-promoted cAMP production. Similarly, most
of the I2aa analogues were inactive, such as those with I2aa at
positions 31-32, 32-33, and 34-35 of both CGRP27-37 and
CGRP29-37 peptides. Only in the case of substitution of I2aa at
positions 33-34 of CGRP27-37 was activity found, and a ∼7
fold increase in potency was observed for inhibition of R-CGRP-
promoted cAMP production.
Finally, substitutions of 4-Ph-I9aa at positions 34-35 and
36-37 of CGRP27-37 and CGRP29-37 resulted in peptide
analogues that failed to inhibit cAMP production by the CGRP1
receptor. Treatment of the CRLR-HEK293 cells with CGRP27-37
analogues (up to 1 µM) in absence of R-CGRP did not result
in significant production of cAMP (data not shown), indicating
that none showed partial agonistic activity toward CGRP1.
furthermore, led to the hypothesis of a polyproline-like structure
71
for [D31,P34,F35]-CGRP27-37
.
The presence of another turn
located lower in the sequence and the possibility of a polyproline
conformation are not, however, supported by our study involving
the systematic use of aza- and indolizidinone amino acids.
Support for a turn conformation was obtained for only the type
II′ beta-turn with Gly33-Pro34 at the i + 1 and i + 2 positions,
as the active antagonist conformation in the parent peptide.
Discussion
The introduction of aza-amino acids into a peptide may
restrict peptide geometry by inducing turn conformations as a
result of electronic interactions including dipole-dipole repul-
sion and resonance stabilization.72,73 Indolidizidone dipeptides,
for their part, create structural constraints by annulation and
steric interactions that also favor turn geometry. Considering
their different modes of action, the aza-scan and indolizidinone-
scan were expected to serve as complementary methods for
identifying the bioactive turn geometry of the active peptides.
Biological activity exhibited by aza-Gly33 (10) and I2aa33-34 (21)
analogues of [D31,P34,F35]CGRP27-37 (1) showing respectively
10- and 7-fold increased antagonism potency, both with similar
efficacy compared to their parent peptide (1), confirmed the
importance of a turn conformation for antagonist activity at this
position. In light of crystallographic and computational data of
Conclusion
A better insight into the structural requirements for antagonist
activity at the CGRP1 receptor of the potent antagonist un-
decapeptide [D31,P34,F35]CGRP27-37 has been obtained by the
use of aza-amino acid and indolizidinone dipeptide surrogates
for identifying turn secondary structures responsible for peptide
activity. The importance of a type II′ beta-turn centered at Gly33-
Pro34 has been illustrated by the respective 10- and 7-fold
increase antagonism potency of the aza-Gly33 (10) and I2aa33-34

1406 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Boeglin et al.
(21) analogues of [D31,P34,F35]CGRP27-37 (1). In addition, a
difference of receptor binding modes has been indicated by the
contrast in potency of aza-Gly33 analogues of 1 and 2. The
application of complimentary organic chemistry tools for
conformational constraint such as aza-amino acids and indo-
lidizidinone dipeptide surrogates has provided two new leads
for the development of calcitonin gene-related peptide antago-
nists as well as insight into their active receptor bound
conformation. Moreover, the improved metabolic stability and
longer duration of action observed previously with aza- and
indolizidinone amino acid analogues suggest that the aza-Gly33
and I2aa33,34 analogues 10 and 21 offer interesting potential for
developing more active antagonist peptide mimics with im-
proved pharmacological profiles.
was dried over MgSO4, filtered, and concentrated. The residue was
purified by flash chromatography using CHCl3/MeOH/AcOH (94/
5/1) as eluent to provided (2S,4R,6R,8S)-9-oxo-8-(N-(Fmoc)-
amino)-4-phenyl-indolizidin-9-one amino acid (330 mg, 63%). 1H
NMR (CHCl3) δ 7.67 (d, 2H, J ) 7.5 Hz), 7.51 (d, 2H, J ) 7.4
Hz), 7.31 (t, 2H, J ) 7.4 Hz), 7.24-7.18 (m, 4H), 7.13 (t, 1H, J
) 7.2 Hz), 7.06 (d, 2H, J ) 7.4 Hz), 5.61 (d, 1H, J ) 5.3 Hz),
4.91 (d, 1H, J ) 5.2 Hz), 4.30 (m, 2H), 4.14 (t, 1H, J ) 6.8 Hz),
3.77 (s, 1H), 2.70 (m, 2H), 2.40 (d, 1H, J ) 13.3 Hz), 2.02 (d, 1H,
J ) 12.6 Hz), 1.81 (m, 1H), 1.58 (q, 1H, J ) 10.7 Hz), 1.58 (q,
1H, J ) 12.4 Hz); 13C NMR (CHCl3) δ 173.2, 172.5, 156.4, 143.7,
141.3, 128.7, 127.7, 127.0, 126.8, 126.6, 125.1, 119.9, 67.0, 52.7,
52.0, 51.4, 47.0, 39.4, 38.1, 35.1, 33.1; [R]20 -14.3° (c 0.006,
D
CHCl3/MeOH: 1/1); MS (ESI) m/z 497.3 (M + H+).
Peptide Synthesis. Aza-peptides were synthesized according to
references 59 and 62. Indolizidinone peptides were synthesized on
a 0.16 mmol scale on Rink linker with an amino caproic acid spacer
attached to BHA (0.4 mmol/g) resin to obtain C-terminal amides
after resin cleavage and deprotection. Side chains of threonine and
aspartic acid were respectively protected as tert-butyl ether and ester.
At the ninth amino acid residue (Pro), the resin was split in two
parts. One part (0.08 mmol) was deprotected with piperidine/DMF
solution (1/1, v/v) and cleaved from the resin using TFA/TIS/H2O
(95/2.5/2.5 v/v/v). The second part (0.08 mmol) was elongated by
two more amino acids and cleaved. Peptides were assembled by
stepwise addition of N-Fmoc amino acids following a typical Fmoc
strategy64 monitored by colorimetric Kaiser’s test.68 Natural amino
acids (300 mol %) were coupled using HBTU (300 mol %) and
DIEA (600 mol %) in DMF for 1 h. Indolizidinone amino acids
were subjected twice to the coupling condition (100 mol % and 50
mol %) using HBTU (100 mol % and 50 mol %) and DIEA (200
mol % and 100 mol %). Final Fmoc deprotection was followed by
treatment with TFA/TIS/H2O (95/2.5/2.5 v/v/v) for 60 min to cleave
the resin and deprotect amino acid side chains. The resin was
washed twice with TFA, and the TFA layers were combined,
concentrated to 1 mL, treated with excess Et2O to precipitate
peptides that were dissolved in a 1:1 acetonitrile/H2O solution, and
lyophilized to white foams that were purified on semipreparative
HPLC/MSQ using an Alltech C18 column (25 cm × 22 mm) with
a 20 mL/min flow rate and a gradient of water:acetonitrile
containing 0.1% of TFA. Purification was made using semiprepara-
tive LC/MS and furnished peptides in 23% average overall yields.
Experimental Section
According to our reported method, (3S,6S,9S)-indolizidin-2-one
N-(Boc)amino acid (Boc-I2aa) was prepared from L-glutamic acid
in 23% overall yield, and (2S,6R,8S)-indolizidin-9-one N-(Boc)-
amino acid (Boc-I9aa) and (2S,4R,6R,8S)-4-phenyl-indolizidin-9-
one N-(Boc)amino acid (Boc-4-Ph-I9aa) were prepared from
L-aspartic acid in 28% and 8% respective overall yield.66,67
Subsequent conversion to their N-Fmoc amino acid counterparts
was performed as described below. Solvents (DMF, CH2Cl2, and
Et2O) were dried using a GlassContour solvent dispensing system.
N,N-Diisopropylethylamine (DIEA) was successively distilled from
ninhydrin, and CaH2 and was kept under argon atmosphere. Solid-
phase synthesis was performed in SPE tubes (3 or 6 mL) using
vortex agitation.
(3S,6S,9S)-2-Oxo-3-(N-(9-fluorenylmethoxycarbonyl)amino)-
1-azabicyclo[4.3.0]nonane-9-carboxylic Acid ((3S,6S,9S)-Fmoc-
I2aa). A solution of (3S,6S,9S)-N-Boc-I2aa (2 g, 6.7 mmol, prepared
according to ref 54) in 50 mL of EtOAc was treated with dry HCl
gas bubbles at room temperature until TLC analysis (eluent 5%
AcOH in EtOAc) showed complete disappearance of the starting
material (Rf ) 0.36). The volatiles were removed in Vacuo, and
the remaining salt was collected and used without further purifica-
tion. A solution of (3S,6S,9S)-I2aa hydrochloride (120 mg, 0.51
mmol) in 10 mL of water was next treated with 84 mg (1 mmol)
of NaHCO3, followed by a solution of succinimidyl 9-fluorenyl-
methyl carbonate (171 mg, 0.51 mmol) in 10 mL of acetone. The
reaction was monitored by TLC (eluent 5% AcOH in EtOAc) until
complete consumption of the starting amine was observed. Citric
acid was added to the reaction mixture, which was subsequently
extracted with EtOAc (2 × 25 mL). The organic layers were
combined, washed with saturated NaCl, and evaporated to a residue
that was purified by column chromatography using 2% AcOH in
EtOAc as eluent. The fractions containing the reaction product were
combined and evaporated to give 175 mg (82%) of Fmoc-I2aa: [R]D
-26.2° (c 1.16, CHCl3); 1H NMR (CDCl3) δ 1.58-1.69 (m, 3 H),
2.00-2.24 (m, 4 H), 2.41 (m, 1 H), 3.63 (br s, 1 H), 4.16 (q, 2 H,
J ) 7.1), 4.27 (m, 2 H), 4.48 (d, 1 H, J ) 8), 5.99 (d, 1 H, J )
6.2), 7.04 (br s, 1 H), 7.23 (t, 2 H, J ) 7), 7.31 (t, 2 H, J ) 7),
7.54 (t, 2 H, J ) 6.5), 7.68 (d, 2 H, J ) 7); 13C NMR (CDCl3) δ
26.5, 26.9, 28.6, 32.0, 47.1, 50.3, 57.1, 58.8, 60.4, 67.1, 119.9,
125.1, 127.0, 127.6, 141.2, 143.7, 143.9, 156.2, 170.3, 173.4; HRMS
calcd for C24H24N2O5Na (MNa+): 443.1583, found: 443.1581.
(2S,4R,6R,8S)-4-Phenyl-indolizidin-9-one N-(Fmoc)amino Acid
(Fmoc-4-Ph-I9aa). (2S,4R,6R,8S)-9-Oxo-8-(N-(Boc)-amino)-4-
phenyl-indolizidin-9-one amino acid (400 mg, 1.07 mmol prepared
according to ref 67) was dissolved in a 1:1 solution of TFA/DCM
and stirred for 2 h. The volatiles were evaporated, and the residue
was dissolved in and evaporated from toluene twice. The amino
acid was treated with DIEA (0.74 mL, 400 mol %) and Fmoc-OSu
(720 mg, 200 mol %) in dioxane (10 mL) for 16 h. The solution
was evaporated to a residue that was partitioned between CHCl3
(50 mL) and 10% citric acid solution (50 mL). The phases were
separated, and brine (25 mL) was added to the aqueous phase, which
was extracted with CHCl3(4 × 25 mL). The combined organic layer
cAMP Measurements. HEK293 cells stably expressing the
human CRLR (Hilairet et al., 2001) were detached using PBS
containing 5 mM EDTA and then resuspended in PBS containing
0.01% D-glucose and 0.75 mM IBMX (Sigma). The cells were then
seeded into cAMP CatchPoint 384 black and clear bottom plates
(Molecular Devices) at a density of 5000 cells/well. To determine
the EC50 of R-cGRP, the cells were challenged with increasing
concentrations of R-cGRP for 15 min at 37 °C. In competition
experiments, the cells were first preincubated with the CGRP (27-
37) peptide analogues for 15 min at 37 °C and then were stimulated
with 0.5 nM of R-cGRP for 15 min at 37 °C. cAMP levels were
measured using the CatchPoint fluorescence-based cAMP kit and
according to the manufacturer’s recommendation (Molecular De-
vices). At least three independent experiments in triplicate were
done for each peptide, and data was analyzed by nonlinear curve-
fitting using the software PRISM 4 from GraphPad.
Acknowledgment. This work has been supported by grants
from the Fonds Que´becois de la Recherche sur la Nature et les
Technologies (FQRNT), the Natural Sciences and Engineering
Research Council of Canada (NSERC), and Valorisation
Recherche Que´bec (VRQ). The authors thank Dalbir S. Sehkon
and Alexandra Furtos for MS analysis. F.H. is a recipient of a
postdoctoral fellowship from the Canadian Institutes of Health
Research (CIHR).

Calcitonin Gene-Related Peptide Analogues
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1407
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