The design and synthesis of potent, selective benzodiazepine sulfonamide bombesin receptor subtype 3 (BRS-3) agonists with an increased barrier of atropisomerization

Article abstract of DOI:10.1016/j.bmc.2012.03.029

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor expressed primarily in the hypothalamus which plays a role in the onset of both diabetes and obesity. We report herein our progress made towards identifying a potent, selective bo

Full text of DOI:10.1016/j.bmc.2012.03.029

Bioorganic & Medicinal Chemistry 20 (2012) 2845–2849
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Bioorganic & Medicinal Chemistry
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The design and synthesis of potent, selective benzodiazepine sulfonamide
bombesin receptor subtype 3 (BRS-3) agonists with an increased barrier
of atropisomerization
Harry R. Chobanian a, , Yan Guo a, Ping Liu a, Thomas J. Lanza Jr. a, Marc Chioda a, Linda Chang a,
⇑
Theresa M. Kelly b, Yanqing Kan b, Oksana Palyha b, Xiao-Ming Guan b, Donald J. Marsh b,
Joseph M. Metzger c, Katie Raustad c, Sheng-Ping Wang c, Alison M. Strack c, Judith N. Gorski c,
Randy Miller d, Jianmei Pang d, Kathy Lyons d, Jasminka Dragovic d, Jian G. Ning e, Wes A. Schafer e,
Christopher J. Welch e, Xiaoyi Gong e, Ying-Duo Gao f, Viktor Hornak f, Marc L. Reitman b,
Ravi P. Nargund a, Linus S. Lin a
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
b Department of Metabolic Disorders, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
c Department of Pharmacology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
d Department of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
e Department of Analytical Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
f Department of Chemistry Modeling and Informatics, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor expressed primarily in the
hypothalamus which plays a role in the onset of both diabetes and obesity. We report herein our progress
made towards identifying a potent, selective bombesin receptor subtype-3 (BRS-3) agonist related to the
previously described MK-77251 Chobanian et al. (2012) that would prevent atropisomerization through
the increase of steric bulk at the C-2 position. This would thereby make clinical development of this class
of compounds more cost effective by inhibiting racemization which can occur over long periods of time at
room/elevated temperature.
Received 14 February 2012
Revised 9 March 2012
Accepted 10 March 2012
Available online 24 March 2012
Keywords:
Bombesin receptor subtype-3 (BRS-3)
Agonist
Ó 2012 Elsevier Ltd. All rights reserved.
Obesity
Atropisomerism
Benzodiazepine sulfonamide
1. Introduction
Mice lacking functional BRS-3 develop metabolic defects and
obesity.3,4
Obesity is a serious and chronic medical condition that has
become a major global health issue. In the United States alone,
obesity rates for adults have doubled and rates for children have
tripled over the last 30 years. Currently, around one third of U.S.
adults and 16% of U.S. children are considered obese.2 In many
cases, the excessive body weight is the root cause of subsequent
co-morbidities, including type 2 diabetes, hypertension, cardiovas-
cular disease, cancer, and arthritis. Our laboratories became inter-
ested in the bombesin receptor subtype-3 (BRS-3) receptor
because of its implication in energy homeostasis and weight loss.
Recently, we disclosed studies which focused on a novel class of
benzodiazepine sulfonamides BRS-3 agonists. This class of mole-
cules was shown to be potent and selective but lacked the pharma-
cokinetic profile necessary for further development. In addition,
pregnane X receptor (PXR) activation also was a major liability that
ultimately needed to be addressed as well.5 In turn, our laborato-
ries discovered MK-77251 which provided the preclinical pharma-
cokinetic properties desired for a development compound. The last
major hurdle for development of MK-7725 and its related com-
pounds is the atropisomerism that plagues this particular class
due to its unusual planar chirality. Any appreciable isomerization
to the inactive enantiomer can be a major roadblock by lowering
the desired exposure of MK-7725 and thus affecting the clinically
efficacious dose.
⇑
Corresponding author.
E-mail address: harry_chobanian@merck.com (H.R. Chobanian).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.029

2846
H. R. Chobanian et al. / Bioorg. Med. Chem. 20 (2012) 2845–2849
O
O
S
O
O
S
Slow @ <50°C
Me
Me
N
N
Me
Me
Complete
Racemization
N
H
N
H
N
N
F3C
Over 4 h @ 100°C
CF3
2a (R)-configuration
2b (S)-configuration
Figure 1. X-ray structure showing atropisomerism of benzodiazepine sulfonamides.
OH
OH
OH
H
N
O
N
H
N
HO2C
Cl
NH2
NH2
b
a
+
N
CF3
N
H
N
H
N
CF3
CF3
3
4
5
6
HO
TfO
O
S
O
S
O
O
OCF3
OCF3
N
N
d
c
HN
HN
80%
N
N
F3C
F3C
7
8
Pure regioisomer
separated by crystallization
Figure 2. Synthesis of triflate intermediate. Reagents and conditions: (a) Sulfolane, 150 °C (87%); (b) BH3ÁTHF, THF (61%); (c) 4-trifluoromethoxyphenylsulfonyl chloride,
pyridine (54%); (d) Tf2O, DCM, TEA (80%).
Typically complete interconversion can be seen after 4 h at
OH
O
100 °C.5 While this can be dealt with in preclinical development,
N
Me
it can pose a problem for long term thermal stability. The major is-
sue posed is that the S-enantiomer is the active enantiomer while
the R-enantiomer is significantly less active on the BRS-3 receptor.
We decided to embark on the synthesis on novel analogs which
could significantly raise the barrier to atropisomerism.
N
Me
O
O
S
OCF3
N
HN
In collaboration with our colleagues in the analytical chemistry
group, we had previously shown that the use of CD spectroscopy as
a tool for measuring the t1/2 for this class of compounds could be
done in an effective manner. The effect of structure of the benzodi-
azepine sulfonamides could be ascertained very quickly and would
lead us to understand exactly what needed to be done to further
increase this barrier. Ideally, we would like to increase this barrier
substantially while further modifying a very sterically hindered
position of the benzodiazepine sulfonamide. We began to synthe-
size a series of new analogs that would be further evaluated by
CD spectroscopy.6
N
1
F3C
MK-7725
hBRS-3 IC50 3 nM (100% act)
mBRS-3 EC50 22 nM (105% act)
The atropisomerism present with this class of molecules is spe-
cific to the conformationally restrained seven-membered diaze-
pine central ring system. Molecular modeling on similar systems
has suggested a barrier of inversion of ꢀ33 kcal/mol ( Fig. 1).

H. R. Chobanian et al. / Bioorg. Med. Chem. 20 (2012) 2845–2849
2847
NC
TfO
TfO
O
S
O
S
O
S
O
O
O
OCF3
OCF3
OCF3
N
N
b
N
a
N
N
N
HN
N
N
F3C
F3C
10
F3C
8
9
c or d
then e
O
N
NC
N
HO
O
S
O
S
O
O
OCF3
OCF3
N
N
f,g,h
10a = gem-dimethyl
10b = cyclobutyl
HN
HN
N
N
F3C
F3C
gem-dimethyl = compound 11a
cyclobutyl = compound 11b
i,j,k,h
X
NH
O
O
S
O
OCF3
N
HN
12a gem-dimethyl = X = H
12b cyclobutyl = X = H
12c cyclobutyl = X = Me
N
F3C
Figure 3. Synthesis of oxadiazole analogs. Reagents and conditions: (a) BnBr, NaOH (50% v/v), benzyltriethylammonium chloride, toluene, 120 °C (75%); (b) TMSCH2CN, ZnF2,
Pd2dba3, S-Phos, DMF, 180 °C (68%); (c) MeI, NaH, THF (91%); (d) diiodopropane, NaH, THF (66%); (e) TMSI, ACN (70%); (f) NH2OH, K2CO3, EtOH (100%); (g) acetoxyisobutyryl
chloride, pyridine, 80 °C, then K2CO3, MeOH (30–52% overall); (h) Chiral separation (Chiralpak OD, EtOH/hexane) (i) DIBAl-H, THF, (72%); (j) NaClO2, NaH2PO4, 2-Me-2-butene,
tBuOH/H2O (64–93%); (k) X-NH2 (X = H or Me), EDC, HOBt, DCM (80–92%).
Table 1
Binding and functional activity at human and mouse BRS-3
Compound
Human BRS-3 binding12
(IC50, nM)
Human BRS-3 agonisma13
(EC50, nM) (activity %)
Mouse BRS-3 agonisma
(EC50, nM) (activity %)
MK-7725
11a
11b
12a
12b
3.6
73 (106)
130 89 (105)
67 9 (105)
76 13 (99)
27 3 (98)
22 7 (100)
22 5 (112)
11 (87)
1.4 0.3
0.6 0.3
0.5 0.1
0.5 0.3
2.1 0.6
10
64 12 (97)
82 32 (97)
26 8 (90)
21 7 (95)
15 5 (106)
31 (96)
12c
13
a
% Act represents the maximum activation of tested compound relative to that of the dY peptide.
conditions giving rise to a regioisomeric ratio of the phenol (1:1)
which could be separated out by crystallization following subse-
quent borane reduction and sulfonylation. The pure regioisomer
7, isolated via crystallization was then treated with trifluorometh-
anesulfonic anhydride giving rise to the corresponding triflate 8.6
2. Chemistry
The synthesis of the key triflate intermediate of interest
commenced with a condensation of 2-chloro-6-trifluoromethyl
nicotinic acid (4) and 2,3-diaminophenol (3) under thermal

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H. R. Chobanian et al. / Bioorg. Med. Chem. 20 (2012) 2845–2849
Table 2
of a potency boost. The secondary amide 12a also showed excellent
in vitro potency across species making it a viable candidate for fur-
ther profiling.
Rat PK data for BRS-3 agonists14
Compound Clp (mL/min/kg) T1/2 (h) PO AUCN
(
lM h kg/mg) Foral (%)
These exciting in vitro results prompted a comparative PK study
of 11a, 11b, 12a–c, and MK-7725 (Table 2). One can surmise that
the linker in compounds 11a and 11b seems to cut the half-life
by ꢀfourfold, however the exposure and bioavailability remain
excellent in only the case of compound 11a. Compound 11b has
MK-7725
11a
11b
12a
12b
16
12
21
33
31
25
11
3.1
2.9
1.6
0.8
1.7
1.60
2.60
0.60
0.30
0.30
0.69
93
100
49
31
32
12c
60
lower oral exposure (0.60 lM h kg/mg) and comparatively worse
bioavailability (49%) than oxadiazole 11a. This could be due to
the decreased measured aqueous solubility of compound 11b ver-
sus 11a. Of the amides 12a–c, each had comparable PK in the rat.
With all this information at hand, we now set out on our ultimate
goal to determine whether any of these compounds would be im-
proved inherently with regards to the atropisomerism issue.
CD spectroscopy is a useful tool for monitoring atropisomer
equilibration particularly for compounds which have chromoph-
ores. In order to achieve this, a timecourse CD spectrum of pure
MK-7725 and its respective enantiomer are subjected to a CD scan.
Once the basal levels of absorption are realized, a time course
experiment can be performed whereby MK-7725 is heated to
60 °C in EtOH over a 3 day period. The respective data obtained
at the 260 nm wavelength is plotted versus time in order to arrive
at a half-life calculation for the interconversion. In the case of MK-
7725, the t1/2 was calculated to 2.2 h. However, once we began to
examine novel derivatives such as 11a, 11b, and 12b an interesting
trend developed (Fig. 4). All three compounds possessed a half-life
greater than 250 h. This represented a major improvement and
makes subsequent development of these particular chemical enti-
ties significantly less complex and more cost-effective.
Atropisomer Interconversion as measured by CD
(60oC in EtOH)
% Initial CD Signal
100
80
MK-7725
11b
12b
60
40
11a
20
0
0
5
10
time (hours)
15
20
Figure 4. CD spectra for specific analogs of interest.
3. Conclusions
At this point, our goal was to introduce a bulky substituent alpha
to the oxadiazole ring present in MK-7725 (Fig. 2). With the desired
triflate 8 in hand, we sought to protect the free amine present. To
accomplish this, we chose phase transfer conditions using benzyl
bromide and benzyltriethylammonium chloride with 50% sodium
hydroxide in toluene. Upon heating to reflux, the desired benzyl
protected intermediate was isolated. Triflate 9 was subjected to a
modified palladium catalyzed coupling of trimethylsilylacetonitrile
using the S-Phos ligand.7,8 The resulting acetonitrile derivative 10
was treated with NaH followed by either MeI or diiodopropane giv-
ing rise to the respective gem-dimethyl nitrile (10a) or the cyclob-
utylnitrile (10b) derivative. Upon surveying a host of debenzylation
conditions, the most reproducible conditions were using TMS-I in
acetonitrile. The debenzylated acetonitrile derivative was con-
verted to the subsequent amide oxime, acylated with acetoxyisobu-
tyryl chloride, and cyclized thermally to furnish the desired 1,2,4-
oxadiazole. Compounds 11a and 11b were obtained in enantiopure
form after deacetylation with K2CO3/MeOH and subsequent chiral
HPLC resolution (Fig. 3).9,10
In summary, we were able to solve a long standing atropisom-
erism problem with the benzodiazepine sulfonamides. This in-
crease in the barrier of atropisomerism would allow for more
rapid development of a solid, stable crystalline form of these com-
pounds. In addition, any racemization at room temperature could
be avoided by building in a linker at the alpha position. The syn-
thetic complexity was increased but with the added benefit of a
>25-fold increase in the barrier of inversion. In addition, to our sur-
prise, these analogs generally maintained or improved upon exist-
ing potency values without a significant change in rat PK. Follow
up studies will be reported in due course.
References and notes
1. Chobanian, H. R.; Guo, Y.; Liu, P.; Chioda, M.; Lanza, T. J.; Chang, L.; Kelly, T. M.;
Kan, Y.; Palyha, O.; Guan, X.; Marsh, D. J.; Metzger, J. M.; Gorski, J. N.; Raustad,
K.; Wang, S.-P.; Strack, A. M.; Miller, R.; Pang, J.; Madeira, M.; Lyons, K.;
Dragovic, J.; Reitman, M. L.; Nargund, R. P.; Lin, L. S. ACS Med. Chem. Lett. 2012,
3. ASAP.
Primary and secondary amide derivatives were prepared via a
three-step sequence consisting of DIBAl-H reduction, Pinnick oxi-
dation,11 and EDC coupling. Enantiomerically pure compounds
12a, 12b, and 12c were then obtained by chiral HPLC resolution.
Compounds 11a and 11b both possessed excellent binding po-
tency (hIC50 = 1.4 and 0.6 nM) and both had very good mouse func-
tional activity (mEC50 = 67 and 76 nM) (Table 1). In comparison to
MK-7725, both compounds show a several fold improvement in
human binding potency in the in vitro assay. Simple amides such
as compounds 12b and 12c showed exquisite potency (hIC50 = 0.5
and 2.1 nM). In comparison to its truncated primary amide cousin,
compound 13, the intrinsic binding potency was improved by
5- to 10-fold. This would indicate not only was the space of impor-
tance as a possible means to increase the barrier of inversion for
the atropisomerism that exists with MK-7725, but is also a source
2. Olshansky, S. J.; Passaro, D. J.; Hershow, R. C.; Layden, J.; Carnes, B. A.; Brody, J.;
Hayflick, L.; Butler, R. N.; Allison, D. B.; Ludwig, D. S. N. Engl. J. Med. 2005, 352,
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3. (a) Ohki-Hamazaki, H.; Watase, K.; Yamamoto, K.; Ogura, H.; Yamano, M.;
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Hampton, L. L.; Battey, J. F.; Moran, T. H. Endocrinology 2008, 149, 971–978.
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Guo, C.; Guzzo, P. R.; Hadden, M.; Henderson, A. J.; Ruenz, M.; Sargent, B.; Yet,
L.; Kelly, T. M.; Palyha, O.; Kan, Y.; Pan, J.; Chen, H.; Marsh, D.; Shearman, L. P.;
Strack, A. M.; Metzger, J. M.; Feighner, S. D.; Tan, C.; Howard, A. D.;
Tamvakopoulos, C.; Peng, Q.; Guan, X.-M.; Reitman, M. L.; Patchett, A. A.;
Wyvratt, M. J.; Nargund, R. P. Bioorg. Med. Chem. Lett. 2010, 20, 1913–1917; (b)
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radioligand bound to the receptor was measured by liquid scintillation
counting of the radioactivity on the filter. The nonspecific binding was
defined as the binding in the presence of 100 nM unlabeled dY-bombesin.
The data, as % inhibition of binding, was plotted vs. the log molar concentration
of receptor ligand (compound). The IC50 was reported as the inflection point of
the resulting sigmoidal curve.
6. Welch, C. J.; Gong, X.; Schafer, W.; Chobanian, H. R.; Lin, L. S.; Biba, M.; Liu, P.;
Guo, Y.; Beard, A. Chirality 2009, 21, 105–109.
13. The functional assay is an aequorin bioluminescence assay. It was performed in
7. Wu, L.; Hartwig, J. F. J. Am. Chem. Soc. 2005, 127, 15824–15832.
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G.; Zaugg, W. Tetrahedron Lett. 2001, 42, 385–389.
10. Baker, R.K.; Chang, L.L.; Chioda, M.; Chobanian, H.R.; Gao, Y.; Guo, Y.; Lin, L.S.;
Liu, P.; Nargund, R.P.; Rupprecht, K.M.; Miao, S. PCT Int. Appl. WO 2008073311,
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11. Ball, M.; Gaunt, M. J.; Hook, D. F.; Jessiman, A. S.; Kawahara, S.; Orsini, P.;
Scolaro, A.; Talbot, A. C.; Tanner, H. R.; Yamanoi, S.; Ley, S. V. Angew. Chem., Int.
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a 96-well format using a Wallac Microbeta luminometer equipped with
microinjector module. Compounds in DMSO (0.5% final concentration) were
titrated in the plates at 2 Â concentration in a volume of 0.1 mL ECB buffer
(20 mM HEPES, pH 7.4, 140 mM NaCl, 20 mM KCl, 1 mM MgCl2, 1 mM CaCl,
5 mM glucose, 0.1 mg/ml BSA). The HEK293AEQ cells from lines expressing
either human, rat or mouse BRS3 (20,000 per well) were charged with
coelenterazine (Molecular Probes) and then injected in 0.1 mL ECB buffer into
the compound containing wells. The bioluminescence was monitored for 30 s,
or alternatively, total bioluminescence was determined over 10 min. The
bioluminescent readings were plotted versus the log molar concentration of
receptor ligands (compounds). The EC50 for activation was reported as the
inflection point of the resulting sigmoidal curve. The percentages of activation
are the maxim activations of tested compounds relative to that of dY-peptide.
12. For human BRS-3 binding assays, 1–4 lg of membrane protein obtained from
NFAT-CHO cells expressing the receptor were incubated with 0.3 pM [125I]-[D-
Tyr6, b-Ala11, Phe13, Nle14]-bombesin (6–14) (125I-dY-peptide) and various
14. Sprague Dawley (SD) rat was used. Plasma clearance (Clp) and half life (T1/2)
concentrations of test compounds in 200
7.2, 5 mM MgCl2, 0.1% BSA). After 2 h incubation at room temperature, the
binding reaction was terminated by filtering through a GF/c filter and washing
l
L of binding buffer (50 mM Tris, pH
calculated following 0.5 mg/kg iv dose. Normalized oral exposure (PO AUCN)
and oral bioavailability (F%) calculated following 1 mg/kg po dose.