Serendipitous oxidation product of BIBN4096BS: A potent CGRP receptor antagonist

Article abstract of DOI:10.1016/j.bmcl.2014.04.033

An oxidation product (5) formed during the synthesis of BIBN-4096BS (1) was found to be a potent CGRP antagonist (IC₅₀ = 0.11 nM). While 5 was found to be ten-fold less potent than 1, another analog 8 with lower molecular weight containing the

Full text of DOI:10.1016/j.bmcl.2014.04.033

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2744–2748
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Serendipitous oxidation product of BIBN4096BS: A potent CGRP
receptor antagonist
a
b
b
b
Bireshwar Dasgupta a, , Edward Kozlowski , Daniel R. Schroeder , John R. Torrente , Cen Xu ,
⇑
Sokhom Pin b, Charlie M. Conway b, Gene M. Dubowchik a, John E. Macor a, Vivekananda M. Vrudhula a,
⇑
a Molecular Sciences and Candidate Optimization, Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, CT 06492, United States
b Neuroscience Discovery Biology, Research and Development, Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, CT 06492, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 February 2014
Revised 8 April 2014
Accepted 9 April 2014
Available online 18 April 2014
An oxidation product (5) formed during the synthesis of BIBN-4096BS (1) was found to be a potent CGRP
antagonist (IC50 = 0.11 nM). While 5 was found to be ten-fold less potent than 1, another analog 8 with
lower molecular weight containing the oxidized fragment demonstrated twenty-fold higher activity than
its parent 7. Alternative conditions which preclude the formation of the oxidation product are described.
The activities of 1, 5, 7 and 8 in functional cAMP assay are also discussed.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
BIBN4096BS
CGRP
Antagonists
Migraines are episodic headaches often characterized by severe
unilateral head pain, photophobia, phonophobia and nausea.1 It
has been hypothesized that migraines are associated with the dila-
tion of cranial blood vessels and activation of the trigeminovascu-
lar system.2 More recent thinking has focused on the importance of
halting peripheral afferent sensitization as key to antimigraine effi-
cacy,3b whereas others see blocking central sensitization as criti-
cal.3c Triptans, which are the current standard of care for
migraines, are 5-HT1B/1D agonists and are believed to function by
their active, nonselective vasoconstriction of these cranial ves-
sels.3a However, because triptans are nonselective vasoconstric-
tors, they are also associated with a number of cardiovascular
side effects and are contraindicated in patients with hypertension
or ischemic heart disease.1 Calcitonin gene-related peptide (CGRP),
an extremely potent vasodilator, is believed to play an important
role in the pathophysiology of migraine4 as five different CGRP
receptor antagonists have been shown to be effective anti-
migraines agents in clinical trials.5 CGRP binds to the CGRP
receptor, which is a G-protein coupled component, calcitonin
receptor-like receptor (CRLR), receptor consisting of a classical
Treatment with anti-migraine drugs, such as sumatriptan
returns CGRP levels to normal, coincident with the alleviation of
headache.7
Several research groups have developed both peptidic and non-
peptidic antagonists for the CGRP receptor.8 It is interesting to note
that a truncated form of CGRP [CGRP(8-37)] itself is a peptidic antag-
onist for the CGRP receptor.9 This compound is potent (Ki = 3.2 nM),
but it is rapidly degraded in human plasma (t1/2 = 20 min) and not
really suitable as a in vivo blocker of CGRP pharmacology.8b Recently
BIBN4096BS (1)10 was identified as a potent antagonist for human
NH2
H
O
N
N
N
N
O
Br
O
H
N
N
N
N
H
O
O
Br
O
HO
H
N
O
O
NH
1
N
N
7-transmembrane and
a receptor activity modifying protein
HN
N
(RAMP).6 Marked elevation of CGRP in serum has been detected
in individuals diagnosed with migraine.4f
N
NH2
B
O
NH2
O
C
Br
HO
⇑
Corresponding authors. Tel.: +1 203 677 7411; fax: +1 203 677 7702 (B.D.); tel.:
+1 203 677 6697 (V.M.V.).
Br
A
E-mail addresses: Bireshwar.Dasgupta@bms.com (B. Dasgupta), Vivekananda.
Vrudhula@bms.com (V.M. Vrudhula).
Scheme 1. Retrosynthetic plan for synthesis of BIBN4096BS.
http://dx.doi.org/10.1016/j.bmcl.2014.04.033
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

B. Dasgupta et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2744–2748
2745
O
CGRP receptor. In binding assays using SK-N-MC cells, 1 demon-
strated 150-fold greater affinity compared to that of the peptide
antagonist CGRP(8-37).10
NH2
H
N
O
O
Br
+
In our medicinal chemistry efforts, we engaged in a program
toward the discovery of new CGRP receptor antagonists as potential
therapeutics for the treatment of migraine.11 Since 1 was a potent
non-peptidic antagonist of CGRPreceptor, we saw itas a starting point
to understand the structural requirements for CGRP receptor antago-
nism. The synthesis of this molecule12b was non-trivial and care had to
be exercised to avoid side reactions and racemization. In this commu-
nication, we report the identification of a benzylic oxidation product5
(Scheme 3) formed unexpectedly during the synthesis of 1. In vitro
activityofthetwocompoundscontainingthisbenzylicoxidationfrag-
ment against CGRP receptor is also reported herein.
N
O
HO
HN
Br
B
A
H
N
N
O
H
N
N
a
O
b
Br
HO
O
2
Br
NH2
H
O
N
H
N
O
N
N
N
O
N
H
N
O
N
N
H
N
H
N
N
HO
O
O
Br
O
Br
HO
c
HO
Br
3
4
Br
+
N-Boc-BIBN-4096BS
O
(1)
HN
O
Even though BIBN-4096BS was largely prepared by the reported
route, several interesting observations are worth noting. The strat-
egy was to separately prepare the three key pieces (Scheme 1), and
assemble them in a convergent synthesis. Since there were two chi-
ral centers in the molecule prone to epimerization, mild reactions
conditions had to be employed to preclude the possibility of
racemization.
N
N
N
NH2
O
C
Initially, when we attempted to couple the pieces A and B using
CDI and 1,2,4-triazole by mixing all the reagents together, the
major product formed was not the desired product 2 (Scheme 2).
Instead a cyclic urethane formed from 3-(piperidin-4-yl)-3,4-
dihydroquinazolin-2-ol as shown below was the major product
as evidenced by LC-MS.
Scheme 2. Coupling of components A, B and C. Reagents and conditions: (a) CDI,
1,2,4-triazole, DMF, 51%; (b) LiOHÁH2O, DME, H2O, 40 °C, 79%; (c) TBTU, HOBt, DMF
(crude, not purified at this stage).
activity of the oxidation product as a CGRP antagonist. Based on
NMR studies, the site of the oxidation was confirmed to be at the
benzylic position of the 3,4-dihydroquinazoline moiety as shown
in 5.14 A known peroxide contaminant in 1,4-dioxane is 2-hydro-
peroxy-1,4-dioxane. We speculate that a contaminant such as
2-hydroperoxy-1,4-dioxane provided the oxidant for converting 1
to 5.
N
N
cyclic urethane by LCMS
N
O
O
While the amount of oxidation product formed was only 12%,
its formation as a byproduct in the final deprotection step and
the difficulty of separating this byproduct from the required prod-
uct prompted us to investigate this deprotection reaction further.
To avoid the oxidation product, TFA and water mixture (90:10,
15 min, ambient temperature) was used for the N-Boc removal.
Under these conditions, no benzylic oxidation product 5 was
observed (Scheme 3).
The HPLC conditions for separation of BIBN-4096BS (1) and 5
and the HPLC traces are shown below in Figures 1 and 2.
We assessed the binding activity of this novel oxidation product
on SK-N-MC membrane tissue (Fig. 3). Binding assays13 were car-
ried out with either 125I-CGRP or 125I-CGRP(8-37), using SK-N-MC
membrane tissue. It was found that 5 (IC50 = 0.11 nM) was 10-fold
less potent than 1 (IC50 = 0.014 nM) (Fig. 4).
To avoid this undesired cyclization product during coupling
with CDI, the tyrosine unit A was pretreated with CDI and 1,2,4-
triazole, and then the dihydroquinolinone fragment B was added
to obtain the desired product 2 (Scheme 2). The ester was then
hydrolyzed to obtain carboxylic acid 3, which after coupling with
the L-lysine component C afforded N-Boc-protected BIBN-4096BS
(4).
In the literature12a pertaining to analogs of 1, two methods
(HCl/dioxane or TFA/dichloromethane) were used for the removal
of the N-Boc in 4. When the N-Boc group on the lysine amine in
4 was removed by treatment with 4 M HCl in dioxane, reverse
phase LC-MS analysis of the reaction mixture showed the desired
BIBN4096BS (1) as the major product (31%) along with a minor
product (12%), which eluted faster than the desired product. The
minor product 5 had mass 14 units higher than 1, suggesting oxi-
dation of a methylene group. There were two benzylic positions in
1, one located on the dibromotyrosine residue and the other on the
3,4-dihydroquinazilinone moiety. We wanted to determine of
these two positions which one underwent oxidation, and the
Compounds 1 and 5 were also tested for their activity in a cAMP
functional assay.13 Compound 1 (EC50 = 0.026 nM) was 6-fold more
active than 5 (EC50 = 0.15 nM). Nonetheless, the oxidized product 5
demonstrated sub-nanomolar activity as
antagonist.
a CGRP receptor

2746
B. Dasgupta et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2744–2748
O
4
200
HN
O
O
H
O
N
N
150
100
50
N
m
A
U
N
H
N
N
N
N
H
O
O
Br
0
HO
Br
0
5
10
15
Minutes
20
25
30
a
NH2
O
Figure 2. HPLC trace of BIBN4096BS. YMC Xterra RPC18 column, 4.6 Â 150 mm,
10 m; A = 0.05% TFA/H2O; B = 0.05% TFA/acetonitrile; 25% B @0.5 mL/min. for
30 min.
l
N
X
N
H
N
N
N
N
H
O
O
H
N
O
CGRP Binding
Br
X =
125
100
75
50
25
0
N
HO
BIBN-4096BS
Br
H
N
O
X =
N
O
5
Scheme 3. N-Boc-deprotection conditions: (a) 4 M HCl in dioxane, CH2Cl2 (BIBN-
4096BS, 31% yield; compound 5, 12% yield).
5
1
We examined analogs of BIBN-4096BS (1) and its oxidation by-
product (5) by replacing the dibromotyrosine in 1 with a simpler
pharmacophore to see if these analogs could retain CGRP receptor
antagonist activity (Scheme 4).15 In compounds 7 and 8 the 3,5-
dibromo-4-hydroxyphenyl residue present in 1 and 5 was replaced
by a smaller benozothiophene unit, shown previously to be active
in other CGRP receptor antagonists.13b The key intermediate 6 was
prepared following similar procedure described for the synthesis of
1 (Scheme 4).12a In this case it was also found that HCl in dioxane
also effected the benzylic oxidation side reaction.
-12
-11
-10
-9
-8
-7
Log [M]
Figure 3. Percent of inhibition in 125I-hCGRP binding assay—hCGRP1 receptor in
SK-N-MC cells of 1 (BIBN4096BS) and 5.
Interestingly, when 7 and 8 were evaluated in our CGRP binding
assay, the oxidized version 8 (IC50 = 2.7 nM) was found to be ten-
fold more potent than 7 (IC50 = 25 nM) (Fig. 5).
110
90
400
300
5
70
50
BIBN4096
200
BIBN-4096
30
100
5
10
0
-10
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
-11
-10
-9
-8
-7
-6
-5
log [compound], M
Figure 1. HPLC trace for BIBN-4096BS and 5. YMC Xterra RPC18 column,
m; A = 5 mM ammonium acetate/H2O, pH 7.2; B = 5 mM
ammonium acetate/10% H2O/90% acetonitrile, pH 7.2; 15–80% B in 20 min, 3 min
hold, @1.0 mL/min.
4.6 Â 150 mm, 3.5
l
Figure 4. Percent of inhibition in CGRP1 receptor (SK-N-MC) cAMP assay—
antagonism of 300 pM CGRP of compounds 1 and 5.
a

B. Dasgupta et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2744–2748
2747
O
110
6
HN
O
O
H
N
90
70
50
30
10
-10
O
N
8
7
N
N
H
N
N
N
N
H
O
O
S
a
NH2
O
-11
-10
-9
-8
-7
-6
log [compound], M
N
X
Figure 6. Percent of inhibition in CGRP1 receptor (SK-N-MC) cAMP assay—
antagonism of 300 pM CGRP of compounds 7 and 8.
N
H
N
a
N
N
N
H
O
O
H
N
O
its oxidation analog 8 also were also both potent CGRP receptor
antagonists.
X =
S
N
7
8
Acknowledgment
H
N
O
The authors would like to thank Dr. Joanne Bronson for critical
reading of the manuscript.
X =
N
O
References and notes
Scheme 4. N-Boc-deprotection conditions: (a) 4 M HCl in dioxane, CH2Cl2 (com-
pound 7, 19%; compound 8, 41%).
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-10
-9
-8
-7
-6
log [compound], M
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SK-N-MC cells of compounds 7 and 8.
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Compounds 7 and 8 were also tested for activity in the cAMP
functional assay (Fig. 6). Compound 7 (EC50 = 96 nM) and 8
(EC50 = 100 nM) showed equivalent activity showing that the oxi-
dized compound 8 was equipotent with 7 as a CGRP receptor
antagonist.
In conclusion, during the eleven step synthesis of 1 we identi-
fied a novel benzylic oxidation product 5 which was found to be
a potent CGRP receptor antagonist. Alternative conditions (90%
TFA, 10% water, ambient temperature) which abrogated such an
oxidation were subsequently utilized. We synthesized 7 as an ana-
log of 1 with a smaller side chain and were able to show that 7 and

2748
B. Dasgupta et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2744–2748
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McMasters, D. R.; Miller-Stein, C.; Moore, E. L.; Mosser, S. D.; Pudvah, N. T.;
Quigley, A. G.; Salvatore, C. A.; Stump, C. A.; Theberge, C. R.; Wong, B. K.;
Zartman, C. B.; Zhang, X.-F.; Kane, S. A.; Graham, S. L.; Vacca, J. P.; Williams, T.
M. Bioorg. Med. Chem. Lett. 2006, 16, 6165; (d) Shaw, Anthony W.; Paone, Daniel
V.; Nguyen, Diem N.; Stump, Craig A.; Burgey, Christopher S.; Mosser, Scott D.;
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S.; Graham, Samuel L.; Vacca, Joseph P.; Williams, Theresa M. Bioorg. Med.
Chem. Lett. 2007, 17, 4795; (e) 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.
14. 1H NMR (500 MHz, MeOD)
d ppm: 8.13 (2H, d, J = 7.6 Hz), 8.01 (1H, d,
J = 7.0 Hz), 7.61–7.67 (1H, m), 7.42–7.46 (2H, m), 7.22 (1H, s), 7.12–7.19 (3H,
m), 5.01–5.12 (1H, m), 4.81 (1H, dd, J = 7.6, 6.10 Hz), 4.44 (1H, t, J = 7.8 Hz),
4.11–4.22 (2H, m), 3.96–4.06 (2H, m), 3.85–3.94 (2H, m), 3.69–3.82 (3H, m),
3.58–3.68 (1H, m), 2.84–3.04 (6H, m), 2.51–2.74 (2H, m), 1.52–1.85 (5H, m),
1.21–1.39 (3H, m); Also analyzed by COSY, HMQC and HMBC and NOSY NMR
for structure elucidation; HRMS: (M+H)+ 882.19; LC–MS: Rt = 1.36 min, (M+H)+
882.22 (method: 0–100% B, 2 min gradient, 3 min run (solvent A 90% water,
10% methanol, 0.1% TFA and solvent B, 10% water, 90% methanol and 0.1% TFA),
k 200 nM, flow 5 mL/min).
15. 1H NMR (500 MHz, MeOD)
d ppm: 8.14 (2H, d, J = 7.3 Hz), 8.01 (1H, d,
J = 7.02 Hz), 7.90 (2H, dd, J = 15.6, 7.9 Hz), 7.61–7.68 (1H, m), 7.40–7.48 (2H,
m), 7.37 (1H, t, J = 7.2 Hz), 7.22 (1H, t, J = 7.5 Hz), 7.10–7.19 (3H, m), 5.00–5.11
(1H, m, J = 11.9, 7.97, 3.97, 3.97 Hz), 4.77 (1H, dd, J = 7.8, 5.9 Hz), 4.67 (1H, t,
J = 7.6 Hz), 4.08–4.21 (2H, m), 3.93–4.03 (2H, m), 3.82–3.89 (2H, m), 3.67–3.79
(3H, m), 3.55–3.67 (1H, m), 3.44 (1H, dd, J = 14.3, 7.6 Hz), 3.28 (1H, dd, J = 14.5,
7.8 Hz), 2.81–2.93 (4H, m), 2.53–2.73 (2H, m), 1.54–1.72 (5H, m), 1.39–1.48
(1H, m), 1.15–1.25 (2H, m). MS: showed (M+H) peak at 766.5 and (MÀH) peak
at 764.4 (solvent: acetonitrile, water, TFA); LC–MS: Rt = 1.43 min, (M+H)+
766.42 [method: same as Ref. 13].
12. (a) Rudolf, K.; Eberlein, W.; Engel, W.; Pieper, H.; Doods, H.; Hallermayer, G.;
Entzeroth, M.; Wienen, W. WO 98/11128 Patent 1998.; (b) Rudolf, K.; Eberlein,