Non-peptide calcitonin gene-related peptide receptor antagonists from a benzodiazepinone lead

Article abstract of DOI:10.1016/j.bmcl.2006.02.051

High-throughput screening of the Merck sample collection identified benzodiazepinone tetralin-spirohydantoin 1 as a CGRP receptor antagonist with micromolar activity. Comparing the structure of 1 with those of earlier peptide-based antagonists such as BIB

Full text of DOI:10.1016/j.bmcl.2006.02.051

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2595–2598
Non-peptide calcitonin gene-related peptide receptor
antagonists from a benzodiazepinone lead
Theresa M. Williams,a,* Craig A. Stump,a Diem N. Nguyen,a Amy G. Quigley,a
Ian M. Bell,a Steven N. Gallicchio,a C. Blair Zartman,a Bang-Lin Wan,a
Kimberly Della Penna,b Priya Kunapuli,c Stefanie A. Kane,b Ken S. Koblan,b
Scott D. Mosser,b Ruth Z. Rutledge,b Christopher Salvatore,b John F. Fay,b
Joseph P. Vaccaa and Samuel L. Grahama
aDepartment of Medicinal Chemistry Merck & Co., West Point, PA 19486, USA
bDepartment of Pain Research, Merck & Co., West Point, PA 19486, USA
cDepartment of Automated Biotechnology, Merck & Co., North Wales, PA 19454, USA
Received 21 December 2005; accepted 16 February 2006
Available online 9 March 2006
Abstract—High-throughput screening of the Merck sample collection identified benzodiazepinone tetralin-spirohydantoin 1 as a
CGRP receptor antagonist with micromolar activity. Comparing the structure of 1 with those of earlier peptide-based antagonists
such as BIBN 4096 BS, a key hydrogen bond donor–acceptor pharmacophore was hypothesized. Subsequent structure activity
studies supported this hypothesis and led to benzodiazepinone piperidinyldihydroquinazolinone 7, CGRP receptor Ki = 44 nM
and IC50 = 38 nM. Compound 7 was orally bioavailabile in rats and is a lead in the development of orally bioavailable CGRP
antagonists for the treatment of migraine.
Ó 2006 Elsevier Ltd. All rights reserved.
Migraine is a neurovascular disorder in which neuropep-
tide release and intracranial blood vessel dilation play an
important role.1 Characteristic symptoms of migraine
include severe headache, nausea, and sensitivity to light
and sound lasting 4–72 h.2 Approximately 12% of the
general population experience migraine headaches, but
since migraine is underdiagnosed and undertreated, this
may be an underestimate.3
Calcitonin gene-related peptide (CGRP) is a 37 amino
acid neuropeptide present in perivascular sensory nerve
fibers and one of the most potent vasodilators known.6
The CGRP receptor is especially abundant in the tri-
geminal vasculature, a network of intracranial vessels
associated with sensory neurons of the trigeminal gan-
glion.7 Activation of trigeminal sensory neurons and
CGRP release are strongly implicated in the pathophys-
iology of migraine headache.8 Therefore, a CGRP
receptor antagonist is an attractive therapeutic target
for the treatment of migraine.9,10
The current standard for the treatment of migraine is 5-
HT1B/1D receptor agonists that form the triptan class of
migraine drugs.4 Part of the triptan mechanism of action
involves direct vasoconstriction of blood vessels through
activation of 5-HT1B receptors.5 Although selective for
intracranial over coronary vessel constriction, triptans
are contraindicated in patients with cardiovascular
disease. A migraine drug that does not cause direct vaso-
constriction would have significant therapeutic value.
In placebo-controlled clinical trials, the potent Boehrin-
ger–Ingelheim CGRP receptor antagonist BIBN 4096
BS was efficacious in alleviating migraine headache
pain.11 Two hours following a 2.5 mg iv dose, 69% of
patients reported significant pain relief. The therapeutic
response was similar to that observed for triptans in pre-
vious clinical trials. No evidence of direct vasoconstric-
tion or effects on hemodynamics was observed with
BIBN 4096 BS.11,12 The Boehringer–Ingelheim clinical
trial is proof-of-concept for treating acute migraine
headache with a CGRP receptor antagonist. Efficacy
Keywords: CGRP receptor antagonist; Benzodiazepinone; CGRP;
CLR/RAMP1; Migraine.
*
Corresponding author. Tel.: +1 215 652 3087; fax: +1 215 652
3971; e-mail: theresa_williams@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.051

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T. M. Williams et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2595–2598
was achieved by blocking the pathological intracranial
response to CGRP and was not a consequence of direct
vasoconstriction, since the latter was not observed.
The CGRP antagonist BIBN 4096 BS was administered
by intravenous infusion in clinical trials. We sought to
identify a suitable lead compound for a program aimed
at developing an orally active CGRP antagonist to treat
acute migraine headache. High-throughput screening
identified benzodiazapinone 1 as a modest inhibitor of
CGRP binding to native human CGRP receptors
(Ki = 4.8 lM, SK-N-MC cell membranes). Follow-up
studies demonstrated that benzodiazapinone 1 was a
functional antagonist of CGRP-stimulated cAMP pro-
duction in SK-N-MC cells (IC50 = 6 lM). Binding and
functional assays using a recombinant form of the hu-
man CGRP receptor obtained similar results. The struc-
ture of 1 was unique compared to previously published
CGRP antagonists based on D-dibromotyrosine (‘Com-
pound 1’)13 or the dipeptide D-dibromotyrosine-L-lysine
(BIBN 4096 BS)14 (Fig. 1). In contrast to amino acid-
based inhibitors, HTS lead 1 linked two heterocycles
through a urea group: the GPCR ‘privileged structure’15
N-methyl 3-aminobenzodiazepinone and the spirohyd-
antoin derived from b-tetralone (spiro ring fusion as a
mixture of isomers).
Figure 2. Overlay of energy minimized piperidinyldihydroquinazoli-
none (blue) and tetralin spirohydantoin (yellow) from Eq. 1.
structures of piperidinyldihydroquinazolinone and one
enantiomer of the tetralin spirohydantoin showed good
alignment between carbonyl amide oxygen–NH pairs
(Fig. 2).
ð1Þ
To test the hypothesis of a common pharmacophore, the
2-aminotetralin spirohydantoin substructure in 1 was re-
placed with piperidinylbenzimidazolone or piper-
idinyldihydroquinazolinone, to form hybrid structures
2 and 3.
The required 3-aminobenzodiazepine15–18 was treated
with p-nitrophenylchloroformate and triethylamine, fol-
lowed by the appropriate 4-substituted piperidine,19 to
give compounds 2–5 and 7–9 (Scheme 1). Compound
6 was obtained by treating 5 with 1 equiv of NaH and
CH3I in DMF.20
The CGRP receptor is heterodimeric and is comprised
of the family B GPCR calcitonin-like receptor (CLR)
and the accessory protein, receptor activity modifying
protein 1 (RAMP1).21,22 Co-expression of CLR and
RAMP1 is required to form a functional CGRP recep-
tor. A human neuroblastoma cell line (SK-N-MC)
expresses the native human CGRP receptor and was
used to determine the binding affinity.23 A cell line
expressing recombinant human CLR/RAMPl exhibited
pharmacology identical to that of the native receptor.
Binding studies were conducted with [I125]CGRP using
E10 (human cloned receptor)24 or SK-N-MC (native hu-
man receptor) cell membranes and results reported as a
Ki. The IC50 for antagonism of CGRP-stimulated
increases in intracellular cAMP was determined in E10
cells expressing the human cloned receptor.
Simple substituent additions to 1 did not increase poten-
cy, but more substantial structural changes were success-
ful. It was noted that potent CGRP antagonists
incorporated another GPCR privileged structure, either
piperidinylbenzimidazolone in the case of ‘Compound
1’,13 or the closely related piperidinyldihydro-quinazoli-
none in BIBN 4096 BS.14 In these compounds, the benz-
imidazolone and dihydroquinazolinone have a C(O)NH
hydrogen bond donor–acceptor pair embedded in the
heterocycle, similar to the hydantoin in 1. This led us
to speculate that ‘Compound 1’, BIBN 4096 BS, and 1
shared a common hydrogen bond donor–acceptor
pharmacophore Eq. 1. An overlay of energy minimized
Scheme 1. Synthesis of substituted piperidine benzodiazepinone ana-
logs. Reagents and conditions: (a) p-nitrophenylchloroformate,
DMSO/THF, TEA, 0 °C; 4-Z-piperidine, TEA.
Figure 1. Previously reported CGRP receptor antagonists.

T. M. Williams et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2595–2598
2597
Hybrid structures were synthesized to link the N-methyl
3-aminobenzodiazepinone component of screening lead
1 to either piperidinylbenzimidazolone (forming 2) or
to piperidinyldihydroquinazolinone (forming 3).
Although benzimidazolone 2 was inactive at the concen-
trations tested, benzodiazepine dihydroquinzolinone
enantiomers 3a (3S-enantiomer) and 3b (3R-enantio-
mer) had low micromolar activity.
showed Ki = 48 nM. In the same cell line, compound 7
demonstrated competitive antagonism of CGRP-stimu-
lated cAMP production. Increasing concentrations of
Overall, the R-enantiomer 3b was approximately 5-fold
more potent than the original lead 1 (Table 1). SAR
studies showed that small lipophilic N-1 benzodiaze-
pinone substituents were well tolerated. Slight increases
in potency were noted for N-ethyl (4) and N-isopropyl
(5) substituents. In partial support of the hydrogen bond
donor–acceptor pharmacophore model, N-alkylation of
the dihydroquinazoline nitrogen was highly detrimental
to activity (compare 5 with N-methyl analog 6),
although steric effects between the ligand and receptor
cannot be ruled out as the cause of poor activity.
Changing the N-1 methyl substituent in 3b to trifluoro-
ethyl increased potency 50-fold. N-Trifluoroethyl benzo-
diazepinone 7 was one of the most potent analogs
prepared, with Ki = 44 nM. In cell culture, 7 blocked
CGRP-stimulated cAMP production with an
IC50 = 38 nM. We found that compounds containing
the dihydroquinazoline substructure required protection
from light and air to prevent dihydroquinazolinone ring
oxidation. The ring expanded 1,3-benzodiazepin-2-one 8
was stable in air and was found to be equipotent to 7.
When the optimized N-1 trifluoroethyl benzodiazepine
substituent in the dihydroquinazolinone series was re-in-
troduced to benzimidazolone 2 (Ki > 20,000 nM), poten-
cy increased more than 10-fold (9, Ki = 2370 nM),
consistent with dihydroquinazolinone structure–activity
relationships.
Figure 3. Concentration–response curves and Schild plot analysis of
compound 7. (a) Concentration–response curves of CGRP-induced
cAMP production in E10 cells expressing human recombinant CLR/
RAMP1 in the presence and absence of compound 7. j = 2560 nM;
m = 1280 nM; , = 640 nM; Ç = 320 nM; d = 160 nM; h = 80 nM;
n = 0 nM. (b) Schild plot showing the effect of compound 7 on CGRP-
induced cAMP production in E10 cells expressing recombinant CLR/
RAMP1. DR is the ratio of CGRP EC50 values in the presence and
absence of compound 7. The X-intercept is equal to the pA2 and the Kb
calculated using the formula pA2 = ꢀlogKb.
Receptor kinetic studies were carried out on compound
7. Competitive binding studies with [125I]CGRP and 7
(E10 membranes, human recombinant CGRP receptor)
Table 1. Binding affinity and antagonist activity for compounds 1–9, compound 1 and BIBN 4096 BS on the human CGRP receptor (CLR/RAMP1)
Compound
R1
BZD C-3
CLR/RAMP1
Kia (nM)
n
CLR/RAMP1
b
IC50 (nM)
n
1
—
CH3
R
4250 275
>13,000
4
5
3
4
4
5
1
7
6
3
57% inh (17 lM)
—
2
2
R,S
S
3a
3b
4
CH3
CH3
8400 800
2200 87
897 118
1060 658
33% inh (100 lM)c
44 17
4450
3640
990
1
1
1
1
R
CH3 CH2
i-Pr
i-Pr
R
5
R,S
R,S
R
1810
—
6
7
CF3 CH2
CF3 CH2
CF3 CH2
38 24
43
1,580
5
1
1
8
R
61 12
2370 321
9
R
Compound 1
BIBN
—
—
—
—
83
0.005 .001
2
5
75
0.100 .058
2
7
a Ki values were determined using E10 cell membranes expressing a human cloned CLR/RAMP1 receptor and are expressed as arithmetic means of n
determinations, along with the standard deveriation.24
b IC50 values were determined using E10 cells expressing a human cloned CLR/RAMP1 receptor and measured the inhibition of CGRP-mediated
cAMP production.24
c Ki value was determined using SK-N-MC cell membranes as previously described.23

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T. M. Williams et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2595–2598
CGRP produced a rightward shift in the dose–response
curve for 7 with no change in maximal response (Fig. 3).
In good agreement with binding experiments, a Schild
plot analysis yielded Kb = 77 nM (pA2 = 7.1). The slope
of the line was 0.9, indicating a 1:1 association of antag-
onist and receptor. In contrast to the human CGRP
receptor, 7 was much less potent on the rat CGRP
receptor, inhibiting only 35% of [125I]CGRP binding at
100 lM. Differences in human and rat RAMP1 se-
quence, most notably at position 74 (Trp in human
and Lys in rat), were responsible for the species differ-
ence in affinity, an effect also observed for BIBN 4096
BS.23 In other studies, 7 was inactive on the human
adrenomedullin receptor CLR/RAMP2.21
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A key objective for the program was to identify a CGRP
antagonist lead that could be optimized for oral bioavail-
ability. To this end, the pharmacokinetic profile of 7 was
determined in rats and dogs. In rats, 7 had an iv
t1/2 = 1.7 h, Cl = 20 mL/min/kg, and had modest but con-
sistent oral bioavailability when dosed as a suspension in
1% aqueous methocel (F = 10%, Cmax = 240 nM,
10 mpk) (n = 3). In dogs, 7 was not detected in plasma
after an oral dose of 2 mpk (1% aqueous methocel, n = 2).
A nonpeptide screening lead 1 was identified that had
micromolar affinity for the human CGRP receptor and
was a functional CGRP antagonist in cells. SAR studies
of benzodiazepinone 1 led to a 100-fold increase in poten-
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a
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ð2Þ
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The authors would like to acknowledge the MRL West
Point analytical chemistry, mass spectroscopy, and
NMR spectroscopy groups for analytical and spectro-
scopic data. We would also like to thank Constantine
Kreatsoulas for preparing the molecular graphics figure,
and Chris Burgey for helpful discussions and manuscript
review.