Discovery and optimization of a novel Neuromedin B receptor antagonist

Article abstract of DOI:10.1016/j.bmcl.2009.05.124

The discovery and parallel synthesis of potent, small molecule antagonists of Neuromedin B receptor based on the ary-hexahydro-dibenzodiazepin-1-one core is described.

Full text of DOI:10.1016/j.bmcl.2009.05.124

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4264–4267
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of a novel Neuromedin B receptor antagonist
a
a
b
b
Jiasheng Fu a, , Stephen J. Shuttleworth , Richard V. Connors , Anne Chai , Peter Coward
*
a Department of Chemistry, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
b Department of Biology, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and parallel synthesis of potent, small molecule antagonists of Neuromedin B receptor
based on the ary-hexahydro-dibenzodiazepin-1-one core is described.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 31 March 2009
Revised 20 May 2009
Accepted 22 May 2009
Available online 6 June 2009
Keywords:
NMBR
Antagonist
Parallel synthesis
Ary-hexahydro-dibenzodiazepin-1-one
In mammals, the bombesin family of G protein-coupled recep-
tors consists of three subtypes:1 Neuromedin B receptor (NMBR,
BB1),2 Gastrin-releasing peptide receptor (GRPR, BB2),3 and BRS-
3.4 While high affinity endogenous ligands for BRS-3 are unknown,
Neuromedin B (NMB) and Gastrin-releasing peptide (GRP) were
discovered in the early 1980s.5,6 NMB and GRP share sequence
homology in the C-terminal region and mediate a range of biolog-
ical mechanisms via action at their receptors. These include CNS-
related responses such as thermoregulation,7 satiety,8 control of
circadian rhythm,9 and modulation of fear and anxiety re-
sponses,10,11 as well as peripheral functions including macrophage
activation12 and gastrointestinal hormone release.13 In addition,
there is considerable literature suggesting a role in control of cellu-
lar proliferation.14
Previously, we and others described the discovery of small mol-
ecule ligands for NMBR.15 In an effort to develop tool compounds
to define how these receptors influence pathological processes,
we report here the identification and optimization of potent and
selective NMBR antagonists. The initial lead in this series, benzodi-
azepine 1 (Fig. 1), was identified as an NMBR antagonist from a
high-throughput screen (HTS). Compound 1 displayed encouraging
potency in a NMB displacement assay (IC50 = 300 nM)16 and was
the discovery of a novel, potent spirocyclic derivative. Data on
the most representative compounds are presented herein.
The synthesis of benzodiazepines is well documented.17 A series
of libraries based on 1 was prepared as described in Scheme 1
using the modified method reported by Blache et al.17b The synthe-
sis began with a condensation between a 2-nitroaniline (A) and a
1,3-cycloalkane dione. The resulting enamine (B) was either meth-
ylated or left unchanged before reduction to the diamine (C).18 For-
mation of the seven-member ring was accomplished via
a
Mannich-type cyclization between the diamine and an aryl alde-
hyde, this cyclization was carried out at room temperature in eth-
anol containing a catalytic amount of acetic acid.19 Alkylation of
the resulting secondary amine (D) was easily effected by treatment
with an aldehyde and Na(AcO)3BH.20 All of the final products were
purified by preparative HPLC.21
An analysis of the preliminary SAR derived from the HTS data
suggested that the methyl substituent on the fused aryl of 1 is re-
CH3
H
CH3
N
selective (10 lM or greater) against a panel of other GPCR targets,
including GRPR and other neuropeptide receptors. The benzodiaz-
epine scaffold is highly amenable to diversification using parallel
synthesis, and we were able to rapidly prepare and evaluate over
200 compounds as part of a series of small libraries to facilitate
H3C
N
O
Cl
H
F
1
IC50
(
125I-NMB) = 0.30 µM
* Corresponding author. Tel.: +1 650 244 2083.
E-mail address: jfu@amgen.com (J. Fu).
Figure 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.124

J. Fu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4264–4267
4265
Table 1
CH3
CH3
N
R2
R2
CH3
CH3
Dean-Stark
NH
H
N
R1
R1
CH3
CH3
O
NO2
NO2
O
R
N
H
O
A
B
Cl
R2 = H
F
R2 = H
CH3I
O
Na2CO3
33%
R2 = CH3
25%-70%
ID #
R
IC
( lM)
125I-NMB) (
50
1
2
3
4
5
6
7
8
9
CH3
CF3
Cl
CF3O
CN
CH3O
COOH
OH
0.30
0.37
0.86
1.3
1.3
2.2
10
CHO
CH3
CH3
R2
N
R3
H2
R1
EtOH, AcOH
65%-80%
Pd/C
27%-85%
NH2
O
10
10
CONH2
C
R2 = H, or CH3
CH3
CH3
R2
N
CH3
O
R2
N
Table 2
CH3
R4
CH3
CH3
H
R1
H
R1
N
N
O
N
H
NaBH(OAc)3
30%-60%
R4
O
R3
H3C
R3
N
H
O
R2
R1
D
Scheme 1.
ID #
R1
R2
IC50 ( lM)
125I-NMB) (
1
10
11
12
13
14
15
16
17
F
Cl
0.30
0.20
0.25
0.28
0.55
1.1
1.3
2.5
6.9
H
H
H
H
H
H
F
CH3S
CF3
Cl
NO2
CH3O
F
35
(+)-enantiomer
IC50 (
125I-NMB) =30 nM
H
F
H
N
IC50 HeLa Cytotoxicity >50 µM
Chiral HPLC
H
H3C
N
H
O
CF3
36
The next series of compounds was prepared to examine the ef-
fects of alkylating the benzodiazepine nitrogen atoms. The data,
summarized in Table 3, show that methylation at R1 resulted in a
dramatic loss of affinity (18), while alkylation at R2 conferred a
marginal increase in potency (19, 20 and 21).
(-)-enantiomer
30
IC50 (
125I-NMB) >10 µM
Scheme 2.
We next turned our attention to the alicyclic portion of the mol-
ecule (Table 4). Significant loss of affinity was observed for the
unsubstituted (25) and ring contracted (29) analogs, indicating
that the cyclohexyl ring and the geminal dimethyl are important
contributors to binding. Moreover, no improvements were ob-
quired for binding affinity. Therefore, in the first library alterna-
tives to the methyl group were examined. As the data in Table 1
show, efforts to replace the methyl group with polar functions
yielded ligands with diminished binding affinity. However elec-
tron-deficient functions were tolerated, as exemplified by the CF3
and Cl analogs 2 and 3. For subsequent libraries, we elected to
maintain CH3 substitution as R.
Table 3
R1
N
CH3
CH3
A second library focused on variants of the exocyclic phenyl
motif. The data in Table 2 summarize the results of this study.
Compounds 12, 15 and 17 nicely illustrate the impact on binding
affinity of the exocyclic aryl substituents of 1. Compound 12, for in-
stance, shows that removal of the fluorine atom has a minimal ef-
fect on binding. However when the chlorine atom is removed, as in
compound 15, binding affinity is diminished fourfold. The reduced
binding affinity of the difluoro compound 16 shows that the elec-
tron withdrawing nature of the substituents is not a key factor
influencing binding. This point is further supported by compound
10, which bears a space-filling substituent on a relatively electron
rich aryl.
H3C
R2
N
O
Cl
F
ID #
R1
R2
IC50 ( lM)
125I-NMB) (
1
18
19
20
21
H
CH3
H
H
H
H
H
CH3
C2H5
i-C4H9
0.30
10
0.17
0.18
0.15

4266
J. Fu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4264–4267
Table 4
Table 5
H
N
H
N
R
H3C
N
H
O
H3C
Cl
N
H
O
R2
F
R1
ID #
R
IC50 ( lM)
125I-NMB) (
ID #
R1
R2
IC50
(
125I-NMB) (
lM)
30
31
32
33
34
H
H
H
F
CF3
Cl
CH3S
CF3
CF3S
0.096
0.24
0.25
0.54
0.64
22
0.28
H
O
23
24
0.45
0.81
2000
1500
1000
500
0
DMSO
O
O
O
1µM (35)
3µM (35)
10µM (35)
25
26
1.4
2.0
O
-14
-12
-10
-8
-6
-4
log[NMB],M
EC50 NMB = 318pM
O
O
O
EC50 NMB (+1µM 35) = 29nM
EC50 NMB (+3µM 35) = 78nM
EC50 NMB (+10µM 35) = 247nM
O
27
28
3.0
5.4
Figure 2.
N
phosphate25, right-shifting a dose response curve of NMB. In addi-
tion, 35 reduced the activity of a sub-maximal dose of NMB, with
an IC50 of 580 nM, a 10-fold improvement over 1 (Fig. 3).
29
10
O
NMB
2500
1 nM NMB + 1
1 nM NMB + 35
2000
1500
1000
500
0
served for the mono-substituted analogs 23, 24, and 26–28. Inter-
estingly, spirocycle 22 showed slightly increased affinity, suggest-
ing that a rigid structure may be favored in this region of the
molecule.
Finally, we revisited exocyclic phenyl substitutions in the con-
text of the spirocyclic derivative 22 (Table 5). Several potent ana-
logues were identified, with compound 30 displaying a threefold
increase in potency compared to the original lead compound 1.
Compound 30 was resolved using preparative chiral HPLC to fur-
nish two optically-pure enantiomers (Scheme 2) (35 and 36).22,23
The (+)-enantiomer 35 was more potent than the racemic mixture,
and 10-fold more potent than 1. Compound 35 showed no cytotox-
-12
-10
-8
-6
-4
log[cmpd],M
icity at concentrations up to 30 lM when incubated with HeLa or
IC50 1 (+ 1nM NMB) = 5.5µM
IC50 35 (+ 1nM NMB) = 580nM
HEK293 cells for 72 h.24 The (ꢀ)-enantiomer, 36, was inactive.
Figure 2 illustrates that compound 35 behaved as an NMBR
antagonist in a signaling assay measuring accumulation of inositol
Figure 3.

J. Fu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4264–4267
18. Joseph, B.; Bethard, A.; Lesur, B.; Guillaumet, G. Synlett 2003, 10, 1542.
4267
In summary, we have identified a novel benzodiazepine-based
NMBR antagonist (35) using a rapid parallel synthesis approach.
This potent antagonist has potential as a tool compound to study
the biology controlled by NMBR.
19. Cortes, C. E.; Hernandez, S. A. M.; Garcia, M. O. J. Heterocycl. Chem. 2002, 39, 55.
20. Castanedo, G. M.; Sailes, F. C.; Dubree, N. J. P.; Nicholas, J. B.; Caris, L.; Clark, K.;
Keating, S. M.; Beresini, M. H.; Chiu, H.; Fong, S.; Marsters, J. C., Jr.; Jackson, D.
Y.; Sutherlin, D. P. Bioorg. Med. Chem. Lett. 2002, 12, 2913.
TM
21. Purification was conducted using a Parallex Flex HPLC system purchased from
Biotage Inc. Final products had 95% purity by HPLC and the structures were
confirmed by LC–MS and 1H NMR.
References and notes
22. Resolution of 30 was accomplished using a Water 600 Tower system equipped
with a Chiralpak AD column (Daicel Chemical Industrial # AD00CJ-DG008,
25 cm ꢁ 2 cm). Protocol: a solution of 30 (10 mg in 0.5 mL isopropyl alcohol)
was injected and isocratic gradient of 60% isopropyl alcohol in hexanes at
20 mL/min was maintained. The fractions corresponding to the two
enantiomers (35 = 5 min; 36 = 20 min) were collected, and concentrated to
afford 35 (2.5 mg) and 36 (2.5 mg). LC–MS analysis confirmed the desired
molecular weights (M+1 = 427.5) of both enantiomers, and purities were
determined by analytical HPLC using a Hewlett Packard series 1050 Tower
system with a Daicel Chemical Industries Chiralpak AD-H column (# AD-H0CE-
DC041, 25 cm ꢁ 0.4 cm), employing an isocratic gradient of 60% isopropyl
alcohol in hexanes at 1 mL/min. Both 35 and 36 were determined to have
enantiomeric excesses greater than 99%. Optical rotation were measured using
1. Moody, T.; Jensen, R. T. Drugs Future 1998, 23, 1305.
2. Wada, E.; Way, J.; Shapira, H.; Kusano, K.; Lebacq-Verheyden, A. M.; Coy, D.;
Jensen, R.; Battey, J. Neuron 1991, 6, 421.
3. Spindel, E. R.; Giladi, E.; Brehm, P.; Goodman, R. H.; Segerson, T. P. Mol.
Endocrinol. 1990, 4, 1956.
4. Fathi, J.; Corjay, M. H.; Shapira, H.; Wada, E.; Benya, R.; Jensen, R.; Viallet, J.;
Sausville, E. A.; Battey, J. F. J. Biol. Chem. 1993, 268, 5979.
5. Minamino, N.; Kangawa, M.; Matsuo, H. Biochem. Biophys. Res. Commun. 1983,
114, 541.
6. McDonald, T. J.; Jornvall, J.; Nilsson, G. Biochem. Biophys. Res. Commun. 1979, 90,
227.
7. Brown, M. R.; Carver, K.; Fisher, L. A.; Fisher, L. A. Ann. N.Y. Acad. Sci. 1988, 547,
174.
a Jasco P-1020 polarimeter (35: ½a D
ꢂ +49.8 (c 0.2, CH3OH). Compound 36: ½aꢂD
8. McCoy, J. G.; Avery, D. D. Peptides 1990, 11, 595.
ꢀ50.3 (c 0.2, CH3OH).
9. Albers, H. E.; Liou, S. Y.; Stopa, E. G.; Zoeller, R. T. J. Neurosci. 1991, 11, 846.
10. Shumyatsky, G. P.; Tsvetkov, E.; Malleret, G.; Vronskaya, S.; Hatton, M.;
Hampton, L.; Battey, J. F.; Dulac, C.; Kandel, E. R.; Bolshakov, V. Y. Cell 2002, 111,
905.
11. Merali, Z.; Bédard, T.; Andrews, N.; Davis, B.; McKnight, A. T.; Gonzalez, M. I.;
Pritchard, M.; Kent, P.; Anisman, H. J. Neuroscience 2006, 26, 10387.
12. DelaFuente, M.; Delrio, M.; Hernanz, A. J. Neuroimmunol. 1993, 48, 143.
13. Tache, Y.; Melchiorri, P.; Negri, L. Ann. N.Y. Acad. Sci. 1988, 517, 1.
14. Gonzalez, N.; Moody, T. W.; Igarashi, H.; Jensen, R. T. Curr. Opin. Endocrinol
Diabetes Obes. 2008, 15, 58.
23. Analytical data for 35: dH (400 MHz, DMSO-d6) 9.02 (1H, s), 7.65 (1H, J = 8.8 Hz,
d), 7.23–7.30 (2 H, m), 6.99 (1 H, J = 10.1 Hz, d), 6.87 (1 H, J = 10.1 Hz, d), 6.50 (1
H, J = 10.1 Hz, d), 6.19 (1 H, s), 6.08 (1 H, J = 7.1 Hz, d), 4.85 (1 H, J = 6.5 Hz, d),
2.74 (1 H, J = 18.5 Hz, d), 2.60 (1 H, J = 18.5 Hz, d), 2.29 (1 H, J = 20.0 Hz, d), 2.13
(1 H, J = 20.0 Hz, d), 1.99 (3 H, s), 1.45–1.65 (6 H, m), 1.27 (1 H, m), 1.06 (2 H,
J = 7.6 Hz, d). M+1 found 427.1; C25H25F3N2O requires 426.5.
24. Protocol for cytotoxicity assay: HeLa cells were seeded at 5 k/well in a 96 well
plate (6 plates-for triplicate 0 and 72 h readings). A threefold dilution series of
each compound was generated, starting at 10 mM. The solution was diluted
eight times to prepare 9 concentrations ranging from 10 mM to 150 nM.
15. (a) Shuttleworth, S. J.; Lizarzaburu, M. E.; Chai, A.; Coward, P. Bioorg. Med.
Chem. Lett. 2004, 14, 3037; (b) Eden, J. M.; Hall, M. D.; Higginbottom, M.;
Horwell, D. C.; Howson, W.; Hughes, J.; Jordan, R. E.; Lewthwaite, R. A.; Martin,
K.; McKnight, A. T.; O’Toole, J. C.; Pinnock, R. D.; Pritchard, M. C.; Suman-
Chauban, N.; Williams, S. C. Bioorg. Med. Chem. Lett. 1996, 6, 2617.
16. Protocol for whole cell radioligand binding assay: Human embryonic kidney
293 (HEK293) cells were stably transfected with expression plasmids encoding
hNMBR. Competition binding assays were performed for 1 h at room
Compound was added to cells (1–100
concentration of compound was 100 M to 15 nM. Zero and 72 h time points
were recorded as follows: (a) 10 L Alamar Blue reagent was added to the
wells, and the samples were incubated at 37 °C for 3 h; (b) fluorescence
intensity was then recorded on an LJL Analyst. The relative growth was
compared to a DMSO control well for each compound.
lL total volume), and the final
l
l
25. Protocol for the inositol phosphate (IP) accumulation assay: hNMBR-
transfected HEK293 cells (2.5 ꢁ 104) were incubated in 96 well plates in
temperature in the presence of 150 pM 125I-[
D-Tyr0]NMB (2200 Ci/mmol,
DMEM High Glucose, w/o
L-glutamine and I-Inositol (US Biological)
Perkin-Elmer Life Sciences) and 1 ꢁ 106 transfected cells in Dulbeco’s Modified
supplemented with 2 g/L sodium bicarbonate, 25 mM Hepes, 2%
glutamine, 10% dialyzed FBS (Gibco), and
1
l
Ci/mL myo-[3H]Inositol
Eagle Media (Mediatech, Inc.) in 100 lL in 96 well plates. Bound ligand was
separated from unbound ligand by filtration using a Filtermate (Packard) and
total counts bound determined on a TopCount NTX reader (Packard). Standard
error of the assay day-to-day was <30%; well-to-well variability was <10%.
17. (a) Sternbach, L. H. J. Med. Chem. 1979, 22, 1; (b) Blache, Y.; Benezech, V.;
Chezal, J.-M.; Boule, P.; Viols, H.; Chavignon, O.; Teulade, J.-C.; Chapat, J.-P.
Heterocycles 2000, 53, 905; (c) Cortés, E. C.; Sanabria, A. M. H.; Mellado, O. G. J.
Heterocycl. Chem. 2002, 39, 55; (d) Cortés, E. C.; Sanabria, A. M. H.; Mellado, O.
G. J. Heterocycl. Chem. 2002, 41, 277.
(82.0 Ci/mmol, Amersham Pharmacia Biotech) overnight at 37 °C. Test
compounds were then added in inositol-free DMEM containing 0.3% BSA
(Sigma) and 10 mM LiCl (Sigma) for 60 min at 37 °C. The cells were lysed
with 20 mM formic acid for 2 h at 4 °C and added to RNA-binding Ysi
scintillation proximity assay beads (Amersham Pharmacia Biotech) for
30 min. Plates were stored overnight at room temperature in the dark and
read the next day on a TopCount NTX.