Development of human calcitonin gene-related peptide (CGRP) receptor antagonists. 1. Potent and selective small molecule CGRP antagonists. 1-[N 2-[3,5-dibromo-N-[[4-(3,4-dihydro-2(1H)-oxoquinazolin-3-yl) -1-piperidinyl]carbonyl]-D-tyrosyl]-L-ly

Article abstract of DOI:10.1021/jm0490641

Although the triptans have greatly improved the acute treatment of migraine headache, there are yet many shortcomings. Therefore, new strategies for the treatment of migraine are needed which offer advantages over current therapy, e.g. triptans. Our novel

Full text of DOI:10.1021/jm0490641

J. Med. Chem. 2005, 48, 5921-5931
5921
Development of Human Calcitonin Gene-Related Peptide (CGRP) Receptor
Antagonists. 1. Potent and Selective Small Molecule CGRP Antagonists.
1-[N2-[3,5-Dibromo-N-[[4-(3,4-dihydro-2(1H)-oxoquinazolin-3-yl)-1-
piperidinyl]carbonyl]-D-tyrosyl]-L-lysyl]-4-(4-pyridinyl)piperazine: The First
CGRP Antagonist for Clinical Trials in Acute Migraine†
Klaus Rudolf,* Wolfgang Eberlein, Wolfhard Engel, Helmut Pieper, Michael Entzeroth,
Gerhard Hallermayer, and Henri Doods
Boehringer Ingelheim Pharma GmbH & Co. KG, Research Division, Birkendorfer Strasse 65, 88397 Biberach/RISS, Germany
Received November 19, 2004
Although the triptans have greatly improved the acute treatment of migraine headache, there
are yet many shortcomings. Therefore, new strategies for the treatment of migraine are needed
which offer advantages over current therapy, e.g. triptans. Our novel approach was based on
the hypothesis that the release of calcitonin gene-related peptide (CGRP) could play a causative
role in migraine headache. Thus we initiated a program aimed at the design and synthesis of
small molecule CGRP receptor antagonists. High throughput screening led to the identification
of (R)-Tyr-(S)-Lys dipeptide-like compounds that showed weak but unequivocal binding to the
human CGRP receptor. Lead optimization afforded highly potent CGRP antagonists, the
prototype being compound 19 (BIBN4096). This compound exhibiting a favorable biological
profile was selected for initial clinical trials. A proof of concept study indicated that intravenous
application of 19 was effective in the treatment of acute migraine headache. This finding
strongly supports our initial working hypothesis that CGRP plays an important role in the
pathophysiology of migraine.
Introduction
far such compounds have not progressed beyond phase
II clinical trials.
Migraine is a very common and painful headache
disorder that is ranked among the world’s most dis-
abling medical illnesses. Approximately 12% of the adult
population in the western society suffer from migraine
attacks.1
Our strategy was to adopt a new approach that was
specifically directed toward the neurovascular aspect of
migraine pathogenesis. For many years it has been
hypothesized that migraine headache is associated with
dilatation of cranial vessels and activation of the
trigeminal vascular system. The major neurotransmit-
ter of the trigeminal vascular system is calcitonin gene-
related peptide (CGRP), a 37 amino acid neuropeptide
belonging to a family of peptides including calcitonin,
adrenomedullin, and amylin. CGRP-containing nerves
innervate blood vessels in various regions of the body,
and those involved in cerebral circulation are especially
densely innervated. Although CGRP has a variety of
effects, its most pronounced action is vasodilatation. It
is indeed one of the most potent endogenous vasodilators
known, e.g. of cerebral and dural vessels.7
In man, stimulation of the trigeminal ganglion
results in the release of CGRP. The hypothesis that
CGRP could be involved in the pathogenesis of migraine
came from observations by Edvinsson and Goadsby,
demonstrating that the levels of the potent vasodilator
CGRP in the jugular blood are elevated during the acute
phase of a migraine attack.8 Inhibition of CGRP-
mediated vasodilatation by CGRP antagonists should
therefore be expected to attenuate vascular headache,
if CGRP is implicated in the pathogenesis of migraine
headache.
Therapy with 5-HT1B/1D agonists, the so-called trip-
tans, is presently considered the most effective treat-
ment of migraine. Although triptans are efficacious and
generally well tolerated, this class of drugs has draw-
backs of therapeutic relevance. Many patients do not
respond to this type of treatment and complete pain
relief is the exception rather than the rule. Between 25
and 40% of responsive patients suffer from recurrent
headaches within several hours. Being 5-HT1B/1D ago-
nists, triptans act as potent vasoconstrictors and are
therefore contraindicated in patients with hypertension
or ischemic heart disease.2 Thus, a clear demand exists
for new antimigraine medication with pronounced im-
provement over current therapies.
Accordingly, research programs targeting different
aspects of migraine pathogenesis have been described
in recent years. Most companies active in this field have
3
4
focused on selective 5-HT1F or 5-HT1D agonists, neu-
rokinin antagonists,5 or endothelin antagonists,6 but so
* Corresponding author: Klaus Rudolf, Boehringer Ingelheim Phar-
ma GmbH & Co. KG, Department of Chemical Research, Birkendorfer
Strasse 65, 88397 Biberach an der Riss, Phone +49/7351/54-8508, Fax
+49/7351/83-8508, E-mail: klaus.rudolf@bc.boehringer-ingelheim.com.
† Dedicated to Prof. Dr. Hans Machleidt on the occasion of his 78th
birthday.
At the outset of our research program rather large
C-terminal fragments of CGRP, e.g., CGRP8-37, had
10.1021/jm0490641 CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/25/2005

5922 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Table 1. Structural Variations of Lead Compounds 1 and 2
Rudolf et al.
a IC50 for inhibition of human CGRP receptor. For details, see Experimental Section.
been described in the literature as antagonists, having
limited potency.9 Due to their peptide nature, these
compounds were limited in their application, especially
in in vivo or clinical investigations. We initiated a
synthetic program for the design and synthesis of small
molecule CGRP receptor antagonists to evaluate the role
of CGRP in experimental migraine models and to
discover if migraine headache is indeed related to the
release of CGRP in man.
A high throughput screen was established using a
human neuroblastoma cell line (SK-N-MC) constitu-
tively expressing the human CGRP receptor, and screen-
ing parts of our corporate compound pool led to the
identification of the (R)-Tyr-(S)-Lys dipeptide-like com-
pounds 1 and 2 displaying hCGRP receptor affinities
in the micromolar range. Since both compounds con-
tained two chiral centers (R)-AA1 and (S)-AA2, we
examined whether the interaction with the hCGRP
receptor was stereospecific. We chose the 3,5-dibromo-
Tyr derivative 1 as a reference compound and synthe-
sized the three corresponding stereoisomers 3 (S,R), 4
(R,R), and 5 (S,S). As can be seen from Table 1, these
compounds are devoid of any hCGRP binding affinity
exhibiting IC50 values above 300 000 nM. This result
indicated that compound 1 interacted with the hCGRP
receptor in a stereospecific manner, with both chiral
centers being involved in this binding process. With this
basis a synthetic program was initiated aimed at

Small Molecule CGRP Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5923
Scheme 1a
Scheme 3a
a (a) 1-(4-Pyridinyl)piperazine, DIEA, TBTU, HOBt, DMF, room
temperature; (b) TFA, CH2Cl2, room temperature; (c) Boc-D-
Phe(3,5-Br2-4-OH)-OH, TBTU, HOBt, DIEA, DMF, room temper-
ature; (d) 2-(2-methoxyphenyl)ethyl isocyanate, THF, room tem-
perature; (e) HBr, AcOH, anisole, room temperature; (f) 2-(3-
methoxyphenyl)ethylamine, CDT, DIEA, THF, reflux.
a (a) R2H, TBTU, HOBt, DIEA, DMF, room temperature; (b)
TFA, CH2Cl2, room temperature; (c) Boc-D- or L-Phe(3,5-Br2-4-X)-
OH, TBTU, HOBt, DIEA, DMF, room temperature; (d) R1CO2H,
TBTU, HOBt, DIEA, DMF, room temperature; (e) HBr, AcOH,
anisole, room temperature; (f) R2H, 4-methylmorpholine, isobutyl
chloroformate, THF, room temperature; (g) Boc-D- or L-Phe(3,5-
Br2-4-X)-OH, 4-methylmorpholine, isobutyl chloroformate, THF,
room temperature; (h) R1CO2H, 4-methylmorpholine, isobutyl
chloroformate, THF, room temperature.
Scheme 4a
Scheme 2a
a (a) H-D-Phe(3,5-Br2-4-OH)-OEt‚HBr, triethylamine, THF, room
temperature; (b) 2 N aq NaOH, THF, room temperature; (c) 21a,
DIEA, TBTU, HOBt, DMF, room temperature; (d) HBr, AcOH,
anisole, room temperature.
a (a) R1CO2H, 4-methylmorpholine, isobutyl chloroformate, THF,
room temperature; (b) 2 N aq NaOH, MeOH/DMF, room temper-
ature; (c) L-Lys(Z) methyl ester hydrochloride, 4-methylmorpho-
line, isobutyl chloroformate, THF, room temperature; (d) R2H,
4-methylmorpholine, isobutyl chloroformate, THF, room temper-
ature; (e) HBr, AcOH, anisole, room temperature.
Results and Discussion
Chemistry. The dipeptide-like compounds listed in
Table 1 were synthesized according to Schemes 1-6,
following methods known from peptide chemistry.
Whereas construction of the peptide backbone of most
compounds started from the C-terminus, compounds 6,
7, 8, and 9 were constructed from their N-termini as
shown in Scheme 6. Two different procedures were used
to introduce the urea moiety characteristic for com-
pounds 11-20. Synthesis of 11 and 12 involved addition
of the free R-amino group of an appropriately protected
amino acid derivative to an isocyanate; for the synthesis
of 13-20 a very general method involved the condensa-
generating compounds with hCGRP receptor affinity in
the submicromolar range using compounds 1 and 2 as
starting structures. Since no reliable information about
the three-dimensional structure of these compounds and
about the binding site of the hCGRP receptor was
available at the beginning of our project, we had to
perform lead optimization by conventional structure
activity studies.

5924 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Rudolf et al.
Scheme 5a
Scheme 6a
a (a) 1-(4-Pyridinyl)piperazine, DIEA, TBTU, HOBt, DMF, room
temperature; (b) H2, Pd/C, KHSO4, MeOH, room temperature; (c)
N-(9-fluorenylmethoxycarbonyl)-3,5-dibromo-D-tyrosine, TBTU,
HOBt, DIEA, THF, room temperature; (d) diethylamine, THF,
room temperature; (e) R1H, CDT, DIEA, THF, reflux; (f) TFA,
CH2Cl2, room temperature; (g) hydrogen chloride/methanol, room
temperature.
tion of a primary or secondary amine with N,N′-
carbonylditriazole and the free R-amino group.
The synthesis of novel piperidine 33, which was
required as a starting material for compound 17, is
illustrated in Scheme 7. 4-Phenylamino-1-phenylmethyl-
piperidine was treated with sodium cyanate in the
presence of trifluoroacetic acid. The benzyl protecting
group of the resulting product was removed by hydro-
genolysis to give 33. Synthesis of novel piperidine 34,
an intermediate required for the preparation of test
compound 20, is depicted in Scheme 8. Boc protected
4-piperidinammonium hydrogencarbonate was treated
with phenacyl bromide, followed by sodium cyanate in
the presence of glacial acetic acid. Removal of the Boc
group by trifluoroacetic acid gave piperidine 34.
Biology. The binding affinities of compounds for the
human CGRP receptor were determined by inhibition
of 125I-CGRP as previously described.11 Species depend-
ent CGRP receptor binding affinities of compounds 13,
18 and 19 are reported in Table 3. CGRP receptor
antagonism for compounds 13, 16, 17, 18 and 19 (Table
2) was determined by measuring the formation of cyclic
AMP in SK-N-MC cells, incubated with CGRP alone or
in the presence of antagonist as depicted for compound
19 in Figure 1.
a (a) Diethylaminotrimethylsilane, 100 °C; (b) R1CO2H, 4-
methylmorpholine, isobutyl chloroformate, THF, -15 to -20 °C;
(c) chlorotrimethylsilane, 4-methylmorpholine, CH2Cl2, room tem-
perature; (d) 30, 4-methylmorpholine, isobutyl chloro-formate,
THF, -15 °C; (e) R2H, 4-methylmorpholine, isobutyl chloroformate,
THF, room temperature; (f) TFA, CH2Cl2, room temperature; (g)
HBr, AcOH, anisole, room temperature; (h) R2H, HOBt, DCC,
DMF/THF, room temperature.
(NT), amino acid-1 (AA1), amino acid-2 (AA2), C-termi-
nus (CT) (Chart I).
Chart 1
Discussion
SAR studies thereafter were based on the assumption
that the contribution of each structural fragment toward
binding affinity was additive. Accordingly, any change
in receptor affinity induced by variation of one fragment,
while keeping all others constant, was taken as a hint
for the significance of this fragment for receptor interac-
tion. Analysis of the C-terminal part comprising com-
pounds 2, 6, 7, 8, and 9 showed quite interesting results.
Despite a large degree of freedom as indicated by
comparable binding affinities of 2 (IC50 40 000 nM) and
The first step of lead optimization strategy was to
screen selected compounds within the corporate com-
pound pool being structurally related to the (R)-Tyr-(S)-
Lys dipeptide-like leads 1 and 2. Compounds 6 to 10
identified by this procedure exhibited CGRP receptor
affinities between 10 300 nM and > 300 000 nM, as
shown in Table 1. To assess the significance of the
individual structural fragments, the lead compounds
were arbitrarily divided into four parts: N-terminus

Small Molecule CGRP Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5925
Scheme 7a
Table 3. Species-Selective CGRP Receptor Binding of
Compounds 13, 18, and 19a
human CGRP
rat CGRP
marmoset CGRP
receptor affinity, receptor affinity, receptor affinity,
compd
IC50 (nM)a
IC50 (nM)b
IC50 (nM)c
13
18
19
200
0.2
0.03
70500
56.7
6.4
n.d.d
2.7
0.06
Binding affinity was determined with SK-N-MC cells expressing
the human CGRP receptor,a rat spleen preparations,b or marmoset
spleen preparations.c 125I-CGRP was used as the radioligand and
was incubated with increasing concentrations of the displacing
compound. The IC50 values were obtained by nonlinear regression
analysis on the basis of a one binding site model. Data reported
are derived from triplicate wells and three independent experi-
ments. Mean IC50 values were determined from 10-point, one-third
log concentration-response curves. d Not determined.
a (a) NaNCO, CF3CO2H, toluene, room temperature; (b) H2,
Pd(OH)2 (Pearlman’s catalyst), methanol, 50 °C.
Scheme 8a
a (a) 4-Amino-1-(1,1-dimethylethoxycarbonyl)piperidine, anhy-
drous sodium acetate, CH2Cl2, room temperature; (b) NaNCO,
glacial acetic acid/water, room temperature; (c) TFA, CH2Cl2, room
temperature.
Table 2. pA2 and IC50 Values of Selected Compounds
a
compd
pA2
IC50 (nM)b
13
16
17
18
19
6.9
8.0
200
44
4.7
0.2
0.03
Figure 1. Concentration response curve for h-RCGRP in
human SK-N-MC cells in the absence or presence of increasing
concentrations of compound 19. Values are mean of three
separate experiments performed in triplicate. Inset: Schild
plot analysis (determination of pA2).
9.1
10.1
11.1
a CGRP receptor antagonism was determined by measuring the
formation of cyclic AMP in SK-N-MC cells. Cells were incubated
with CGRP alone or in the presence of at least four concentrations
of antagonist. pA2 values were determined by means of Schild-
Plot analysis. The values are the mean of three individual
experiments performed in triplicate. b Binding affinity was deter-
mined with SK-N-MC cells expressing the human CGRP-receptor
using 125I-CGRP as the radioligand. Data reported is derived from
triplicate wells and three independent experiments. Mean IC50
values were determined from 10-point, one-third log concentra-
tion-response curves.
from a comparison of 1 (IC50 17 000 nM) with 11 (IC50
13 000 nM). Replacement of the 3-phenylpropyl group
in 1 with a (2-methoxyphenyl)ethylamino moiety (11),
thereby creating a rigid urea bridge into the side chain
seemed to be also acceptable for hCGRP receptor
affinity. Finally, as 1 (X ) OH) showed a 2-fold prefer-
ence in binding affinity over compound 2 (X ) NH2),
we decided to perform the next synthetic steps exclu-
sively with derivatives containing the 3,5-dibromo-(R)-
Tyr moiety.
6 (IC50 50 000 nM), the presence of an aromatic system
at a certain distance to (S)-Lys seemed not to be
On the basis of our hypothesis that the binding
affinity is the sum of individual fragment contributions,
we envisaged that combining the optimal elements
identified so far would lead to compounds with consider-
ably improved binding affinity, which indeed proved to
be the case. Modification of 1 by replacing the NT with
the (2-methoxyphenyl)ethylamino group and the CT
with the 4-(4-pyridinyl)piperazine moiety led to the
identification of 12 (IC50 1000 nM) that exhibited a 17-
fold improved affinity toward the human CGRP recep-
tor.
Having substantiated our hypothesis, we decided to
focus next on the structural modification of the N-
terminus leaving AA1, AA2, and CT unchanged.
In this context the presence of an urea bridge in 12
seemed to be more favorable than a carboxamide moiety
present in 1 to 10. We performed a variation of the chain
sufficient for receptor recognition, since 7 (IC50
>
300 000 nM) showed no hCGRP receptor binding at all.
In contrast, the incorporation of a rigidifying element
bearing a negatively polarized group at the end of the
aromatic fragment such as the 4-(4-pyridinyl)-piper-
azine moiety of 9 (IC50 10 300 nM) seemed to be very
suitable for a favorable interaction with the CGRP
receptor, accounting for the more than 30-fold improved
receptor affinity as compared to the 4-phenylpiperazine
derivative 7 (IC50 > 300 000 nM).
For the establishment of SAR in the N-terminal part
a comparison of compounds 10 and 11 with our lead 2
proved also interesting. The binding affinities of 2 (IC50
40 000 nM) and 10 (IC50 196 000 nM) differed by a factor
of 5 indicating that the presence of an aromatic system
at a certain distance to the dibromo-Tyr seemed to be
important for receptor recognition. Another clue came

5926 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Rudolf et al.
length between the distal N-atom of the urea bridge and
the phenyl group as well as variation of the aromatic
substitution pattern. Variation of the chain length
showed no improvement in activity (data not shown),
whereas a shift of the methoxy group from the ortho to
the meta position led to the discovery of compound 13
(IC50 200 nM) exhibiting a 5-fold increase in hCGRP
receptor affinity.
and indeed, 19 (IC50 0.03 nM) and 20 (IC50 0.05 nM)
exhibited a significant increase in affinity. In fact,
compound 19 (BIBN4096) possesses higher affinity for
the human CGRP receptor than the endogenous ligand
CGRP and 150-fold higher affinity compared to the
peptidic antagonist CGRP8-37
.
SAR established during the described optimization
work constituted a platform from which the design and
synthesis of novel CGRP antagonists of reduced size and
structural complexity could begin. This work already
disclosed in a series of patent applications10 will be
published soon.
Submicromolar compound 13 was used to address two
important questions. First, whether the compounds
obtained so far were hCGRP antagonists devoid of
agonistic activity and second whether there was species
dependent CGRP receptor binding. Compound 13 proved
to be a competitive antagonist without any intrinsic
agonistic activity exhibiting a pA2 value of 6.9 (Table
2). Furthermore, 13 displayed a 350-fold higher affinity
to human CGRP receptor compared to those of rat (rat
spleen, IC50 70 500 nM; Table 3). Next we reasoned that
binding affinity could be further improved by introduc-
ing conformational constraints in the flexible linear
linker chain between the distal N-atom of the urea
bridge and the phenyl group at the end of this fragment.
Replacement of the linear ethylene chain by a rigidifying
piperazinyl or piperidine group yielded 14 (IC50 226 nM)
and 15 (IC50 250 nM), which showed similar binding
affinities as 13 and clearly indicated that the proximal
NH group of the urea bridge was not essential for
receptor binding. The existence of bulk tolerance in the
spatial vicinity of the restraint linker and the aromatic
system opened interesting possibilities for further de-
sign efforts, and we next extended the type of substit-
uents in the 4-position of the piperidinyl ring to an
aromatic system that was incorporated into a bicyclic
ring system, as exemplified by a 2(3H)-benzoxazolone
system (16, IC50 44 nM, pA2 ) 8.0; Table 2), showing a
5-fold increase in affinity. Next we investigated the
effect of a hydrogen bond donor group such as an
aminocarbonyl system on affinity. This idea was real-
ized in compound 17, which showed high binding (IC50
4.7 nM, pA2 ) 9.1; Table 2) emphasizing the importance
of this region for receptor binding. On the basis of the
results of 16 and 17, we next synthesized the benz-
imidazolidinone 18 in which the NH hydrogen donor
system was fixed into a planar system. Compound 18
exhibited a hCGRP receptor affinity in the subnano-
molar range (IC50 0.2 nM) and represented a break-
through in our work.
In view of the favorable binding data of this compound
its pharmacological and physicochemical properties
have been examined in detail and will be published
separately. The results so far obtained demonstrate that
18 is a high affinity and selective hCGRP antagonist
(IC50 0.2 nM, pA2 ) 10.1; Table 2); the excellent water
solubility of some of its salts makes it an ideally suited
tool for pharmacological and biological investigations.
Next, we investigated whether the spatial positioning
of the aryl part of the benzimidazolone system could be
further optimized. To get a better insight into this
problem a set of benzimidazolone-like structures were
designed. Comparing energy-minimized partial struc-
tures with the benzimidazolone system indicated that
3,4-dihydro-2(1H)-quinazolinone and 1,3-dihydro-4-
phenyl-2(2H)-imidazolone systems display the putative
key interaction points in a similar spatial arrangement,
In view of its outstanding binding properties, com-
pound 19 was selected for further comprehensive phar-
macological, ADME, physicochemical and toxicological
evaluations. A detailed report about the basic pharma-
cology of this compound has been recently published.11
The results clearly show that 19 is a pure antagonist
(pA2 ) 11.1; Figure 1) for the hCGRP receptor and is
selective against a broad panel of receptors and en-
zymes. Moreover, 19 reverses CGRP-mediated vasodi-
lation in human cerebral vessels and inhibits neurogenic
vasodilation in a surrogate animal model of migraine
pathophysiology.11 On the basis of these encouraging
results, 19 has been selected for clinical investigations.
As oral bioavailability of 19 proved to be rather low in
animal experiments (Fpo (rat, dog) < 1%),12 proof of
concept studies have been performed by intravenous
administration.
The outcome of clinical phase I and phase II studies
with 19 has recently been published.13,14 The clinical
data clearly proved that 19 is efficacious for the treat-
ment of migraine headache and thus establishes that
CGRP antagonism is a safe and novel approach for the
treatment of acute migraine headache.
Conclusion
A high throughput screening effort provided di-
peptide-like compounds which displayed weak but un-
equivocal inhibition of binding to the hCGRP receptor.
These compounds, characterized by a 3,5-dibromo-(R)-
Tyr-(S)-Lys dipeptide fragment as the central pharma-
cophore, were used to design inhibitors with higher
affinity to the receptor. Our strategy was to leave the
dipeptidic scaffold unchanged and concentrate mainly
on the C- and N-terminal parts of these molecules. The
introduction of rigidifying structural elements into both
terminal fragments together with an exact spatial
positioning of the decisive pharmacophoric groups led
to highly potent hCGRP antagonists, the prototype
being compound 19 (Chart 2). Compound 19 reverses
CGRP-induced vasodilation in human cerebral vessels
and has proved active in an animal model related to
migraine. In view of these favorable results, 19 was
selected for clinical trials. In phase I studies following
single intravenous administration, 19 shows good toler-
ability and appears to be safe.13 In a proof of concept
study, performed in a multicenter, double-blind, ran-
domized clinical trial, 19 was effective in acute treat-
ment of migraine headache attacks and is without
cardiovascular side effects. 14
In summary, it can be concluded that CGRP plays an
important role in the pathophysiology of migraine and
that CGRP receptor antagonism is a valid approach for

Small Molecule CGRP Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5927
dimethylethoxycarbonyl)-N6-(phenylmethoxycarbonyl)-D-lysine-
2-phenethylamide, prepared analogously to its (S)-enantiomer
in a yield of 96% [Anal. (C27H37N3O5: 483.6) C, H, N], was
treated with TFA as above. The amorphous title compound
(98%) was obtained after evaporation of the solvent and
trituration with ethyl acetate, followed by diethyl ether. Anal.
(C24H30F3N3O5: 497.51) C, H, N.
Chart 2
N2-[N-(1,1-Dimethylethoxycarbonyl)-3,5-dibromo-D-
tyrosyl]-N6-(phenylmethoxycarbonyl)-L-lysine-2-phen-
ethylamide (22a). N-Methylmorpholine (1.20 g, 12 mmol) and
isobutyl chloroformate (1.7 g, 12.5 mmol) were added succes-
sively to a solution of N-[(1,1-dimethylethoxy)carbonyl]-3,5-
dibromo-D-tyrosine (5.2 g, 11.8 mmol) in 300 mL tetrahydro-
furan (THF), while stirring and maintaining a reaction
temperature of -15 °C. After stirring at -15 °C for further
15 min, 21a (4.6 g, 12 mmol) was added, batchwise, and the
mixture was stirred at -15 °C for another 2 h, kept overnight
at room temperature, diluted with 1.5 l of aqueous 0.5 M
potassium hydrogensulfate and extracted exhaustively with
ethyl acetate. The combined organic phases were dried and
concentrated to dryness in vacuo. Column chromatography on
silica, eluant dichloromethane/ethanol 9/1 (v/v), afforded the
desired 22a (7.5 g, 79%). Anal. (C36H44Br2N4O7: 804.57) C, H,
Br, N.
N2-[4-Amino-3,5-dibromo-N-(1,1-dimethylethoxycarbon-
yl)-D-phenylalanyl]-N6-phenylmethoxycarbonyl)-L-lysine-
2-phenethylamide (22b). Synthesized from 4-amino-N-[(1,1-
dimethylethoxy)carbonyl]-3,5-dibromo-D-phenylalanine and 21a
as described above for 22a. Amorphous solid. Yield: 82%. Anal.
(C36H45Br2N5O6: 803.58) C, H, Br, N.
the acute treatment of migraine headache. However,
further clinical studies are required before the true
potential of this novel class of drug can be established.
N2-[N-(1,1-Dimethylethoxycarbonyl)-3,5-dibromo-L-
tyrosyl]-N6-(phenylmethoxycarbonyl)-D-lysine-2-phen-
ethylamide (22c). Synthesized from N-[(1,1-dimethylethoxy)-
carbonyl]-3,5-dibromo-L-tyrosine, TBTU, HOBt, diisopropyl-
ethylamine (DIEA) and 21b as described above. Colorless
crystals. Yield: 94%. ESI-MS: (M + H)+ ) 803/805/807 (Br2);
(M + Na)+ ) 825/827/829 (Br2). Anal. (C36H44Br2N4O7: 804.57)
C, H, Br, N.
Experimental Section
Chemistry. Procedures for the preparation of all final
products are presented below along with representative pro-
cedures for all methods used in the preparation of intermedi-
ates. All solvents and reagents were used without purification
as acquired from commercial sources.
Nuclear magnetic resonance (NMR) spectra were recorded
on Bruker spectrometers at 80 MHz, 200, 400, or 600 MHz
for proton (1H) and 150 MHz for carbon (13C) in the solvent
indicated. Chemical shifts are reported in parts per million
relative to tetramethysilane. Melting points were recorded on
a HWS SG 2000 melting point apparatus and are uncorrected.
Elemental analyses were performed on a Heraeus CHN Rapid
Elemental Analyzer and are within 0.4% of the calculated
values. Mass spectra were recorded on the following instru-
ments, using the stated ionization methods: Finnigan MAT
8230 mass spectrometer, electron impact ionization; Finnigan
TSQ 700 mass spectrometer, electrospray ionization.
N2-[N-(1,1-Dimethylethoxycarbonyl)-3,5-dibromo-D-
tyrosyl]-N6-(phenylmethoxycarbonyl)-D-lysine-2-phen-
ethylamide (22d). Synthesized from N-[(1,1-dimethylethoxy)-
carbonyl]-3,5-dibromo-D-tyrosine, TBTU, HOBt, DIEA and 21b
as described above. Colorless crystals. Yield: 81%. ESI-MS:
(M + H)+ ) 803/805/807 (Br2); (M + Na)+ ) 825/827/829 (Br2).
Anal. (C36H44Br2N4O7: 804.57) C, H, Br, N.
N2-[N-(1,1-Dimethylethoxycarbonyl)-3,5-dibromo-L-
tyrosyl]-N6-(phenylmethoxycarbonyl)-L-lysine-2-phen-
ethylamide (22e). Synthesized from N-[(1,1-dimethylethoxy)-
carbonyl]-3,5-dibromo-L-tyrosine, TBTU, HOBt, DIEA and 21a
as described above. Colorless crystals. Yield: 77%. ESI-MS:
(M + H)+ ) 803/805/807 (Br2); (M + Na)+ ) 825/827/829 (Br2).
Anal. (C36H44Br2N4O7: 804.57) C, H, Br, N.
N6-(Phenylmethoxycarbonyl)-l-lysine-2-phenethyl-
amide (21a). To a mixture of Boc-Lys(Z)-OH (6.0 g, 15.7
mmol), diisopropylethylamine (2.0 g, 16 mmol), TBTU (5.2 g,
16 mmol), HOBt (2.2 g, 15.7 mmol) and 30 mL dimethyl-
formamide (DMF) was added dropwise, with stirring, 2-phen-
ethylamine (1.90 g, 15.7 mmol), dissolved in 13 mL of DMF,
and the mixture was stirred overnight at room temperature.
The solvent was removed in vacuo, and the residue was taken
up in ethyl acetate. The ethyl acetate phase was then washed
three times successively with 20 mL of saturated aqueous
saline solution, dried over sodium sulfate, evaporated down
in vacuo and triturated with diisopropyl ether to afford N2-
(1,1-dimethylethoxycarbonyl)-N6-(phenylmethoxycarbonyl)-L-
lysine-2-phenethylamide (7.0 g, 92%) as colorless crystals.
Anal. (C27H37N3O5: 483.6) C, H, N. To this intermediate,
dissolved in 50 mL of dichloromethane, were added 10 mL of
trifluoroacetic acid (TFA) and the reaction mixture was stirred
2 h at room temperature. After neutralization by addition of
saturated aqueous sodium hydrogencarbonate solution, the
organic phase was dried and evaporated down in vacuo to give
the desired compound as a colorless highly viscous oil (5.5 g,
99%). Anal. (C22H29N3O3: 383.48) C, H, N.
N2-[3,5-Dibromo-N-(4-phenyl-1-oxobutyl)-D-tyrosyl]-L-
lysine-2-phenethylamide Hydrobromide (1). Treatment of
22a with TFA in dichloromethane as described for 21a gave
amorphous N2-[3,5-dibromo-D-tyrosyl]-N6-(phenylmethoxycar-
bonyl)-L-lysine-2-phenethylamide (58%) that was coupled with
benzenebutanoic acid in the presence of isobutyl chloroformate
and N-methylmorpholine as described above to afford colorless
N2-[3,5-dibromo-N-(4-phenyl-1-oxobutyl)-D-tyrosyl]-N6-(phenyl-
methoxycarbonyl)-L-lysine-2-phenethylamide (81%), mp 203-
205 °C. Anal. (C41H46Br2N4O6: 850.65) C, H, Br, N. A mixture
of this intermediate (3.7 g, 4.35 mmol), 25 mL of glacial acetic
acid, 30 mL of a 33% solution of hydrogen bromide in glacial
acetic acid and 5 mL of anisole was stirred overnight at
ambient temperature. The reaction mixture was stirred into
diethyl ether, the solvents were decanted and the sticky
precipitate formed was triturated with water, suction filtered
and dried in a vacuum drier at 50 °C to afford the title
compound as colorless water-insoluble crystals (2.1 g, 61%).
EI-MS: M+ ) 714/716/718 (Br2). Anal. (C33H40Br2N4O4‚HBr:
797.42) C, H, Br, N.
N6-(Phenylmethoxycarbonyl)-d-lysine-2-phenethyl-
amide trifluoroacetate (21b). The intermediate N2-(1,1-
N2-[4-Amino-3,5-dibromo-N-(4-phenyl-1-oxobutyl)-D-
phenylalanyl]-L-lysine-2-phenethylamide Hydrobromide

5928 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Rudolf et al.
(2). The intermediate N2-[4-amino-3,5-dibromo-N-(4-phenyl-
1-oxobutyl)-D-phenylalanyl]-N6-(phenylmethoxycarbonyl)-L-
lysine-2-phenethylamide was prepared from 22b in two steps
as described above (96%), mp 190-192 °C. Anal. (C41H47-
Br2N5O5: 849.65) C, H, Br, N. Treatment with hydrogen
bromide in glacial acetic acid as above gave the desired
hydrobromide 2 (85%). ESI-MS: (M + H)+ ) 714/716/718 (Br2);
(M + Br)- ) 792/794/796/798 (Br3). EI-MS: M+ ) 713/715/
717 (Br2). Anal. (C33H41Br2N5O3‚HBr: 796.43) C, H, Br, N.
drochloride, N-methylmorpholine and isobutyl chloroformate
were reacted as described above to give N2-[4-amino-3,5-
dibromo-N-(3-phenyl-1-oxopropyl)-D-phenylalanyl]-N6-(phenyl-
methoxycarbonyl)-L-lysine methyl ester (92%); mp 183-186
°C Anal. (C33H38Br2N4O6: 746.49) C, H, Br, N. The title
compound was prepared from this intermediate by saponifica-
tion with aq sodium hydroxide solution as described above
(94%); mp 150-153 °C. Anal. (C32H36Br2N4O6: 732.46) C, H,
Br, N.
N2-[3,5-Dibromo-N-(4-phenyl-1-oxobutyl)-L-tyrosyl]-D-
lysine-2-phenethylamide (3). Reaction of 22c with TFA as
above gave N2-[3,5-dibromo-L-tyrosyl]-N6-(phenylmethoxy-
carbonyl)-D-lysine-2-phenethylamide trifluoroacetate (67%)
[Anal. (C33H37Br2F3N4O7: 818.47) C, H, Br, N], which was
transformed with benzenebutanoic acid, TBTU and HOBt
to colorless crystals of N2-[3,5-dibromo-N-(4-phenyl-1-oxo-
butyl)-L-tyrosyl]-N6-(phenylmethoxycarbonyl)-D-lysine-2-phen-
ethylamide (50%). ESI-MS: (M + Na)+ ) 871/873/875 (Br2).
Anal. (C41H46Br2N4O6: 850.64) C, H, Br, N. Removal of the
benzyloxycarbonyl group by hydrogen bromide in glacial acetic
acid as above and final treatment with base gave the title
compound 3 (39%). Anal. (C33H40Br2N4O4: 716.5) C, H, Br, N.
N2-[4-Amino-3,5-dibromo-N-(3-phenyl-1-oxopropyl)-D-
phenylalanyl]-L-lysine-2-phenethylamide Hydrochloride
(10). The acid 25 was reacted with 2-phenethylamine in the
presence of N-methylmorpholine and isobutyl chloroformate
as described above to afford N2-[4-amino-3,5-dibromo-N-
(3-phenyl-1-oxopropyl)-D-phenylalanyl]-N6-(phenylmethoxycar-
bonyl)-L-lysine-2-phenethylamide (81%); mp 232-234 °C. Anal.
(C40H45Br2N5O5: 835.62) C, H, Br, N. This intermediate was
treated with a solution of hydrogen bromide in acetic acid as
described above; the obtained hydrobromide was converted via
its base into the desired title hydrochloride (89%); amorphous
solid. ESI-MS: (M + H)+ ) 700/702/704 (Br2). EI-MS: M+
)
699/701/703 (Br2). Anal. (C32H40Br2ClN5O3: 737.95) C, H, Br,
Cl, N.
N2-[3,5-Dibromo-N-(4-phenyl-1-oxobutyl)-D-tyrosyl]-D-
lysine-2-phenethylamide (4). Starting from 22d, the title
compound was synthesized following the procedures described
above. Anal. (Calcd for C33H40Br2N4O4: 716.5) C, H, Br, N.
N2-[3,5-Dibromo-N-(4-phenyl-1-oxobutyl)-L-tyrosyl]-L-
lysine-2-phenethylamide (5). Starting from 22e, the title
compound was synthesized following the procedures described
above. Anal. (Calcd for C33H40Br2N4O4: 716.5) C, H, Br, N.
1-[N6-(Phenylmethoxycarbonyl)]-L-lysyl]-4-(4-pyridinyl)-
piperazine (21c). Starting from Boc-Lys(Z)-OH and 1-(4-
pyridinyl)-piperazine, the title compound was synthesized in
two steps as described above for 21a and 21b. Anal.
(C23H31N5O3: 425.52) C, H, N.
1-[N2-(3,5-Dibromo-D-tyrosyl)-N6-(phenylmethoxycar-
bonyl)-L-lysyl]-4-(4-pyridinyl)piperazine (26). As described
above for 21a, the title compound was prepared by coupling
of 21c with N-[(1,1-dimethylethoxycarbonyl]-3,5-dibromo-D-
tyrosine, followed by treatment with TFA. Anal. (C32H38-
Br2N6O5: 746.49) C, H, Br, N.
(R)-4-Amino-3,5-dibromophenylalanine Methyl Ester
Hydrochloride (23). To a solution of d-4-aminophenylalanine
hydrate (125.0 g, 0.631 mol) in a mixture of 2.1 l of glacial
acetic acid, 300 mL of water and 2 mL of concentrated
hydrobromic acid a solution of anhydrous bromine (64.2 mL,
1.261 mol) in 100 mL of glacial acetic acid was added dropwise,
with stirring and while maintaining a reaction temperature
of 20 to 25 °C. The mixture was stirred for 1 h at ambient
temperature, diluted with 2.2 l of water, and adjusted to pH
5 by addition of ca. 2.2 l of 10 N aq sodium hydroxide solution,
while cooling with a mixture of crushed ice and methanol. The
precipitated crystals were suction filtered, thoroughly washed
with water and dried in vacuo to afford the intermediate (R)-
4-amino-3,5-dibromophenylalanine (171.2 g, 80%); mp 265-
266 °C (decomposition). To -10 °C cold anhydrous methanol
(600 mL) was added dropwise thionyl chloride (27.4 mL, 0.377
mol), while maintaining a reaction temperature below -5 °C,
and the resulting solution combined batchwise with the above
intermediate (85 g, 0.251 mol). The mixture was stirred for a
further 90 min at 50 °C and overnight at room temperature,
then evaporated down in vacuo and the residue dissolved in
500 mL of hot methanol. After cooling, 1.5 L of diethyl ether
was added with stirring. The resulting precipitate was suction
filtered and dried in vacuo to yield the title compound (82.9 g,
85%), mp 223-224 °C (decomposition). Anal. (C10H13Br2-
ClN2O2: 388.48) C, H, Br, N.
1-[N2-[3,5-Dibromo-N-[[[2-(2-methoxyphenyl)ethyl]-
amino]carbonyl]-D-tyrosyl]-L-lysyl]-4-(4-pyridinyl)piperazine
(12). To a solution of 26 (1.0 g, 1.34 mmol) in 80 mL of
tetrahydrofuran (THF) was added 2-methoxyphenethyl iso-
cyanate (0.28 g 1.6 mmol), and the mixture was stirred for 3
days at room temperature. The reaction mixture was evapo-
rated down in vacuo, and the residue was purified by column
chromatography (MN-silica gel 60, Macherey-Nagel, 70-230
mesh ASTM, eluant: dichloromethane/methanol/cyclohexane/
ammonia ) 350/75/75/10 (v/v/v/v)). 0.5 g (40%) of a colorless
amorphous solid was obtained. ESI-MS: (M + H)+ ) 922/924/
926 (Br2). Anal. (C42H49Br2N7O7: 923.69) C, H, Br, N. This
intermediate (0.5 g, 0.54 mmol), presumably 1-[N2-[3,5-
dibromo-N-[[[2-(2-methoxyphenyl)ethyl]-amino]carbonyl]-D-
tyrosyl]-N6-(phenylmethoxycarbonyl)-L-lysyl]-4-(4-pyridinyl)-
piperazine, was treated with hydrogen bromide in glacial acetic
acid as described above for 22a and converted to its base in
the usual manner to yield the title compound 12 as a colorless,
amorphous solid (0.2 g, 47%). ESI-MS: (M + H)+ ) 788/790/
792 (Br2); (M + 2H)2+ ) 394/395/396 (Br2). Anal. (C34H43-
Br2N7O5: 789.56) C, H, Br, N.
1-[N2-[3,5-Dibromo-N-[[[2-(3-methoxyphenyl)ethyl]-
amino]carbonyl]-D-tyrosyl]-L-lysyl]-4-(4-pyridinyl)piperazine
(13). A tetrahydrofuran solution (50 mL) of 26 (1.0 g, 1.34
mmol) was added dropwise over a period of 60 min to a
suspension of CDT (0.22 g, 1.34 mmol) in THF (30 mL) while
cooling to -5 °C and stirring. The reaction mixture was then
stirred for 3 h at ambient temperature and mixed with a
solution of 3-methoxybenzeneethanamine (0.20 g, 1.34 mmol)
in THF (5 mL). Then the mixture was refluxed for 2 h and
stirred overnight at ambient temperature. The reaction mix-
ture was evaporated down in vacuo. Column chromatography
of the residue on silica, eluant dichloromethane/cyclohexane/
methanol/ammonia 7/1.5/1.5/0.2 (v/v/v/v), afforded 0.5 g (40%)
of 1-[N2-[3,5-dibromo-N-[[[2-(3-methoxyphenyl)ethyl]amino]-
carbonyl]-D-tyrosyl]-N6-(phenylmethoxycarbonyl)-L-lysyl]-4-(4-
pyridinyl)piperazine as an amorphous foam. ESI-MS: (M +
H)+ ) 922/924/926 (Br2); (M + Na)+ ) 944/946/948 (Br2). Anal.
(C42H49Br2N7O7: 923.69) C, H, Br, N. This intermediate (0.5
g, 0.54 mmol) was treated with hydrogen bromide in glacial
4-Amino-3,5-dibromo-N-(3-phenyl-1-oxopropyl)-D-phenyl-
alanine (24a). The intermediate 4-amino-3,5-dibromo-N-(3-
phenyl-1-oxopropyl)-D-phenylalanine methyl ester was pre-
pared from 23, benzenepropanoic acid, N-methylmorpholine
and isobutyl chloroformate by the method described above: mp
155-156 °C. Yield: 95%. A mixture of this intermediate (46.0
g, 0.095 mol), 700 mL of THF and 48 mL 2 N aq sodium
hydroxide solution was stirred for 3 h at room temperature,
then adjusted to pH 2.5 by addition of 0.5 M aq potassium
hydrogensulfate solution and extracted twice with ethyl
acetate. The combined ethyl acetate phases were dried and
evaporated down. The residue was triturated with diethyl
ether, suction filtered, washed with ether and dried in a
circulating air drier to give the title compound (40.3 g, 90%);
mp 148-150 °C. Anal. (C18H18Br2N2O3: 470.16) C, H, Br, N.
N2-[4-Amino-3,5-dibromo-N-(3-phenyl-1-oxopropyl)-D-
phenylalanyl]-N6-(phenylmethoxycarbonyl)-L-lysine (25).
24a, N6-(phenylmethoxycarbonyl)-L-lysine methyl ester hy-

Small Molecule CGRP Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5929
acetic acid as described above for 22a and converted to its base
in the usual manner to give the title compound 13 as a
colorless, amorphous solid (0.42 g, 99%). ESI-MS: (M + H)+
) 788/790/792 (Br2); (M + 2H)2+ ) 395.7. Anal. (C34H43-
Br2N7O5: 789.56) C, H, Br, N.
(M + 2H)2+ ) 415/416/417. Anal. (C36H46Br2N8O5: 830.61) C,
H, Br, N. Instead of methanolic hydrogen chloride solution,
TFA in dichloromethane may be used to split off the tert-
butoxycarbonyl group.
1-[N2-[3,5-Dibromo-N-[[4-(2-methoxyphenyl)-1-piper-
idinyl]carbonyl]-D-tyrosyl]-L-lysyl]-4-(4-pyridinyl)piperazine
Bis(trifluoroacetate) (15). As described for compound 14,
the title compound was prepared from 4-(2-methoxyphenyl)-
piperidine, CDT, and 29. ESI-MS: (M + H)+ ) 828/830/832
(Br2); (M + 2H)2+ ) 414/415/416.7. Anal. (Calcd for C41H49-
Br2F6N7O9: 1057.67) C, H, Br, N.
3,5-Dibromo-N-[[[2-(2-methoxyphenyl)ethyl]amino]car-
bonyl]-D-tyrosine (27). To a suspension of ethyl 3,5-dibromo-
D-tyrosinate hydrobromide (20.0 g, 44.65 mmol) in THF (300
mL) was added successively triethylamine (4.52 g, 44.65 mmol)
and the solution of 2-methoxyphenethyl isocyanate (8.0 g, 53.7
mmol) in THF (50 mL), and the mixture was stirred overnight
at ambient temperature. The precipitate was filtered off, the
filtrate was evaporated down in vacuo and the residue was
added to 2 N aq sodium hydroxide solution (200 mL, 400 mmol)
and stirred overnight. The reaction mixture was diluted with
water (200 mL) and extracted four times with 100 mL of ethyl
acetate each. The aqueous phase was acidified to pH 6 with
diluted aq hydrochloric acid and the resulting colorless pre-
cipitate dried in a circulating air drier at 60 °C to afford the
title compound (17.0 g, 74%) as colorless crystals; mp >196
°C (dec). ESI-MS: (M - H)- ) 513/515/517 (Br2). Anal. (C19H20-
Br2N2O5: 516.18) C, H, Br, N.
1-[N2-[3,5-Dibromo-N-[[4-[2(3H)-oxobenzoxazol-3-yl]-1-
piperidinyl]carbonyl]-D-tyrosyl]-L-lysyl]-4-(4-pyridinyl)-
piperazine Bis(trifluoroacetate) (16). As described for
compound 14, the title compound was prepared from 4-[2(3H)-
oxobenzoxazol-3-yl]piperidine, CDT, and 29. ESI-MS: (M +
H)+ ) 855/857/859 (Br2); (M + 2H)2+ ) 428/429/430. Anal.
(C41H46Br2N8O10: 1084.65) C, H, Br, N.
1-[N2-[N-[[4-[N-(Aminocarbonyl)phenylamino]-1-piper-
idinyl]carbonyl]-3,5-dibromo-D-tyrosyl]-L-lysyl]-4-(4-pyrid-
inyl)piperazine Bis(trifluoroacetate) (17). As described for
compound 14, the title compound was prepared from 4-[N-
(aminocarbonyl)-phenylamino]piperidine, CDT, and 29. ESI-
MS: (M + H)+ ) 856/858/860 (Br2); (M + 2H)2+ ) 428/429/
430.6 (Br2). Anal. (C41H49Br2F6N9O9: 1085.68) C, H, Br, N.
1-[N2-[3,5-Dibromo-N-[[4-(1,3-dihydro-2(2H)-oxobenz-
imidazol-1-yl)-1-piperidinyl]carbonyl]-D-tyrosyl]-L-lysyl]-
4-(4-pyridinyl)piperazine (18). As described for compound
14, the title compound was prepared from 4-(1,3-dihydro-
2(2H)-oxobenzimidazol-1-yl)piperidine, CDT, and 29. ESI-
MS: (M + H)+ ) 854/856/858 (Br2); (M + 2H)2+ ) 427/428/
429.6. Anal. (C37H45Br2N9O5: 855.62) C, H, Br, N.
N2-[3,5-Dibromo-N-[[[2-(2-methoxyphenyl)ethyl]amino]-
carbonyl]-D-tyrosyl]-L-lysine-2-phenethylamide
(11).
21a and 27 were reacted as described above for 21a to give
N2-[3,5-dibromo-N-[[[2-(2-methoxyphenyl)ethyl]amino]carbon-
yl]-D-tyrosyl]-N6-(phenylmethoxycarbonyl)-L-lysine-2-phenethyl-
amide Anal. (C41H47Br2N5O7: 881.65) C, H, Br, N. This
intermediate was treated with hydrogen bromide in glacial
acetic acid as described above for 22a and converted to its base
in the usual manner to give the title compound 11 as a
colorless, amorphous solid. ESI-MS: (M + H)+ ) 746/748/750
(Br2). Anal. (C33H41Br2N5O5: 747.52) C, H, Br, N.
1-[N2-[3,5-Dibromo-N-[[4-(3,4-dihydro-2(1H)-oxoquinazo-
lin-3-yl)-1-piperidinyl]carbonyl]-D-tyrosyl]-L-lysyl]-4-(4-
pyridinyl)piperazine (19). As described for compound 14,
the title compound was prepared from 4-(3,4-dihydro-2(1H)-
oxoquinazolin-3-yl)piperidine, CDT, and 29. ESI-MS: (M + H)+
) 868/870/872 (Br2); (M + 2H)2+ ) 434/435/436.7. Anal.
(C38H47Br2N9O5: 869.65) C, H, Br, N.
1-[N6-(1,1-Dimethylethoxycarbonyl)-L-lysyl]-4-(4-
pyridinyl)piperazine (28). A solution of intermediate 1-[N6-
(1,1-dimethylethoxycarbonyl)-N2-(phenylmethoxycarbonyl)-L-
lysyl]-4-(4-pyridinyl)piperazine (120 g, 0.2283 mol) [EI-MS: M+
) 525. Anal. (C28H39N5O5: 525.64) C, H, N], prepared from
Z-Lys(Boc)-OH and 1-(4-pyridinyl)piperazine as described
above for 21a, in a mixture of methanol (1 L) and 1 M aq
potassium hydrogensulfate (240 mL) was hydrogenated in the
presence of 10% palladium/charcoal (30 g) at room temperature
and 3 bar of hydrogen pressure until the uptake of hydrogen
had ceased. After working up in the usual way the title
compound 28 was obtained as a colorless oil; 87.0 g, 97%. Anal.
(C20H33N5O3: 391.51) C, H, N.
1-[N2-[3,5-Dibromo-N-[[4-(1,3-dihydro-4-phenyl-2(2H)-
oxoimidazol-1-yl)-1-piperidinyl]carbonyl]-D-tyrosyl]-L-
lysyl]-4-(4-pyridinyl)piperazine (20). As described for com-
pound 14, the title compound was prepared from 4-(1,3-
dihydro-4-phenyl-2(2H)-oxoimidazol-1-yl)piperidine, CDT, and
29. ESI-MS: (M + H)+ ) 880/882/884 (Br2); (M + 2H)2+
)
440.5/441.5/442.5. Anal. (C39H47Br2N9O5: 881.66) C, H, Br, N.
4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-phenyl-
alanine (30). A suspension of 4-amino-3,5-dibromo-D-phenyl-
alanine (138.4 g, 0.41 mol) in diethylaminotrimethylsilane (250
mL) was heated to ca. 75 °C, while the diethylamine being
formed was distilled off. When the theoretical amount of
diethylamine had been collected, the reaction was interrupted
and the surplus of diethylaminotrimethylsilane was removed
using a vacuum rotatory evaporator. The residue was taken
up in THF (250 mL) to give reaction mixture A. To a solution
of benzenebutanoic acid (67.2 g, 0.41 mol) in dichloromethane
(650 mL) were added dropwise isobutyl chloroformate (53,2
mL, 0.41 mol) and N-methylmorpholine (45 mL, 0.41 mol),
while cooling to -15 to -20 °C. The mixture was kept at this
temperature for another 3 h. Then reaction mixture A was
poured into this solution drop by drop while stirring and
maintaining the same temperature. After stirring at -15 °C
for a further 2 h, the mixture was allowed to come back slowly
to room temperature. The precipitate was filtered off, and the
filtrate concentrated in vacuo. The residue was taken up in
dichloromethane (500 mL) and stirred into aq concentrated
potassium hydrogensulfate solution. The formed crystalline
product was suction filtered, washed twice with acetone and
air-dried to give 134 g (68%) of the title compound; mp 214
°C. Anal. (C19H20Br2N2O3: 484.18) C, H, Br, N.
1-[N2-(3,5-Dibromo-D-tyrosyl)-N6-(1,1-dimethylethoxy-
carbonyl)-L-lysyl]-4-(4-pyridinyl)piperazine (29). As
described above for 21a, N-(9-fluorenylmethoxycarbonyl)-
3,5-dibromo-D-tyrosine was coupled with compound 28 to
give 1-[N2-[3,5-dibromo-N-(9-fluorenylmethoxycarbonyl)-D-
tyrosyl]-N6-(1,1-dimethylethoxycarbonyl)-L-lysyl]-4-(4-pyrid-
inyl)piperazine. A mixture of this intermediate (50 g, 53.5
mmol) and 300 mL of diethylamine was heated to 60 °C with
stirring; 100 mL of methanol were added and stirring was
continued for a further 5 h at 60 °C. The reaction mixture was
evaporated down in vacuo and the residue was purified by
chromatography (silica gel, ethyl acetate/methanol 6/4 (v/v))
to afford compound 29 as a colorless foam (26 g, 68%). EI-
MS: M+ ) 710/712/714 (Br2). Anal. (C29H40Br2N6O5: 712.47)
C, H, Br, N.
1-[N2-[3,5-Dibromo-N-[[4-(2-methoxyphenyl)-1-piper-
azinyl]carbonyl]-D-tyrosyl]-L-lysyl]-4-(4-pyridinyl)piper-
azine (14). Analogously to the method described above for
compound 13, 1-(2-methoxyphenyl)piperazine, CDT, and 29
were transformed to 1-[N2-[3,5-dibromo-N-[[4-(2-methoxy-
phenyl)-1-piperazinyl]carbonyl]-D-tyrosyl]-N6-(1,1-dimethyl-
ethoxycarbonyl)-L-lysyl]-4-(4-pyridinyl)piperazine. To this in-
termediate (1.35 g, 1.45 mmol) was added methanol saturated
with hydrogen chloride (30 mL), and the mixture was stirred
overnight at ambient temperature, then evaporated down in
vacuo and the residue purified by chromatography (silica gel,
ethyl acetate/methanol/ammonia 7/3/0.5 (v/v/v)) to give the title
compound 14 (41%). ESI-MS: (M + H)+ ) 829/831/833 (Br2);
N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-
phenylalanyl]-N6-(phenylmethoxycarbonyl)-L-lysine (31).
To a suspension of H-Lys(Z)-OH (16.8 g, 0.06 mol) in dichloro-
methane (150 mL) were added dropwise, while cooling with

5930 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19
Rudolf et al.
ice-water, trimethylchlorosilane (13.1 g, 0.12 mol) and N-
methylmorpholine (12.1 g, 0.12 mol). This mixture A was
stirred at ambient temperature for a further 12 h. A solution
of 30 (29.0 g, 0.06 mol) in THF (300 mL) was cooled to -15
°C, cautiously admixed with isobutyl chloroformate (8.2 g, 0.06
mol) and N-methylmorpholine (0.06 mol) and kept at the same
temperature for 30 min. To this solution was added slowly,
while maintaining a reaction temperature of ca. -15 °C, the
above mixture A. Stirring at this temperature was continued
for 2 h, whereupon the mixture was allowed to come back to
room temperature, then poured into cold aq 0.5 M potassium
hydrogensulfate solution (2 L) and exhaustively extracted with
dichloromethane. The combined organic extracts were dried
over sodium sulfate and concentrated in vacuo to give 37.0 g
(87%) of the title compound; mp 168-172 °C. Anal. (C33H38-
Br2N4O6: 746.49) C, H, Br, N.
The remaining colorless crystals (2.92 g, 34%) were identified
to be 4-[N-(aminocarbonyl)phenylamino]-1-phenylmethyl-
piperidine; mp 158-162 °C. ESI-MS: (M + H)+ ) 310. Anal.
(C19H23N3O: 309.41) C, H, N. A solution of this intermediate
(13.0 g, 0.042 mol) in methanol (150 mL) was hydrogenated
at 50 °C and in the presence of palladium(II) hydroxide
[Pearlman’s catalyst] (12 g). When the hydrogen uptake had
ended, the catalyst was removed and the filtrate concentrated
in vacuo to give after trituration with diethyl ether the desired
title compound as colorless crystals (9.2 g, 100%). ESI-MS: (M
+ H)+ ) 220. Anal. (C12H17N3O: 219.28) C, H, N.
4-(1,3-Dihydro-2(2H)-oxo-4-phenyl-imidazol-1-yl)pi-
peridine Trifluoroacetate (34). To a solution of 1-(1,1-
dimethylethoxycarbonyl)-4-piperidinammonium hydrogen-
carbonate (55.794 g, 0.213 mol) and diisopropylethylamine
(87.365 mL, 0.5 mol) in dichloromethane (500 mL) was added
dropwise, while stirring, phenacyl bromide (50.00 g, 0.251 mol).
After stirring a further 7 h at ambient temperature, sodium
cyanate (28.665 g, 0.441 mol) was added. The mixture was
acidified with acetic acid, while cooling with a mixture of
crushed ice and water, and stirred overnight at ca. 0 °C,
whereupon the mixture was allowed to come back to room
temperature, thoroughly washed with water, dried over so-
dium sulfate and concentrated in vacuo. The residue was
triturated with ether, suction filtered and dried at ambient
temperature in a circulating air drier to afford the intermedi-
ate 4-(1,3-dihydro-2(2H)-oxo-4-phenyl-imidazol-1-yl)-1-(1,1-
dimethylethoxycarbonyl)piperidine (51.0 g, 70%). EI-MS: M+
) 343. Anal. (C19H25N3O3: 343.19) C, H, N. This was converted
into the title compound following the procedure described
above for 21a. Anal. (C16H18F3N3O3: 357.33) C, H, N.
Determination of Binding Affinity with SK-N-MC
Cells Expressing Human CGRP-Receptor. SK-N-MC cells
were cultivated in Dulbecco’s modified Eagle Medium. The
medium of confluent cultures was removed. The cells were
washed twice with PBS-buffer (Gibco 041-04190 M), detached
by the addition of PBS-buffer, mixed with 0.02% EDTA, and
isolated by centrifuging. After resuspension in 20 mL of
Balanced Salts Solution [BSS (in mM): NaCl 120, KCl 5.4,
NaHCO3 16.2, MgSO4 0.8, NaHPO4 1.0, CaCl2 1.8, D-glucose
5.5, HEPES 30, pH 7.40] the cells were centrifuged twice at
100g and resuspended in BSS. After the cell number had been
determined, the cells were homogenized using an Ultra-Turrax
and centrifuged for 10 min at 3000g. The supernatant was
discarded and the pellet was recentrifuged, resuspended (1 mL/
1 000 000 cells) in Tris buffer (10 mM Tris, 50 mM NaCl, 5
mM MgCl2, 1 mM EDTA, pH 7.40) and enriched with 1%
bovine serum albumine and 0.1% bacitracine. The homogenate
was frozen at -80 °C. The membrane preparations were stable
for more than 6 weeks under these conditions.
After thawing, the homogenate was diluted 1:10 with assay
buffer (50 mM Tris, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA,
pH 7.40) and homogenized for 30 s with an Ultra-Turrax. 230
µL of the homogenate was incubated at ambient temperature
for 180 min with 50 pM of 125I-iodotyrosyl-calcitonin-gene-
related peptide (Amersham) and increasing concentrations of
the test substances in a total volume of 250 µL. Incubation
was ended by rapid filtration using GF/B-glass fiber filters
treated with polyethyleneimine (0.1%) by means of a cell
harvester. The protein-bound radioactivity was measured
using a gamma counter. The nonspecific binding is defined as
the radioactivity bound in the presence of 1 µM of human
R-CGRP during incubation.
N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-
phenylalanyl]-N6-(phenylmethoxycarbonyl)-L-lysine-phen-
ylamide (32a). Prepared as described above for 22a from 31
and aniline; mp 206-215 °C. Anal. (C39H43Br2N5O5: 821.6) C,
H, Br, N.
1-[N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-
phenylalanyl]-N6-(phenylmethoxycarbonyl)-L-lysyl]-4-
phenylpiperazine (32b). Prepared as described above for 22a
from 31 and 1-phenylpiperazine; mp 170-175 °C. ESI-MS: (M
+ H)+ ) 889/891/893 (Br2). Anal. (C43H50Br2N6O5: 890.7) C,
H, Br, N.
1-[N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-
phenylalanyl]-N6-(phenylmethoxycarbonyl)-L-lysyl]-4-
(1,1-dimethylethoxycarbonyl)piperazine (32c). Prepared
as described above for 22a from 31 and 1-(1,1-dimethylethoxy-
carbonyl)piperazinehydrochloride.Anal.(C42H54Br2N6O7: 914.72)
C, H, Br, N.
1-[N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-
D-phenylalanyl]-N6-(phenylmethoxycarbonyl)-L-lysyl]pi-
perazine Trifluoroacetate (32d). Prepared as described
above for 21a from 32c and TFA. Anal. (C39H47Br2F3N6O7:
928.63) C, H, Br, N.
1-[N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-
phenylalanyl]-N6-(phenylmethoxycarbonyl)-L-lysyl]-4-(4-
pyridinyl)piperazine (32e). Prepared as described above for
22a from 31 and 1-(4-pyridinyl)piperazine. ESI-MS: (M + H)+
) 890/892/894 (Br2). Anal. (C42H49Br2N7O5: 891,69) C, H, Br,
N.
N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-
phenylalanyl]-L-lysine-phenylamide Hydrobromide (6).
Prepared, as described above for 1, from 32a and hydrogen
bromide. ESI-MS: (M + H)+ ) 686/688/690 (Br2). EI-MS: M+
) 685/687/689 (Br2). Anal. (C33H37Br2N5O3‚HBr: 768.38) C, H,
Br, N.
1-[N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-
phenylalanyl]-L-lysyl]-4-phenyl-piperazine Hydrobro-
mide (7). Prepared as described above for 1 from 32b and
hydrogen bromide. ESI-MS: (M + H)+ ) 755/757/759 (Br2).
Anal. (C35H44Br2N6O3‚HBr: 837.48) C, H, Br, N.
1-[N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-
phenylalanyl]-L-lysyl]-piperazine Dihydrobromide (8).
Prepared as described above for 1 from 32d and hydrogen
bromide. ESI-MS: (M + H)+ ) 679/681/683 (Br2). Anal. (C29H40-
Br2N6O3‚2HBr: 842.3) C, H, Br, N.
1-[N2-[4-Amino-3,5-dibromo-N-(1-oxo-4-phenylbutyl)-D-
phenylalanyl]-L-lysyl]-4-(4-pyridinyl)piperazine (9). Pre-
pared as described above for 1 from 32e and hydrogen bromide.
ESI-MS: (M + H)+ ) 756/758/760 (Br2); (M + 2H)2+ ) 378.6/
379.6/380.6 (Br2). Anal. (C34H43Br2N7O3: 757.56) C, H, Br, N.
4-[N-(Aminocarbonyl)phenylamino]piperidine (33). To
the suspension of 4-phenylamino-1-phenylmethylpiperidine
(7.3 g, 0.027 mol), toluene (100 mL) and sodium cyanate (1.8
g, 0.027 mol) was added dropwise, while stirring, TFA (3.1 g,
0.027 mol). Stirring was continued for 2 h, same amount as
above both of sodium cyanate and TFA added and stirring
continued overnight at ambient temperature. The mixture was
concentrated in vacuo, the residue washed successively with
water, aq sodium hydroxide solution, water and diethyl ether.
The concentration-binding curves were analyzed using a
computer-aided nonlinear curve adaptation. All data are the
mean of at least three independent experiments performed in
triplicate (SEM < 5%).
Determination of Binding Affinity for Rat and Mar-
moset CGRP-Receptor. In separate studies, homogenates
of marmoset or rat spleen were used instead of SK-N-MC cells
(see above) to determine the binding affinities of compounds
for rat and marmoset CGRP receptors.
Determination of CGRP Antagonism in SK-N-MC
Cells. SK-N-MC cells (1 million cells) were washed twice with

Small Molecule CGRP Receptor Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 19 5931
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250 µL of incubation buffer (Hanks’s HEPES, 1 mM 3-isobutyl-
1-methylxanthine, 1% BSA, pH 7.4) and preincubated at 37
°C for 15 min. After the addition of CGRP (10 µL) as agonist
in increasing concentrations (10-11 to 10-6 M) or additionally
of substance in three to four different concentrations, incuba-
tion was continued for a further 15 min. Intracellular cAMP
was then extracted by the addition of 20 µL of 1 M HCl and
centrifugation (2000g, 4 °C for 15 min). The supernatants were
frozen in liquid nitrogen and stored at -20 °C. The cAMP
contents of the samples were determined by radioimmuno-
assay (Amersham), and the pA2 values of antagonistically
acting substances were determined graphically.
(10) (a) Rudolf, K.; Eberlein, W.; Engel, W.; Pieper, H.; Doods, H.;
Hallermayer, G.; Entzeroth, M.; Wienen, W. Preparation of
modified amino acids and their use as calcitonin gene-related
peptide antagonists in pharmaceutical compositions. PCT Int.
Appl. 1998, WO9811128 (b) Eberlein, W.; Engel, W.; Rudolf, K.;
Doods, H.; Hallermayer, G.; Bauer, E. Preparation of 3-(1-
cyclopropylcarbonyl-4-piperidinyl)-3,4-dihydro-2(1H)-quinazoli-
nones as calcitonin gene-related peptide receptor antagonists.
PCT Int. Appl. 2001, WO2001032648. (c)Rudolf, K.; Eberlein,
W.; Engel, W.; Doods, H.; Hallermayer, G.; Bauer, E. Preparation
of naphthalenes, piperidines, imidazoles, and quinazolines as
calcitonin gene-related peptide receptor antagonists. PCT Int.
Appl. 2001, WO2001032649. (d) Rudolf, K.; Eberlein, W.; Dreyer,
A.; Mueller, S. G.; Doods, H.; Bauer, E. Preparation of piperidine-
substituted amino acids for use in treatment of CGRP-mediated
disorders. PCT Int. Appl. 2001, WO2001049676. (e) Rudolf, K.;
Mueller, S. G.; Stenkamp, D.; Lustenberger, P.; Dreyer, A.;
Bauer, E.; Schindler, M.; Kirsten, A.; Doods, H. Preparation of
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receptor antagonists for the treatment of migraine headaches.
PCT Int. Appl. 2004, WO2004037810.
Acknowledgment. We thank Guenter Baisch,
Reinhard Borkert, Gerhard Breuer, Alexander Dreyer,
Helmut Egger, Eva Hammer, Klaus Heinrich, Beate
Hirner, Walter Hudler, Siegfried Kolb, Elisabeth Lieb-
hardt, Britta Missler, Angelika Raible-Po¨schl, Stefanie
Rausenberger, Hilde Wiedenmayer, and Fritz Zitzler for
their skillful technical assistance.
Supporting Information Available: Nuclear magnetic
resonance (NMR) spectra and combustion analyses of all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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