Synthesis and evaluation of substituted 4-alkoxy-2-aminopyridines as novel neuropeptide Y1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.01.049

A series of substituted 4-alkoxy-2-aminopyridines 2, which were formally derived from neuropeptide Y1 antagonist 1 by replacing the morpholino portion with alkoxy groups, were synthesized and evaluated as neuropeptide Y Y1 receptor antagonists. Primary structure-activity relationships and identification of potent 4-alkoxy derivatives are described.

Full text of DOI:10.1016/j.bmcl.2004.01.049

Bioorganic& Medicinal Chemistry Letters 14 (2004) 1761–1764
Synthesis and evaluation of substituted 4-alkoxy-2-aminopyridines
as novel neuropeptide Y1 receptor antagonists
Nagaaki Sato,* Takunobu Shibata, Makoto Jitsuoka, Toshiyuki Ohno,
Toshiyuki Takahashi, Tomoko Hirohashi, Tetsuya Kanno, Hisashi Iwaasa,
Akio Kanatani and Takehiro Fukami
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd, Okubo 3, Tsukuba 300-2611, Japan
Received 2 December 2003; accepted 14 January 2004
Abstract—A series of substituted 4-alkoxy-2-aminopyridines 2, which were formally derived from neuropeptide Y1 antagonist 1 by
replacing the morpholino portion with alkoxy groups, were synthesized and evaluated as neuropeptide Y Y1 receptor antagonists.
Primary structure–activity relationships and identification of potent 4-alkoxy derivatives are described.
# 2004 Elsevier Ltd. All rights reserved.
Neuropeptide Y (NPY) is the most potent orexigenic
peptide identified to date.1À3 Chronicadministration of
NPY into the brain results in body weight gain with
we attempted to further extend the structural diversity
of this series. The structure–activity relationships (SAR)
for the 6- and N-substituents of the morpholino-2-
aminopyridine core of compound 1 have been investi-
gated in detail over the past few years;14 however, the
4-morpholino portion remains to be explored. Explora-
tion of the 4-substituent was initiated by the replace-
ment of the morpholino group with various alkoxy
groups. In this work, we report the synthesis and the
preliminary SAR of the 4-alkoxy-2-aminopyridine deriv-
atives 2.
hyperphagia, reduces energy expenditure, and increases
4,5
lipogenicatcivity in the liver and adipose tissue.
In
addition, NPY-deficient ob/ob mice were less obese and
exhibited reduced food intake when compared with ob/
ob mice.6 From these findings, NPY has been inferred to
be a major regulator of energy balance. Accumulating
evidence shows that NPY-mediated feeding might be
regulated by multiple hypothalamicNPY receptor sub-
types.7À13 The Y1 receptor is considered to be a major
feeding receptor. Compound 1 is one of the most pro-
mising ligands (Fig. 1), with in vivo Y1 specificity
demonstrated by feeding experiments using Y1 receptor
deficient mice.13 With this promising structure in hand,
Two possible precursors 3 and 4 (Scheme 1) were con-
sidered on the basis of three reliable chemical reactions
(A, B, and C). The alkylation of hydroxypyridine 3 (A)
appeared not be suitable due to the limited availability
Figure 1. Structures of compound 1 and 2.
* Corresponding author. Tel.: +81-29-877-2000; fax: +81-29-877-2029; e-mail: satoung@banyu.co.jp
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.049

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N. Sato et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1761–1764
of alkylating reagents. For the Mitsunobu reaction (B)
and the aromaticnucleophilicsubstitution reaction (C),
commercially available alcohols can be utilized as the
source of the alkyl group.15 In the substitution reaction
(C), a methanesulfonyl group was considered to be a
better leaving group than the frequently utilized chloro
group, since the reactive methanesulfonyl group can be
masked as a sulfide group in the course of the synthesis.
Considering operational simplicity and ease of purifica-
tion associated with arising side products, the substitu-
tion reaction (C) was thought to be ideal for the present
study. Thus, we decided to develop a synthesis of the 4-
methanesulfonylpyridine 4 (Scheme 2). The starting
bromide 5 smoothly reacted with sodium thiomethoxide
to give the corresponding thiomethoxy ether. The two
symmetrical esters were differentiated by half-reduction
using NaBH4 in the presence of CaCl2 to afford the
hydroxy ester 6 in 72% yield over 2 steps. The hydroxy
group of 6 was protected as its tetrahydropyranyl
(THP) ether. The ester 7 was hydrolyzed to the corres-
ponding carboxylic acid, which was treated with diphe-
nylphosphoryl azide (DPPA) followed by thermal
Scheme 1.
Scheme 2. Reagents and conditions: (a) (i) CH3SNa, DMF, 0 ꢀC, 10 min, (ii) NaBH4, CaCl2, EtOH, rt, 3 h, 72%; (b) DHP, PPTS, CHCl3, 50 ꢀC,
1.5 h; (c) (i) 1 N aqueous NaOH–MeOH, 40 ꢀC, 1 h, (ii) DPPA, Et3N, 1,4-dioxane, rt, 1 h, (iii) t-BuOH, 1,4-dioxane, reflux, 0.5 h, 57% from 6;
ꢀ
ꢀ
.
(d) (i) p-TsOH, EtOH, 40 C, 1 h, (ii) SO3 Py, DMSO, Et3N, rt, 0.5 h, (iii) (MeO)2P(O)CH2CO2Me, NaH, THF, 0 C, 0.5 h, 70%; (e) (i) NaBH4,
NiCl2, THF–MeOH, rt, 0.5 h, (ii) 4N aqueous NaOH, THF–MeOH, 40 ꢀC, 2 h, 83%; (f) 2-amino-3-pentanone, EDCI, HOBt, N-methylmorpholine,
DMF, rt, 15 h, 87%; (g) Lawesson’s reagent, toluene, reflux, 6 h, 79%; (h) NaH, DMF, 0 ꢀC, 0.5 h, then 1-chloro-3-(chloromethyl)-5-nitrobenzene,
0 ꢀC, 1 h, 100%; (i) (i) KMnO4, 20% aqueous AcOH–acetone, rt, 10 min, (ii) Fe, NH4Cl, MeOH–H2O, reflux, 2.5 h, 77%; (j) i-PrOCOCl, DMAP,
CHCl3, rt, 2 h, 99%; (k) TFA, rt, 10 min, 99%.
Scheme 3.

N. Sato et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1761–1764
1763
rearrangement in refluxing 1,4-dioxane in the presence
of tert-butylalcohol to furnish tert-butoxycarbonyl-
aminopyrdine 8 in 57% yield over 4 steps. The hydroxy
group of 8 was unmasked with pyridinium p-toluene-
sulfonate (PPTS) and oxidized to the corresponding
aldehyde with sulfurtrioxide–pyridine complex in DMSO,
which was subjected to the Horner–Emmons reaction
with trimethyl phosphonoacetate and NaH to provide
the a,b-unsaturated ester 9 in 70% yield from 8. The
reduction of the double bond of 9 with a combination of
NaBH4 and NiCl2 followed by saponification gave 10 in
83% yield. The carboxylic acid 10 and 2-amino-3-pen-
tanone were coupled, and treatment of the resulting
ketoamide 11 with Lawesson’s reagent effected
cyclization, affording thiazole 12 in 79% yield. 1-Chloro-
3-(chloromethyl)-5-nitrobenzene, which was derived
from chlorination of 1-chloro-3-(hydroxymethyl)-5-nitro-
benzene with thionyl chloride, was coupled with 12 in
the presence of NaH to furnish 13 in quantitative yield.
Oxidation of the thiomethoxy group of 13 to the
corresponding methanesulfonyl group was best-accom-
plished using potassium permanganate, and the sub-
sequent reduction of the nitro group with Fe afforded
14 in 77% yield in 2 steps. Treatment of 14 with iso-
propyl chloroformate in the presence of 4-(dimethyl-
amino)pyridine (DMAP) followed by cleavage of the
tert-butoxycarbonyl (Boc) group under acidic condi-
tions afforded the key intermediate 4. With methane-
sulfonylpyridine 4 in hand, we examined the substitution
of the methanesulfonyl group by alkoxide anions. The
substitution reaction of 4 turned out to be sluggish; the
desired substituted product was obtained in at most
20% yield. It was subsequently determined that the
protected precursor 15 participates in the substitution
reaction under mild conditions (Scheme 3). The desired
alcohol (5 equiv) and 15 were combined in anhydrous
DMSO, which was treated with 5 equiv of t-BuOK at
ambient temperature. The starting 4-methanesulfo-
nylpyridine 15 was completely consumed within 10 min,
giving the desired 4-alkoxypyridine with 30–80% yield.
Deprotection of the Boc group with TFA afforded the
target alkoxy compound 2. The crude products were
purified by HPLC (YMC-Pack Pro C18 (50Â30 mm
I.D.) with liner gradient system of H2O–CH3CN–TFA
75:25:0.1 to 25:75:0.1 over 8 min and at a flow rate of 40
mL/min) and tested as TFA salts.
Table 1. Y1Binding affinitiesof4-alkoxy-2-aminopyridinederivativesa
Compd
RO
Binding affinityb
(IC50, nM)
[Ca2+]i responsec
(IC50, nM)
1
2a
2b
Morpholino
MeO
EtO
1.4Æ0.2
234Æ9
33Æ6
17Æ2
16Æ4
35Æ4
63Æ3
2.9Æ0.2
d
d
d
d
d
d
d
2c
2d
2e
2f
2g
2h
72Æ10
4.7Æ1.4
64Æ7
d
2i
12Æ1
8.4Æ1.0
17Æ3
2j
59Æ9
d
2k
2l
d
55Æ3
2m
2n
2o
2p
2q
2r
5.5Æ1.7
11Æ1
33Æ3
d
d
d
11Æ2
29Æ1
4.4Æ2.2
237Æ27
42Æ2
d
Our syntheticroute allowed us to prepare the sub-
stituted 4-alkoxy derivatives incorporating a variety of
commercially available alcohols (Table 1).16À18 The
normal alkyl derivatives (2a–g) were first prepared in
order to estimate the size of the binding pocket. The
propoxy and butoxy derivatives (2c and d) displayed the
highest binding affinities. The derivatives with longer or
shorter chain length showed a decrease in the binding
affinity. It should be noted that the hexenyloxy deriva-
tive 2h had a considerably higher binding affinity
(IC50=4.7 nM), despite its rather long chain length. The
structural constraint imparted by the internal double
bond might be responsible, which may provide an
opportunity for further optimization. With the approx-
imate size of the binding pocket in mind, the branched
alkoxy (2i–m) and cycloalkyloxy (2n–q) derivatives were
d
d
2s
2t
2u
30Æ3
20Æ2
6.5Æ1.2
46Æ6
a The values represent the meanÆSE for n=3.
b Human recombinant Y1 receptors in CHO/dhFrÀ cell membranes,
[
125I]PYY; see ref 13.
c Antagonistic activities (human recombinant Y1 receptors in CHO/
dhFrÀ cells) at 1 nM NPY stimulation; see ref 13.
d Not determined.

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N. Sato et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1761–1764
examined. Branching was found to be effective to
enhance potency. The 1-ethylpropoxy derivative 2m was
3-fold more potent than the parent propoxy derivative
2c. The cyclization strategy was not found to result in
significant improvement with respect to potency; how-
ever, the pyranyloxy derivative 2q was found to have a
high binding affinity (IC50=4.4 nM), a 7-fold improve-
ment over the cyclohexyloxy derivative 2p. The sig-
nificantly improved binding affinity is attributed to the
oxygen atom of the pyranyl ring that is probably acting
as a hydrogen bonding acceptor. Finally phenyl sub-
stituted derivatives were examined. It is important to
note that phenoxy derivatives could not be prepared
since phenoxide anions did not participate in the current
substitution reaction. Substitution in the phenyl moiety
was not observed to contribute to the enhanced binding
affinity (2r–t); however, the binding affinity of the pyr-
idyl derivative 2u was relatively higher than the corres-
ponding phenyl derivative 2t. This slight increase in
binding affinity is probably ascribed to electrostatic
effects, rather than the hydrogen bonding acceptor
property of the pyridine ring. The antagonisticactivities
of the selected high-affinity compounds were mea-
sured by their ability to inhibit NPY-induced [Ca2+]i
increases in CHO/dhFrÀ cells, which expressed the
recombinant human Y1 receptor.16 In this [Ca2+]i
functional assay, 2h, j, m, q, and u dose-dependently
inhibited the [Ca2+]i increase (See Table 1 for their IC50
values).
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16. See ref 13 for the experimental conditions for the binding
and functional assays described herein.
In summary, a number of substituted 4-alkoxy-2-ami-
nopyridine derivatives 2 were synthesized and evaluated
for their binding affinities to the human NPY Y1
receptor. Several potent antagonists were identified
among them. The primary SAR was elucidated, reveal-
ing that a number of alkoxy substituents are capable of
replacing the morpholino portion of compound 1. In
addition, the activities of the potent derivatives 2h, j, m,
q, and u are of interest for use in in vivo studies. The
pharmacokinetics and brain penetrability of these deri-
vatives in rodents remain to be addressed for further in
vivo evaluation of the present alkoxy type of Y1
antagonists.
17. All of the compounds tested for Y1 binding are >95%
pure. Analytical HPLC analyses were performed under
the following conditions: Wakopak combi ODS fast
(30Â2.0 mmI.D.) with liner gradient system of H2O–
CH3CN–TFA 95:5:0.1 to 5:95:0.1 over 6 min and at a
flow rate of 0.8 mL/min.
1
18. Selected spectral data: 2q TFA salt: H NMR (400 MHz,
CDCl3) d 1.26 (3H, t, J=7.4 Hz), 1.30 (6H, d, J=6.3 Hz),
1.70 (2H, m), 1.87 (2H, m), 2.34 (3H, s), 2.74 (2H, q,
J=7.4 Hz), 3.19 (2H, t, J=7.6 Hz), 3.43 (2H, t, J=7.6
Hz), 3.54 (2H, ddd, J=2.8, 8.8, 12.2 Hz), 3.91 (2H, td,
J=4.4, 12.2 Hz), 4.37 (2H, d, J=5.4 Hz), 4.46 (1H, m),
4.99 (1H, sept, J=6.3 Hz), 5.69 (1H, d, J=2.2 Hz), 6.23
(1H, d, J=2.2 Hz), 6.97 (1H, s), 7.22 (1H, s), 7.28 (1H, s),
7.54 (1H, s), 10.38 (1H, t, J=5.4 Hz); MS (ESI) m/z 573.2
[M+H]+. 2u TFA salt: 1H NMR (400 MHz, CDCl3) d
1.28 (6H, d, J=6.3 Hz), 1.33 (3H, t, J=7.4 Hz), 2.14 (2H,
m), 2.23 (3H, s), 2.80 (2H, q, J=7.4 Hz), 2.98 (2H, t,
J=7.2 Hz), 3.21 (2H, dd, J=6.7, 9.1 Hz), 3.63 (2H, dd,
J=6.7, 9.1 Hz), 4.06 (2H, t, J=6.0 Hz), 4.41 (2H, d,
J=5.6 Hz), 4.97 (1H, sept, J=6.3 Hz), 5.71 (1H, d, J=2.2
Hz), 6.37 (1H, d, J=2.2 Hz), 6.97 (1H, s), 7.25 (1H, s),
7.40 (1H, s), 7.52 (1H, s), 7.77 (1H, dd, J=5.6, 8.0 Hz),
8.13 (1H, d, J=8.0 Hz), 8.66 (1H, dd, J=1.4, 5.6 Hz),
8.74 (1H, d, J=1.4 Hz), 10.24 (1H, t, J=5.6 Hz); MS
(ESI) m/z 608.2 [M+H]+.
Acknowledgements
We thank Dr. Steven A. Weissman (Merck Research
Laboratories) for critical review of the manuscript.
References and notes
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