The synthesis and SAR of calcitonin gene-related peptide (CGRP) receptor antagonists derived from tyrosine surrogates. Part 2

Article abstract of DOI:10.1016/j.bmcl.2013.01.011

Various substituted indazole and benzoxazolone amino acids were investigated as d-tyrosine surrogates in highly potent CGRP receptor antagonists. Compound 3, derived from the 7-methylindazole core, afforded a 30-fold increase in CGRP binding potency compared with its unsubstituted indazole analog 1. When dosed at 0.03 mg/kg SC, compound 2 (a racemic mixture of 3 and its (S)-enantiomer) demonstrated robust inhibition of CGRP-induced increases in mamoset facial blood flow up to 105 min. The compound possesses a favorable predictive in vitro toxicology profile, and good aqueous solubility. When dosed as a nasal spray in rabbits, 3 was rapidly absorbed and showed good intranasal bioavailability (42%).

Full text of DOI:10.1016/j.bmcl.2013.01.011

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1870–1873
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis and SAR of calcitonin gene-related peptide (CGRP)
receptor antagonists derived from tyrosine surrogates. Part 2 q
⇑
Xiaojun Han , Rita L. Civiello, Charles M. Conway, Deborah A. Cook, Carl D. Davis, Andrew P. Degnan,
Xiang-Jun Jiang, Robert Macci, Neil R. Mathias, Paul Moench, Sokhom S. Pin, Richard Schartman,
Laura J. Signor, George Thalody, George Tora, Valerie Whiterock, Cen Xu, John E. Macor,
⇑
Gene M. Dubowchik
Department of Molecular Sciences and Candidate Optimization (MSCO), Bristol-Myers Squibb Research & Development, 5 Research Parkway, Wallingford, CT 06492, USA
Department of Neuroscience Biology, Bristol-Myers Squibb Research & Development, 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:
Various substituted indazole and benzoxazolone amino acids were investigated as D-tyrosine surrogates
Received 2 November 2012
Revised 28 December 2012
Accepted 2 January 2013
Available online 24 January 2013
in highly potent CGRP receptor antagonists. Compound 3, derived from the 7-methylindazole core, affor-
ded a 30-fold increase in CGRP binding potency compared with its unsubstituted indazole analog 1.
When dosed at 0.03 mg/kg SC, compound 2 (a racemic mixture of 3 and its (S)-enantiomer) demonstrated
robust inhibition of CGRP-induced increases in mamoset facial blood flow up to 105 min. The compound
possesses a favorable predictive in vitro toxicology profile, and good aqueous solubility. When dosed as a
nasal spray in rabbits, 3 was rapidly absorbed and showed good intranasal bioavailability (42%).
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
CGRP receptor antagonist
Calcitonin gene-related peptide (CGRP)
Migraine headache
Tyrosine surrogates
CYP 3A4 inhibition
Triptans, the current standard of care for migraine, are 5-HT1B/
receptor agonists. Their efficacy depends on a vasoconstrictive
Although oral formulations are usually the most convenient
route for delivering medicines, intranasal delivery would seem ide-
ally suited for the treatment of migraine. A nasal spray can be ex-
pected to achieve a more rapid onset of action in comparison with
traditional oral formulations.8,9 Moreover, while nausea can limit
the usefulness of oral medicines in migraneurs, intranasal dosing
offers the possibility of treating even those patients suffering from
migraine-related emesis.10 However, an effective intranasal formu-
lation imposes a number of stringent compound requirements.
First, the molecule must have excellent plasma protein-adjusted
binding potency because of the need to deliver a fairly low dose
into the nasal cavity.11 Also, the compound should have very high
aqueous solubility as the entire dose must be delivered in a small
1D
effect on blood vessels. Although selective for intracranial over cor-
onary vessel constriction, triptans are contraindicated in patients
with cardiovascular disease and hypertention.2 Thus, there is a
clear need for acute migraine therapies without cardiovascular lia-
bilities. Calcitonin gene-related peptide (CGRP) is a 37-amino acid
neuropeptide that is expressed in trigeminal ganglia nerves, and
has been implicated in the pathogenesis of migraine.3 Increased
levels of CGRP are observed during a migraine attack, and intrave-
nous (iv) administration of CGRP can induce migraine in migrai-
neurs.4 The potent CGRP receptor antagonist, BIBN4096BS
(olcegepant), when doses iv demonstrated antimigraine efficacy
that was minimally equivalent to the triptans, without cardiovas-
cular or other serious adverse effects.5 Orally administered CGRP
receptor antagonists have also recently demonstrated clinical util-
ity in acute migraine.6 In this context, we undertook a medicinal
chemistry effort to identify a potent CGRP antagonist that could
be delivered by another convenient route of administration.7
volume of water or aqueous buffer (6100 l
L/nostril).12
Previously, we have reported the identification and SAR of novel
CGRP receptor antagonists derived from heterocyclic tyrosine sur-
rogates.1 Compound 1 (Fig. 1) was found to be a potent antagonist
with a reasonable cytochrome P450 (CYP) profile, and it showed
activity in a primate model of CGRP-induced facial blood flow
when dosed subcutaneously. In this Letter, we report the SAR ef-
forts that led to compound 3, a highly potent CGRP receptor antag-
onist that was the direct precursor to BMS-694153, our first
potential intranasal clinical candidate.13 We also describe the
activity of 3 in ex vivo and in vivo models relevant to migraine.13
q
See Ref. 1.
Corresponding authors.
E-mail addresses: xiaojun.han@bms.com (X. Han), gene.dubowchik@bms.com
⇑
(G.M. Dubowchik).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.011

X. Han et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1870–1873
1871
R = H, X = H, Compound
1
The results of this study are summarized in Table 1. We were
pleased to see that the racemic amino acid with a 7-Me substitu-
tion on the indazole (2) showed excellent binding potency
(Ki = 0.012 nM). As expected from our previous studies, activity re-
sided primarily with the R-enantiomer (3, Ki = 0.0073 nM), which
was 30-fold more potent than compound 1. It was also encourag-
ing to see that inhibition of CYP3A4 metabolism of BZR, our stron-
gest isoform interaction in this series, was unchanged in
hCGRP Ki (nM)
0.23
CYP3A4-BFC IC50 (µM) >40
CYP3A4-BZR IC50 (µM) 4.0
O
N
N
O
N
NH
X
R = Me, X = H, Compound3
hCGRP Ki (nM)
CYP3A4-BFC IC50 (µM) 36.0
CYP3A4-BZR IC50 (µM) 6.0
NH
O
N
0.0073
R
R = Me, X = F,BMS-694153
hCGRP Ki (nM)
CYP3A4-BFC IC50 (µM) 26.0
CYP3A4-BZR IC50 (µM) 6.7
N
HN
0.013
comparison with compound 1 (CYP 3A4 BZR IC50, 6.0 vs 4.0 lM,
respectively, Table 1). This effectively improved our safety margin
versus CYP 3A4 by greater then 30-fold. Replacement of the 7-Me
in 2 with -Cl (5) or -Et (6) resulted in comparable CGRP receptor
binding activity but increased CYP inhibition (3A4 BZR IC50’s 0.18
Figure 1.
and 0.45 lM for 5 and 6, respectively, Table 1). The corresponding
As a surrogate of tyrosine, the indazole-containing amino acid
in compound 1 gave us the best balance of CGRP receptor antago-
nist potency and acceptable CYP inhibition.1 Therefore, we first
decided to study the effects of substitution on the indazole core,
and then study the effects of substitution on related heterocycles.
7-i-Pr analog (7) was significantly less potent (Ki = 0.22 nM), and
the disubstituted compounds 8 and 9 were modestly less potent
(Ki = 0.048 and 0.032 nM, respectively, Table 1). These results sug-
gested that the 7-Me indazole was the best fit for the
binding pocket in the CGRP receptor.
D-amino acid
Table 1
SAR of central amino acid moieties
H
O
N
N
O
H
N
N
N
O
N
AA Moiety
Compd AA moiety (stereochemistry) Ki (nM)
CYP3A4 (IC50
4.0 (>40)
,
l
M) BZR (BFC)a Compd AA moiety (stereochemistry) Ki (nM) CYP3A4 (IC50
, lM) BZR (BFC)
Cl
N
N
N
1
2
0.23
9
0.032
0.039
1.6 (3.5)
17 (>4 0)
H
N
H
(R)
(R)
4
4
3
3
5
5
O
2
O
N
1
2
0.012
1.1 (24)
10
6
N
6
1
N
H
7
H
7
Cl
( )
(R)
O
O
N
N
3
4
5
0.0073 6.0 (36)
11
12
13
0.023
0.055
7.2 (>40)
5.5 (32)
NA
N
H
N
H
(R)
(S)
Br
I
(R)
O
O
N
H
27
NA
N
H
(R)
(R)
O
O
N
N
H
0.011
0.18 (13)
11
N
H
CN
Cl
( )
( )
O
N
O
N
H
N
H
6
7
0.0050 0.45 (25)
14
15
0.28
0.14
5.0 (>40)
2.3 (18)
Cl
(R)
(R)
O
N
O
N
H
0.22
0.37 (NA)
6.9 (>40)
N
H
Br
( )
3
4
5
N
2
N
N
1
8
0.048
16
0.015
7.3 (>40)
6
N
H
N
H
7
(R)
( )
a
BZR = 7-benzyloxy-resorufin and BFC = 7-benzyloxy-4-trifluoromethylcoumarin.

1872
X. Han et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1870–1873
Previous results from our laboratories indicated that ben-
140
120
100
80
Compound 2, 0.03g/Kg
zooxazolone and benzotriazole moieties showed CGRP receptor
antagonist potencies and CYP inhibition profiles that were compa-
rable to indazole 1.1 Thus we prepared a small series of halogen-
substituted benzoxazolones. The 7-Cl (10, Ki = 0.039 nM), 7-Br
(11, Ki = 0.023 nM), and 7-I (12, Ki = 0.055 nM) analogs all demon-
strated very good CGRP receptor binding, albeit somewhat inferior
to 3 (Ki = 0.0073 nM), with CYP3A4 inhibition that became undesir-
ably more potent with increasing size of the halogen substituent.
Substitution at the 6-position of the benzoxazolones with Cl (14)
**p < 0.01 n = 5 marmosets
60
40
**
**
20
0
1
and Br (15) both resulted in
a significant loss of potency
Baseline +15 min +60 min +105 min
(Ki = 0.28 and 0.14 nM, respectively, Table 1) compared with the
corresponding 7-substituted analogues (10 and 11), but similar
to the unsubstituted benzoxazolone (compound 25 in Ref. 1,
Ki = 0.27 nM). Interestingly, the 7-CN analog (13) was almost
300-fold less potent than the 7-Cl compound (10), suggesting a
very strict requirement for hydrophobicity in that small binding
pocket in the CGRP receptor. Compound 16, the 7-Me substituted
benzotriazole, was comparable to 3 in terms of CGRP receptor
binding potency and CYP inhibition.
Functional activity of new compounds was determined by mea-
suring concentration-dependent inhibition of CGRP-stimulated
cAMP production in SK-N-MC cells.13 All compounds shown in Ta-
ble 1 demonstrated full antagonism of the human CGRP receptor
(for 3, EC50 = 0.017 nM). Compound 3 was chosen for further study.
First, we examined the ability of 3 to antagonize CGRP-induced
blood vessel dilation in an assay utilizing ex vivo human intracra-
nial arteries.13 In this experiment, each vessel ring was mounted
between two wire hooks and attached to a force transducer that
measured arterial tone. Vessels were first contracted with 10 mM
Figure 2. Marmoset facial blood flow.
Our marmoset facial blood flow model was used to assess
in vivo efficacy of our CGRP receptor antagonists.13 In this model,
marmosets were anesthetized and facial blood flow was measured
and increased by iv administration of haCGRP (10 lg/kg) at 45 min
intervals (ꢀ30, 15, 60, and 105 min). The effect of antagonist 2 (the
racemate of 3), delivered subcutaneously (SC) at 0 min, on
CGRP-induced changes in facial blood flow was measured by laser
Doppler flowmetry. In this study, compound 2 (0.03 g/kg, SC)
demonstrated strong inhibition at 60 and 105 min (Fig. 2).
Comparing efficacy versus exposure of 2 at 15 min (4.7 nM),
60 min (13 nM) and 105 min (11 nM), showed that plasma levels
of 2 above 10 nM were associated with robust in vivo activity, con-
sistent with what we had previously seen for BMS-694153.13
CGRP-induced increases in marmoset facial blood flow were simi-
lar across all time points in vehicle-treated animals.
Intranasal administration of 3 was explored in rabbits. The
aqueous solubility of amorphous 3ꢁHCl in the pH range 4.0–6.2
was between 23 and 13 mg/mL. When intranasally dosed as a
spray at a concentration of 10 mg/mL, the HCl salt of 3 was rapidly
and efficiently absorbed from the nasal cavity of rabbits. Measur-
able plasma levels were seen as early as 2 min and Tmax occurred
within 10 min. The intranasal bioavailability of a 0.6 mg/kg dose
of a 10 mg/mL solution dose of 3 was 42%.
KCl, then fully dilated with 1 nM haCGRP. Dilation was then re-
versed by the cumulative addition of increasing concentrations of
3 in half-log amounts. In this study, compound 3 potently reversed
CGRP-induced dilation on human intracranial arteries with an
EC50 = 0.050 nM. Commensurate with its improved binding affin-
ity, compound 3 was 24-fold more potent than compound 1
(EC50 = 1.2 nM) in this assay.
H
O
N
1. LiOH/THF/MeOH/H2O
(90%)
N
CO2Me
NH2
CDI/THF, then
O
H
N
3
N
N
2. PyBOP/DIEA/DMF
O
MeO
N
H
NH
N
O
17
19
HN
HN
N
N
N
H
18
(60%)
(70%)
MeO2C
NHCbz
N
HN
N
HN
NaH,tBuLi
KOtBu
24
P(O)(OMe)2
NaNO2, HCl
Br
H2N
Br
N
Br
CHO
N
DMSO
89%
then t-BuSH
then DMF
65%
TMG
97%
t-Bu
N
87%
20
21
22
23
Br
CO2Me
NH2
CO2Me
CO2Me
NH2
Pd/C, H2
100%
N
N
H2N
N
NHCbz
N
H
N
H
H
27
25
26
28
CO2Me
NH2
CO2Me
NH2
Br
Br
Br
CO2Me
NH2
N
N
N
N
H
N
H2N
H2N
H2N
N
H
H
Cl
Cl
29
30
31
32
33
34
Scheme 1. Synthesis of compound 3 and amino esters 26, 28, 30, 32 and 34.

X. Han et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1870–1873
1873
Compound 3 had a favorable CYP profile with IC50’s >40
CYP isoforms 1A2, 2C19, 2C9 and 2D6. Its IC50’s for CYP3A4 were
36 M (BFC) and 6.0 M (BZR). Compound 3 was negative in the
Ames test, and it demonstrated only low levels of hERG channel
inhibition and PXR activation.
lM for
References and notes
1. For Part I, see: Han, X.; Civiello, R. L.; Conway, C. M.; Cook, D. A.; Davis, C. D.;
Macci, R.; Pin, S. S.; Ren, S. X.; Schartman, R.; Signor, L. J.; Thalody, G.;
Widmann, K. A.; Xu, C.; Chaturvedula, P. V.; Macor, J. E.; Dubowchik, G. M.
Bioorg. Med. Chem. Lett. 2012, 22, 4723.
l
l
2. Silberstein, S. D. Lancet 2004, 363, 381.
The representative syntheses of compound 3 and the amino
acids required for compounds 2–16 is outlined in Scheme 1. The
coupling of amino ester 1714 and quinazolinone 1815 afforded urea
19. Saponification of the methyl ester of 19 with LiOH gave the car-
boxylic acid, which was coupled with 1,40-bipiperidine to afford
compound 3 (Scheme 1).
The synthesis of amino ester 26 began with the treatment of
aniline 20 with NaNO2, followed by quenching of the resulting dia-
zonium ion with t-BuSH to afford 21. Reaction of 21 with KOtBu in
DMSO gave indazole bromide 22,16 which was converted to alde-
hyde 23 by reaction with NaH/tBuLi and quenching with DMF.
The olefination17 of aldehyde 23 using phosphonate 24 provided
enamide 25. Hydrogenation of 25 afforded amino ester 26. In a
similar manner, amino esters 28, 30, 32 and 34 were prepared from
the corresponding anilines 27, 29, 31 and 33 (Scheme 1). The
synthesis of other amino esters used in the compounds shown in
Table 1 have been reported previously.
3. (a) Goadsby, P. J. Drugs 2005, 65, 2557; (b) Edvinsson, L. Cephalalgia 2004, 24,
611; (c) Williamson, D. J.; Hargreaves, R. J. Microsc. Res. Tech. 2001, 53, 167.
4. Lassen, L. H.; Haderslev, P. A.; Jacobson, V. B.; Iversen, H. K.; Sperling, B.; Olesen,
J. Cephalalgia 2002, 22, 54.
5. Olesen, J.; Diener, H. C.; Husstedt, I. W.; Goadsby, P. J.; Hall, D.; Meier, U.;
Pollentier, S.; Lesko, L. M. N. Engl. J. Med. 2004, 350, 1104.
6. Oral CGRP receptor antagonist medicinal chemistry efforts have been reported
in: (A) For MK-0974 Paone, D. V.; Shaw, A. W.; Nguyen, D. N.; Burgey, C. S.;
Deng, J. Z.; Kane, S. A.; Koblan, K. S.; Salvatore, C. A.; Mosser, S. D.; Johnston, V.
K.; Wong, B. K.; Miller-Stein, C. M.; Hershey, J. C.; Graham, S. L.; Vacca, J. P.;
Williams, T. M. J. Med. Chem. 2007, 50, 5564; For MK-3207 (b) Bell, I. M.;
Gallicchio, S. N.; Wood, M. R.; Quigley, A. G.; Stump, C. A.; Zartman, C. B.; Fay, J.
F.; Li, 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. ACS Med. Chem.
Lett. 2010, 1, 24.
7. (a) IDDB, Thomson Scientific reports that the development of BIBN4096BS (IV
delivery) has been discontinued.; (b) Merck
discontinuation of the clinical development program for MK-3207 (oral
delivery) on Sept. 10, 2009.
& Co., Inc. announced
8. Behl, C. R.; Pimplaskar, H. K.; Sileno, A. P.; DeMeireles, J.; Romeo, V. D. Adv. Drug
Deliv. Rev. 1998, 29, 89.
9. Rapoport, A. Cephalalgia 2001, 21, 13.
In summary, we explored substituted indazole and benzoxazo-
10. Rapoport, A. M.; Bigal, M. E.; Tepper, S. J.; Sheftell, F. D. CNS Drugs 2004, 18, 671.
11. Wermeling, D. P.; Miller, J. L.; Rudy, A. C. Drug Deliv. Technol. 2002, 2, 56.
12. Merkus, P.; Guchelaar, H.-J.; Bosch, D. A.; Merkus, F. W. H. M. Neurology 2003,
60, 1669.
13. Degnan, A. P.; Chaturvedula, P. V.; Conway, C. M.; Cook, D. A.; Davis, C. D.;
Denton, R.; Han, X.; Macci, R.; Mathias, N. R.; Moench, P.; Pin, S. S.; Ren, S. X.;
Schartman, R.; Signor, L. J.; Thalody, G.; Widmann, K. A.; Xu, C.; Macor, J. E.;
Dubowchik, G. M. J. Med. Chem. 2008, 51, 4858. For biology experimental
procedures to measure [125I]CGRP binding, CYP inhibition, reversal of CGRP-
induced dilation in ex vivo human intracranial artery, Schild analysis, in vivo
efficacy in marmoset facial blood flow, and pharmacokinetics properties, see
the Supplementary data in this Letter.
14. For the synthesis of the amino acids not shown in Scheme 1, see: (a) Han, X.;
Civiello, R. L.; Fan, H.; Wu, D.; Gao, Q.; Chaturvedula, P. V.; Macor, J. E.;
Dubowchik, G. M. J. Org. Chem. 2008, 73, 8502; (b) Han, X.; Jiang, X.-J.; Civiello,
R. L.; Degnan, A. P.; Chaturvedula, P. V.; Macor, J. E.; Dubowchik, G. M. J. Org.
Chem. 2009, 74, 3993.
lone amino acids as
D-tyrosine surrogates in CGRP receptor
antagonists. We found that compound 3, derived from the
7-methylindazole core, afforded a 30-fold increase in CGRP binding
potency compared with the corresponding unsubstituted indazole
1. When dosed at 0.03 mg/kg SC, 2 (racemate of 3) demonstrated
robust inhibition of CGRP-induced increases in mamoset facial
blood flow out to 105 min. Compound 3 also possessed a favorable
predictive in vitro toxicology profile. However, despite showing
good IN bioavailability in rabbits, its aqueous solubility of 3 was
not deemed sufficient for continued development. Further efforts
resulted in the discovery of BMS-694153, a compound with supe-
rior aqueous solubility.13
15. Rudolf, K.; Eberlein, W.; Engel, W.; Pieper, H.; Entzeroth, M.; Hallermayer, G.;
Doods, H. J. Med. Chem. 2005, 48, 5921.
Acknowledgment
16. Dell’Erba, C.; Novi, M.; Petrillo, G.; Tavani, C. Tetrahedron 1994, 50, 3529.
17. Schmidt, U.; Griesser, H.; Leitenberger, V.; Lieberknecht, A.; Mangold, R.; Myer,
R.; Riedl, B. Synthesis 1992, 487.
We thank the members of Discovery Analytic Sciences for their
help in the chiral purification and characterization of compounds
contained herein.