Design and synthesis of potent antagonists containing rigid spirocyclic privileged structures for the CGRP receptor

Article abstract of DOI:10.1016/j.bmcl.2012.05.118

We report the synthesis of rigid spirocyclic systems as conformationally constrained variants of the Ala-Phe-NH₂ dipeptide amide C-terminus of the calcitonin gene-related peptide (CGRP). CGRP receptor antagonists containing these moieties displ

Full text of DOI:10.1016/j.bmcl.2012.05.118

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4719–4722
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of potent antagonists containing rigid spirocyclic
privileged structures for the CGRP receptor
Prasad V. Chaturvedula a, , Sokhom Pin b, George Tholady b, Charlie M. Conway b, John E. Macor a,
⇑
Gene M. Dubowchik a
a Departments of Molecular Sciences and Candidate Optimization, Bristol-Myers Squibb R&D, 5 Research Parkway, Wallingford, CT 06492, USA
b Departments of Neuroscience Discovery Biology, Bristol-Myers Squibb R&D, 5 Research Parkway, Wallingford, CT 06492, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis of rigid spirocyclic systems as conformationally constrained variants of the Ala-
Phe-NH2 dipeptide amide C-terminus of the calcitonin gene-related peptide (CGRP). CGRP receptor
antagonists containing these moieties displayed potent affinity, functional antagonism and excellent oxi-
dative stability. Structure–activity relationship studies demonstrated the relative importance of hydro-
gen bond donor/acceptor functionalities and the preferred orientation of an aromatic ring. Antagonists
showed potent and full reversal of CGRP-induced dilation of ex vivo human intracranial arteries.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 18 April 2012
Revised 16 May 2012
Accepted 18 May 2012
Available online 9 June 2012
Keyword:
CGRP receptor antagonists
Migraine is a neurovascular disorder that afflicts more than 12%
of adults and is often characterized by severe headache, nausea,
and sensitivity to external stimuli lasting more than 4 h.1 The cur-
rent standard of therapy involves treatment with 5-HT1B/1D ago-
nists (triptans) that cause direct vasoconstriction of blood vessels
through activation of 5-HT1B receptors.2 Because of their vasocon-
strictive properties, triptans are contraindicated in patients with
cardiovascular disease. Thus, a migraine drug that is devoid of car-
diovascular liabilities is expected to have a significant advantage
over existing agents.3
Calcitonin gene-related peptide (CGRP) is a 37-amino acid neu-
ropeptide widely distributed in both the peripheral and central
nervous system (CNS), principally localized in afferent and central
neurons and displaying a number of biological effects, including
potent vasodilation.4 Activation of trigeminal sensory neurons
and increases in serum levels of CGRP are strongly implicated in
the pathophysiology of migraine.5 Clinical proof of concept for
CGRP receptor antagonists as an effective migraine therapy has
been demonstrated with intravenous administration of olcegepant
(BIBN-4096) 1 and through oral administration of a structurally
distinct antagonists, telcagepant (MK-0974), MK-3207 and BI
44370 TA.6
amino acid core; a GPCR (G-protein coupled receptor) privileged
structure containing an aromatic moiety attached to the central
amino acid at the N-terminus; and a basic or hydrophobic moiety
attached to the central core unit at the C-terminus. Based on the
simplicity and good potency of these antagonists we prepared
compounds 3–5 (Fig. 1).
Encouraged by the initial potencies of 3–5, we set out to iden-
tify: (i) additional novel GPCR privileged substructures that prefer-
ably contained fewer amide bonds and degrees of freedom for
improved affinity and a more favorable pharmacokinetic profile;
(ii) novel amino acids that provided improved binding affinity
and selectivity; and (iii) a key recognition element that defined
the bio-active conformation of CGRP antagonists. This strategy
led us to design and synthesize constrained CGRP receptor antago-
nists with reduced peptide character that should have improved
oral exposure. In this Letter we would like to disclose our initial
results of this research program. We and others8 hypothesized that
the piperidinyldihydroquinazolinone in 4 might mimic the Ala-
Phe-NH2 at the C-terminus of the CGRP peptide (Fig. 2). A recent
crystal structure of the ectodomain of the CGRP receptor in com-
plex with two known CGRP antagonists confirmed the importance
of this key pharmacophore containing a hydrogen bond donor–
acceptor pair and an aromatic moiety.9 In light of the known
liability of facile benzylic oxidation associated with the piper-
idinyldihydroquinazolinone heterocycle in 4,10 our investigation
began with the design of alternate GPCR privileged scaffolds that
were less prone or free from benzylic oxidation.
In this context, we initiated a research program aimed at iden-
tifying a CGRP receptor antagonist suitable for rapid onset of mi-
graine relief. At the outset of our program, there existed a
limited number of small molecule CGRP antagonists (e.g., 1–2,
Fig. 1).7 Their key structural features were: an aromatic central
Spiropiperidine systems containing hydrogen bond donor and
acceptor moieties constrained in a five- or six-membered ring with
a fused or pendant aromatic moiety were initially considered as
⇑
Corresponding author. Tel.: +1 203 677 6699; fax: +1 203 677 7702.
E-mail address: prasad.chaturvedula@bms.com (P.V. Chaturvedula).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.118

4720
P. V. Chaturvedula et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4719–4722
NH2
H
N
Synthesis of compound 6
O
N
Bn
N
Bn
N
N
O
N
O
a
H
N
N
N
N
H
H2N
CN
O
O
O
b
Br
N
1: IC50 = 0.02 nM
Bn
N
H
N
NH
OH
c, d
Br
N
O
H
O
N
N
CN
O
HN
R
N
H
NH
6
H2N
O
S
R
2
O
Synthesis of compound 7
Bn
N
NH
H
N
X
N
e, f
O
H
NH
N
N
N
3: IC50 = 9 nM
O
O
O
O
N
7
NH
S
N
S
g
S
N
N
4: IC50 = 3 nM
NH
HO
NHBoc
NHBoc
O
N
O
O
8
h, i
O
H
N
S
N
N
O
O
N
N
5: IC50 = 4 nM
N
H
N
H
N
HN
S
O
O
NH
Figure 1. Known CGRP antagonists.
Scheme 1. Reagents and conditions: (a) NaCN, NH4Cl, MeOH, 25 °C, 12 h, 91%; (b)
RCOCl, Et3N, DMAP, CH2Cl2, 25 °C, 12 h, 72–91%; (c) 30% H2O2, 6 M NaOH, reflux,
3 h, 54–68%; (d) HCl in dioxane, 25 °C, 3 h, 95%; (e) N-phenylurea, 100 °C,
polyphosphoric acid, 3 h, 45%; (f) 10% Pd on carbon, MeOH, 6.0 M HCl (cat),
H2,(1 atm), 12 h, 96%; (g) 4-piperidinopiperidine, PyBop, Et3N, CH2Cl2, 91%; (h)
dioxane–HCl, 25 °C, 4 h, 95%; (f) N,N0-disuccinimidyl carbonate, diisopropylethyl-
amine, CH2Cl2, 25 °C, 3 h.
O
N
NH
H
N
NH2
N
N
O
H
O
spiropiperidines (R = Ph, cyclohexyl, 3-pyridyl, 4-pyridyl) 6 in
52–75%yields. Spiropiperidine 7 was prepared by reacting 1-ben-
zyl-4-piperidone with N-phenylurea and polyphosphoric acid at
100 °C followed by debenzylation in 45% overall yield. Treatment
Figure 2. Key pharmacophore common in -Ala-Phe-NH2 of CGRP(8–37) peptide
and dihydroquinazolinone.
of Boc-(D)-3-benzothienylalanine and 4-piperidinopiperidine with
PyBOP provided 8 in excellent yield. The amino protecting group
was removed under acidic conditions (TFA). The amine was then
coupled to various spiropiperidines mediated by disuccinimidyl
carbonate to give the desired compounds 9–20 (Fig. 3) in 70–90%
yields.
All target compounds were tested in human neuroblastoma SK-
N-MC cells that endogenously expressed the CGRP receptor with
an identical sequence to the cloned human CGRP receptor. The
ability of compounds to compete for the radioligand [125I]CGRP
endogenous peptide human alpha CGRP was measured using a
radioligand competition assay to determine IC50’s. Functional
antagonism for selected compounds was determined by measuring
mimetics of the Ala-Phe-NH2 dipeptide amide and piperidinyldihy-
droquinazolinone replacements.
Convergent syntheses of the desired targets are shown in
Scheme 1. The spiropiperidine intermediates used in the synthesis
of CGRP receptors antagonists were prepared using known litera-
ture methods.11 Novel spiropiperidines were prepared according
to the synthetic scheme shown in Scheme 1. 1-Benzyl-4-piperi-
done was converted to
a-amino nitrile under standard Strecker
conditions in 85% yield. The amine was reacted with various acid
chlorides in the presence of triethylamine to give amides in 80–
95% yields. Hydrolysis of the nitrile with 30% hydrogen peroxide/
6 M sodium hydroxide followed by debenzylation provided the

P. V. Chaturvedula et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4719–4722
4721
O
O
H
N
N
N
O
N
S
O
O
NH
HN
NH
N
N
N
11: IC50 = 29 nM
EC50 = 15 nM
O
10: IC50 = 23 nM
9: IC50 = 170 nM
EC50 = 12 nM
H
N
O
H
N
N
N
O
NH
N
N
N
12:
13: IC50 = 860 nM
IC50 = 150 nM
EC50 = 130 nM
14: IC50 = 20 nM
EC50 = 13 nM
O
O
O
NH
N
N
NH
N
NH
N
N
N
N
15:
16: IC50 = 8500 nM
17: IC50 = 1400 nM
IC50 = 330 nM
O
O
O
Figure 4. Reversal of CGRP-induced artery dilation.
N
HN
NH
O
NH
NH
N
N
Reversal of CGRP-induced dilation of intracranial arteries is be-
lieved to be an ex vivo measure which models the clinical treat-
ment conditions where migraine-related CGRP release precedes
initiation of acute therapy. Accordingly, Compounds 14, 19 and
20 were tested for their ability to reverse CGRP-induced dilation
in human intracranial artery preparations.12 Pre-contracted vessels
were dilated using CGRP and then the dilation was reversed with a
CGRP receptor antagonist. In this protocol,12 antagonist treatment
reverses CGRP-induced artery dilation in a dose dependent man-
ner. In the tracings shown in Figure 4, compounds 14, 19 and 20
were tested at 10, 100 and 1000 nM. Compounds 14, 19 and 20
showed potent and full reversal of CGRP-induced dilation of
ex vivo human intracranial arteries with EC50 values in the range
19–65 nM. In general the rank order of activity tracked well across
radioligand binding, cell-based functional and ex vivo human ves-
sel assays (e.g., 14 and 19), although there were a few exceptions
(e.g., 20).
N
N
19: IC50 = 20 nM
20: IC50 = 5 nM
18: IC50 = 210 nM
EC50 = 31 nM
EC50 = 3 nM
Figure 3. Structure–activity relationships of CGRP antagonists containing spirocy-
clic moieties.
their ability to inhibit CGRP-stimulated formation of cyclic AMP in
attached whole SK-N-MC cells (EC50).12
CGRP-receptor binding data obtained for the compounds 9–20
is presented in Fig. 3. Homologation of 9 to give 10 enhanced CGRP
receptor binding potency. The reversal of donor (NH) and acceptor
(C@O) moieties in 12 versus 10 led to a decrease in affinity that
was restored by homologation between the carbonyl and phenyl
ring in 11. This preference for a single atom separation between
the carbonyl and phenyl group was further demonstrated by the
poor affinity of quinazolinone 13. The spirocyclic imidazolinone
14 with a pendant phenyl group was eightfold more potent com-
pared with its fused congener 9. Substituting the aromatic moiety
with cyclohexyl (16), 4-pyridinyl (17) or 3-pyridyl (18) led to a sig-
nificant loss of binding potency suggesting the preference for a
neutral aromatic group at C2 of the imidazolinone. In an effort to
maximize the putative hydrogen bond donor/acceptor ability of
spiropiperidines with the CGRP receptor, compounds 19 and 20
were synthesized. Compound 20 was found to be the most potent
in the series in line with the expected highest electron density on
the urea carbonyl oxygen (best H-bond acceptor in the group).
Selected CGRP receptor antagonists containing the benzothio-
phene moiety and spirocyclic substructures were profiled in vari-
ous assays for metabolic stability, oxidative stability, cytochrome
P450 inhibition and Caco-2 permeability. In general, these com-
pounds showed excellent metabolic stability, oxidative stability,
and favorable CYP inhibition profiles (IC50’s >40
lM) with the
exception of CYP 3A4 (IC50’s <1 M). However, Caco-2 cell perme-
l
ability was poor for these compounds, most likely because of a
high degree of amide bond polarity and/or P-glycoprotein efflux.
Specific profiling data for compound 20 is shown here. The metab-
olism of compound 20 was evaluated in pooled human and rat li-
ver microsomes. The rate of metabolism at 3
lM was low in both
species after 10 min (human: 82% compound remaining; rat:

4722
P. V. Chaturvedula et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4719–4722
C.; Lynch, J. J.; Moore, E. L.; Mosser, S. D.; Prueksaritanont, T.; Regan, C. P.;
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CYP1A2, 2C9, 2C19 with IC50’s >40 M, but was a potent inhibitor
of CYP3A4 with an IC50 of 0.1 M, most likely hydrophobic benzo-
l
l
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thiophene moiety contributing to CYP3A4 recognition.
In summary, we have identified novel CGRP receptor antago-
nists by incorporating rigid spirocyclic moieties that mimic the
Ala-Phe-NH2 C-terminus of CGRP into a known benzothiophene-
containing core structure. SAR investigations helped to define the
optimal spatial relationship between the –(C@O)NH– hydrogen
bond acceptor–donor pair and the phenyl ring. Selected CGRP
antagonists displayed potent receptor binding, functional potency,
and oxidative stability. These compounds also caused reversal of
CGRP-induced dilation of isolated human intracranial arteries, an
ex vivo migraine model, suggesting potential efficacy as anti-mi-
graine agents.
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