Identification of a novel RAMP-independent CGRP receptor antagonist

Article abstract of DOI:10.1016/j.bmcl.2011.09.056

Identification of an HIV integrase inhibitor with micromolar affinity for the CGRP receptor led to the discovery of a series of structurally novel CGRP receptor antagonists. Optimization of this series produced compound 16, a low-molecular weight CGRP rec

Full text of DOI:10.1016/j.bmcl.2011.09.056

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6705–6708
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a novel RAMP-independent CGRP receptor antagonist
C. Blair Zartman a, , Ian M. Bell a, Steven N. Gallicchio a, Samuel L. Graham a, Stefanie A. Kane b,
⇑
John J. Mallee b, Ruth Z. Rutledge b, Christopher A. Salvatore b, Joseph P. Vacca a, Theresa M. Williams a
a Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
b Department of Pain Research, Merck Research Laboratories, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Identification of an HIV integrase inhibitor with micromolar affinity for the CGRP receptor led to the dis-
covery of a series of structurally novel CGRP receptor antagonists. Optimization of this series produced
compound 16, a low-molecular weight CGRP receptor antagonist with good pharmacokinetic properties
in both rat and dog. In contrast to other nonpeptide antagonists, the activity of 16 was affected by the
presence of divalent cations and showed evidence of an alternative, RAMP-independent CGRP receptor
binding site.
Received 16 August 2011
Revised 14 September 2011
Accepted 15 September 2011
Available online 21 September 2011
Keywords:
CGRP
Ó 2011 Elsevier Ltd. All rights reserved.
Hydroxypyridine
RAMP-independent
HIV integrase
Calcitonin gene-related peptide (CGRP), a 37-amino acid neuro-
peptide first discovered in the early 1980’s, is produced by alterna-
tive splicing of the calcitonin gene and is widely distributed in the
central and peripheral nervous systems.1,2 The CGRP receptor is a
heterodimer comprised of the G-protein-coupled receptor (GPCR),
calcitonin receptor-like receptor (CLR) plus the receptor activity-
modifying protein 1 (RAMP1) (Fig. 1).3 The same CLR protein is also
known to associate with other receptor activity-modifying proteins
(RAMP2 or RAMP3) to produce high affinity receptors for the pep-
tide adrenomedullin. Similarly, the related calcitonin receptor
(CTR) can dimerize with RAMP1 to form a high affinity amylin
receptor.4
Profiling of the HIV integrase inhibitor L-870,810,13 compound
5 (Fig. 2), revealed that it possessed micromolar affinity for the
CGRP receptor. Since this compound represented a distinct chem-
otype from other known CGRP receptor antagonists, further inves-
tigation seemed warranted. Screening of other HIV integrase
inhibitors identified a number of other active compounds and sug-
gested that the cyclic sultam was important for antagonism of the
CGRP receptor (data not shown). With these preliminary data in
B
In many tissues, CGRP-containing nerves are closely associated
with blood vessels and CGRP is known to be a potent vasodilator.2
Additionally, a number of lines of evidence have pointed to a key
role for CGRP in migraine pathophysiology and this has led to
interest in the development of small molecule antagonists of the
CGRP receptor as a possible treatment for migraine and pain.5
Boehringer Ingelheim reported the first selective small molecule
CGRP receptor antagonist olcegepant 1 (Fig. 2), which exhibited
impressive affinity for human CGRP receptors (Ki = 14 pM).6 Intra-
venous administration of olcegepant provided clinical proof of con-
cept for CGRP receptor antagonists in the treatment of migraine7
and these findings have been subsequently corroborated with the
orally bioavailable compounds telcagepant (MK-0974) 2,8,9 MK-
3207 310,11 (Fig. 2) and BI 44370 TA.12
A
CLR
RAMP1
C
Figure 1. Schematic representation of CGRP receptor. CLR is shown in light gray
and RAMP1 in dark gray. Putative binding sites for various ligands are labeled as A
(CGRP), B (most small molecule antagonists), and C (compound 16).
⇑
Corresponding author.
E-mail address: blair_zartman@merck.com (C.B. Zartman).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.056

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C. B. Zartman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6705–6708
a preference for fluoro substitution of the aromatic ring (compare
compounds 5, 6 and 9 in Table 1). In particular, the 3,5-dif-
luorobenzylamide (compound 10) offered improved potency com-
pared with benzylamide 6. In addition to lipophilic, fluoroaromatic
substituents, moderately polar groups were also tolerated, as
exemplified by the pyridine 7 and the tert-butyl ester 8.
Exploration of the naphthyridine core (Table 1) revealed some
of the key pharmacophoric elements for compounds of this class.
The inactivity of compound 11 highlights the importance of the ap-
pended hydroxyl group. Replacement of the naphthyridine core
with an isoquinoline (12) or quinoline (13) also produced com-
pounds with diminished activity. The ester analog 14 lost all activ-
ity, implying that the more basic amide carbonyl of 10 may be
required for activity.
In the context of the 3,5-difluorobenzyl amide moiety, SAR
around a truncated pyridine core was also explored (Table 2). Sim-
ple deletion of the western pyridine ring from naphthyridine 10 led
to a 20-fold loss in receptor affinity (compound 15), but incorpora-
tion of a bromine atom provided compound 16, which possessed
similar potency to the naphthyridine analog. As in the naphthyri-
dine series, the hydroxyl substituent appeared to be critical for
activity and methylation (17) caused a significant loss in potency.
Also in analogy with the naphthyridines, the glycine tert-butyl es-
ter derivative 18 possessed similar receptor affinity to the benzyl
amides. With compound 18 in hand, an array of glycine amides
were explored leading to the most active compound in this series,
compound 21 (Ki = 710 nM).
Figure 2. CGRP receptor antagonists olcegepant (1), telcagepant (2), MK-3207 (3),
compound 4 and L-870,810 (5).
The lack of activity observed with compounds 11, 13, 14 (Ta-
ble 1) and 17 (Table 2) was of significant interest. One interpreta-
tion of these data was that the presence of the hydroxyl moiety and
the coplanarity of the basic amide carbonyl and the naphthyridine
ring were crucial for activity. Taken together with the fact that the
original lead 5 evolved from the known metal-chelating diketo acid
hand, we initiated an effort to characterize and optimize this novel
lead series.
As noted previously, the lead compound 5 had micromolar
affinity for the CGRP receptor (Ki = 7300 nM). Evaluation of a num-
ber of alternatives to the 4-fluorobenzylamide moiety of 5 revealed
Table 1
Selected CGRP receptor antagonists and naphthyridine analogs
O
S
N
Z
O
X
R
Y
O
Compd
R
X
Y
Z
CGRP binding Ki, nMa,b
CGRP cAMP IC50, nMa,c
2
3
4
—
—
—
—
—
—
—
—
—
—
—
—
0.78 0.05 (10)e
0.024 0.001 (3)f
9.3 3.1 (7)
2.2 0.29 (8)d,e
0.12 0.02 (6)d,f
41 12 (11)
F
H
5
N
N
OH
7300 1700 (3)
28,000 (1)
N
6
7
8
N
N
N
N
N
N
OH
OH
OH
16,000 (2)
14,000 (2)
8000 (1)
NA
NA
>10,000 (1)
9
N
N
N
N
OH
OH
5200 1400 (4)
1900 (1)
>10,000 (1)
2600 (1)
10
F
H
N
11
12
N
N
N
H
>100,000 (1)
26,000 (1)
NA
NA
CH
OH

C. B. Zartman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6705–6708
6707
Table 1 (continued)
Compd
R
X
Y
Z
CGRP binding Ki, nMa,b
CGRP cAMP IC50, nMa,c
13
CH
N
OH
>100,000 (1)
NA
NA
14
N
N
OH
>100,000 (1)
a
Number of replicates given in parentheses (NA, not tested).
b
c
d
e
f
Ki values for inhibition of 125I-hCGRP binding determined using native human receptor in SK-N-MC cell membranes.14
Inhibition of CGRP-stimulated cAMP production in SK-N-MC cells.14
Inhibition of CGRP-stimulated cAMP production in HEK293 cells stably expressing CLR/RAMP1.14
Data from Ref. 14.
Data from Ref. 15.
pharmacophore found in many HIV integrase inhibitors13 this sug-
gested that metal binding was critical for the interaction of these
compounds with the CGRP receptor. The HIV integrase inhibitors
related to lead compound 5 are thought to bind to divalent cations
in the active site of the enzyme. We noted that our standard CGRP
receptor binding assay contained 5 mM MgCl2 and hypothesized
that an interaction with this metal ion may be important for the
activity of compounds like 5.
In order to investigate this possibility, CGRP and a number of
representative antagonists were analyzed in the binding assay
both in the presence and absence of 5 mM MgCl2 (Table 3). The
small molecule CGRP receptor antagonist 4, a truncated analog of
olcegepant, was essentially unaffected by removal of magnesium
chloride from the assay. In contrast, it was clearly evident that
the hydroxypyridine 16 required Mg2+ for binding to the CGRP
receptor and its Ki value shifted >35-fold in the absence of added
magnesium. Related analogs, such as 21, also lost significant recep-
tor affinity without added MgCl2, suggesting that the binding of
this novel class of antagonists to the CGRP receptor requires
magnesium ions, in contrast to other known small molecule CGRP
receptor antagonists. The native agonist CGRP and the peptide
antagonist CGRP (8–37) were unaffected by the presence or ab-
sence of magnesium ions.
Radioligand binding studies utilizing chimeric receptors gener-
ated by exchanging regions of the CLR with corresponding regions
of the calcitonin receptor (CTR), a related class II G-protein-coupled
receptor, followed by coexpression with RAMP1 afforded further
insight into the binding of these hydroxypyridine-based CGRP
receptor antagonists.16 Compound 16 (designated ‘‘compound 4’’
in Ref. 16) was shown to bind to CGRP by interaction with trans-
membrane region 7 (TM7) of CLR independent of RAMP1, while
small molecule antagonists related to olcegepant were shown to
bind RAMP1-dependently to the amino terminus of CLR (Fig. 1).16
Because 16 interacted with the CGRP receptor in a RAMP-indepen-
dent manner, it exhibited a different selectivity profile to that ob-
served for many CGRP receptor antagonists, such as telcagepant
(2). For example, while 2 possessed good selectivity against the hu-
man adrenomedullin receptor CLR/RAMP2 (Ki >100 lM), 16 did not
Table 2
Selected hydroxypyridine compounds
Compd
R
X
Y
CGRP binding Ki, nMa,b
CGRP cAMP IC50, nMa,c
15
H
OH
45,000 (1)
NA
16
17
Br
Br
OH
3300 1100 (11)
>100,000 (1)
2700 (2)
NA
OCH3
18
19
Br
Br
OH
OH
1700 500 (6)
11,000 (2)
9800 (2)
>10,000 (1)
20
21
Br
Br
OH
OH
1800 (2)
710 (2)
2400 (1)
2100 (1)
a
b
c
Number of replicates given in parentheses (NA, not tested).
Ki values for inhibition of 125I-hCGRP binding determined using native human receptor in SK-N-MC cell membranes.14
Inhibition of CGRP-stimulated cAMP production in SK-N-MC cells.14

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C. B. Zartman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6705–6708
oral bioavailability in rat and dog, with low plasma clearance and
good plasma half-lives in both species.
Table 3
Effect of magnesium on CGRP and selected CGRP receptor antagonists
In conclusion, a novel series of CGRP receptor antagonists has
been characterized. Optimization of naphthyridine 5 led to the or-
ally bioavailable hydroxypyridine 16, which possessed a 10-fold
improvement in functional potency. Compound 16 interacted with
the CGRP receptor in a magnesium-dependent fashion at a binding
site that involved TM7 and was independent of RAMP1. As such,
this CGRP receptor antagonist is both structurally and functionally
distinct from previously reported small molecule antagonists of
this receptor.
Compd
Ki, nMa 5 mM MgCl2
Ki, nMa 0 mM MgCl2
Shift
4
16
21
9 (2)
8 (2)
0.9
>35
6.7
0.8
1
2700 (3)
840 (1)
5.5 (1)
0.01 (1)
>100,000 (3)
5600 (1)
4.5 (1)
CGRP (8–37)
CGRP
0.01 (1)
a
Number of replicates given in parentheses. Ki values determined in the presence
and absence of 5 mM MgCl2 for the inhibition of 125I-CGRP binding to SK-N-MC cell
membranes.
Acknowledgments
The authors thank Yvonne M. Leonard, Cynthia Miller-Stein and
Audrey Wallace for animal dosing and pharmacokinetic analysis;
the MRL West Point analytical, mass spectrometry and NMR spec-
troscopy groups for analytical and spectroscopy data; and Neville
Anthony, Scott Ceglia, Robert Gomez and John Wai for providing
compounds 5, 6 and 9 and for helpful discussions.
References and notes
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Scheme 1. Synthesis of compound 16. Reagents and conditions: (a) TEA, meth-
anesulfonyl chloride, THF, 5 °C, 95%; (b) N,N-diisopropylamine, 1,10-phenanthro-
line, n-BuLi in hexanes, THF, À20 °C to rt, 77%; (c) H2SO4, CH3OH, 79%; (d) Br2, H2O,
79%; (e) Cu(I)O, pyridine, 130 °C; then EDTA, CH2Cl2, H2O; (f) H2SO4, CH3OH, 34%
over two steps; (g) NBS, CH3Cl, 80 °C, 47%; and (h) 3,5-difluorobenzylamine,
toluene, reflux, 42%.
9. Ho, T. W.; Ferrari, M. D.; Dodick, D. W.; Galet, V.; Kost, J.; Fan, X.; Leibensperger,
H.; Froman, S.; Assaid, C.; Lines, C.; Koppen, H.; Winner, P. K. Lancet 2008, 372,
2115.
Table 4
Pharmacokinetic data for compound 16
10. Bell, I. M.; Gallicchio, S. N.; Wood, M. R.; Quigley, A. G.; Stump, C. A.; Zartman,
C. B.; Fay, J. F.; Li, C.-C.; Lynch, J. J.; Moore, E. L.; Mosser, S. D.; Prueksaritanont,
T.; Regan, C. P.; Roller, S.; Salvatore, C. A.; Kane, S. A.; Vacca, J. P.; Selnick, H. G.
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Species
F (%)
iv t1/2 (h)
Cl (mL/min/kg)
Vdss (L/kg)
Rat
Dog
26a
13b
5.4c
7.1d
1.1
5.1
0.4
3.0
11. Hewitt, D. J.; Aurora, S. K.; Dodick, D. W.; Goadsby, P. J.; Ge, Y.; Bachman, R.;
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Fisher, T. E.; Embry, M.; Guare, J. P.; Egbertson, M. S.; Vacca, J. P.; Huff, J. R.;
Felock, P. J.; Witmer, M. V.; Stillmock, K. A.; Danovich, R.; Grobler, J.; Miller, M.
D.; Espeseth, A. S.; Jin, L.; Chen, I.-W.; Lin, J. H.; Kassahun, K.; Ellis, J. D.; Wong,
B. K.; Xu, W.; Pearson, P. G.; Schleif, W. A.; Cortese, R.; Emini, E.; Summa, V.;
Holloway, M. K.; Young, S. D. PNAS 2004, 101, 11233.
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E. L.; Mosser, S. D.; Burgey, C. S.; Paone, D. V.; Shaw, A. W.; Graham, S. L.; Vacca,
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a
b
c
Dosed at 10 mpk in 1% methylcellulose.
Dosed at 1 mpk in 1% methylcellulose.
Dosed at 2 mpk in DMSO.
d
Dosed at 0.5 mpk in DMSO.
(Ki = 7500 nM).14 Additionally, 2 had reduced affinity for non-pri-
mate CGRP receptors such as the rat receptor (Ki = 1200 nM), and
much of this species selectivity was dictated by sequence differ-
ences in RAMP1, notably by residue 74.17,18 Unsurprisingly, since
16 does not appear to interact with RAMP1, it did not exhibit sig-
nificantly lower affinity for the rat CGRP receptor (Ki = 6800 nM).16
The synthesis of compound 16, which is representative of the
methodology used to synthesize the related analogs in Table 2, is
shown in Scheme 1.19 The cyclic sultam was synthesized in two
steps20 from commercially available 3-bromopropan-1-amine
hydrobromide and the methyl 6-bromo-3-hydroxypyridine-2-
carboxylate was obtained in two steps from commercially avail-
able 3-hydroxypyridine-2-carboxylic acid. An Ullmann coupling
of the two followed by bromination with NBS and direct amidation
of the ester with amines yielded compound 16 and closely related
analogs. Amidation with tert-butyl glycinate followed by hydroly-
sis and standard amide couplings allowed for the synthesis of re-
lated amides 19–21.
16. Salvatore, C. A.; Mallee, J. J.; Bell, I. M.; Zartman, C. B.; Williams, T. M.; Koblan,
K. S.; Kane, S. A. Biochemistry 2006, 45, 1881.
17. Miller, P. S.; Barwell, J.; Poyner, D. R.; Wigglesworth, M. J.; Garland, S. L.;
Donnelly, D. Biochem. Biophys. Res. Commun. 2010, 391, 437.
18. Moore, E. L.; Gingel, J. J.; Kane, S. A.; Hay, D. L.; Salvatore, C. A. Biochem. Biophys.
Res. Commun. 2010, 394, 141.
19. All final compounds were characterized by 1H NMR, HPLC and HRMS.
Additional synthetic details are provided in: Bell, I. M.; Gallicchio, S. N.;
Zartman, C. B. WO 2005/009962.
20. Lee, J.; Askin, D. Department of Process Research, Merck Research Laboratories,
personal communication.
The pharmacokinetic properties of compound 16 were evalu-
ated in rat and dog. As detailed in Table 4, 16 exhibited moderate