Design and synthesis of substituted N-methylbenzamide analogues derived from SR 48,968 as neurokinin-2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.06.053

A series of N-methylbenzamide analogues (2-18) that is structurally derived from SR 48,968, a potent neurokinin-2 (NK₂) receptor antagonist (pK_b 9.1), has been obtained using asymmetric synthesis. Isothiocyanato-N-methylbenzamide (10-12) and bromoacetamido-N-methylbenzamide derivatives (16-18) have been designed to serve as potential electrophilic affinity labels. Nitro-N-methylbenzamide (4-6) and acetamido-N-methylbenzamide (13-15) were designed to serve as the nonelectrophilic controls for these ligands. Functional assay results using guinea pig trachea indicate that electrophilic N-methylbenzamide analogues exhibit potent but surmountable NK₂ receptor antagonist activity. Several members of this series (2, 3, 7-9) exhibit potent NK₂ receptor antagonist potencies with pK _b values in the range of 9.1-9.7. para-Fluoro substituted analogue 3 was found to be highly potent with a pK_b of 9.7.

Full text of DOI:10.1016/j.bmcl.2004.06.053

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4779–4782
Design and synthesis of substituted N-methylbenzamide
analogues derived from SR 48,968 as neurokinin-2 receptor
antagonists
Shih-Chung Huang,a Bradley J. Undemb and Vijaya L. Korliparaa,
*
aDepartment of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions,
St. Johnꢀs University, Jamaica, NY 11439, USA
bJohns Hopkins Asthma and Allergy Center, Baltimore, MD 21224, USA
Received 7 May 2004; accepted 16 June 2004
Available online 19 July 2004
Abstract—A series of N-methylbenzamide analogues (2–18) that is structurally derived from SR 48,968, a potent neurokinin-2
(NK2) receptor antagonist (pKb 9.1), has been obtained using asymmetric synthesis. Isothiocyanato-N-methylbenzamide (10–12)
and bromoacetamido-N-methylbenzamide derivatives (16–18) have been designed to serve as potential electrophilic affinity labels.
Nitro-N-methylbenzamide (4–6) and acetamido-N-methylbenzamide (13–15) were designed to serve as the nonelectrophilic controls
for these ligands. Functional assay results using guinea pig trachea indicate that electrophilic N-methylbenzamide analogues exhibit
potent but surmountable NK2 receptor antagonist activity. Several members of this series (2, 3, 7–9) exhibit potent NK2 receptor
antagonist potencies with pKb values in the range of 9.1–9.7. para-Fluoro substituted analogue 3 was found to be highly potent with
a pKb of 9.7.
Ó 2004 Elsevier Ltd. All rights reserved.
The mammalian neurokinins, namely, substance P (SP),
neurokinin A (NKA), and neurokinin B (NKB) are neuro-
peptides of 10-11 amino acid residues in length. They share
a common C-terminal sequence Phe-X-Gly-Leu-Met,
wherein X is typically an aromatic or branched aliphatic
amino acid.1 These peptides are associated with a variety
of biological activities such as pain transmission, neuro-
genic inflammation, anxiety, smooth muscle contraction,
salivary secretion, and activation of the immune system.
The SP, NKA, and NKB bring about their effects through
activation of the G-protein-coupled receptors neurokinin-1
(NK1), neurokinin-2 (NK2), and neurokinin-3 (NK3),
respectively.2 The presence of NK2 receptors in tissues
throughout the body is well established, and, in recent
years, evidence has been presented for the presence of
NK2 receptors in the central nervous system.3 A number
of potential therapeutic indications of NK2 receptor antag-
onists have been proposed, which include the treatment of
diseases such as asthma, inflammatory bowel disorders,
rheumatoid arthritis, pain, emesis, and psychiatric
disorders.4
Electrophilic affinity labels bind covalently to the recep-
tors and are useful in the structural and functional char-
acterization of receptors at the molecular level.5,6 The
availability of such ligands is helpful in complementing
molecular biological studies where evidence for impor-
tant amino acids in the binding site is obtained indirectly
by site-directed mutagenesis studies. Potent and selective
electrophilic affinity labels have demonstrated their use-
fulness as pharmacologic tools in studying a number of
receptor systems including opioid, muscarinic, adrener-
gic, adenosine, serotonin, and benzodiazepine recep-
tors.5 A diazirinyl-analogue of SR 48,968 (IC50 of
6.7nM) has been previously reported as a potent photo-
affinity label for the NK2 receptors, which upon photo-
lysis is expected to generate a highly reactive carbene.7
However, to our knowledge no NK2 receptor selective
electrophilic affinity labels have been reported to date.
SR 48,968 (1a) is a potent and selective NK2 receptor
antagonist (Fig. 1).8 In this study, we have employed
the racemic 4-hydroxy-4-phenyl piperidine analogue
*
Corresponding author. Tel.: +1-718-990-5369; fax: +1-718-990-
1877; e-mail: korlipav@stjohns.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.053

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S.-C. Huang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4779–4782
O
HO
O
AcHN
N
*
N
N
N
CH3
CH3
Cl
Cl
Cl
Cl
(1a) Ki 0.5 nM
(1b) Ki 1.0 nM
Figure 1. Potent neurokinin-2 antagonists: (1a) SR 48,968. (1b) Racemic 4-hydroxy-4-phenyl piperidine derivative of SR 48,968.
(1b) of SR 48,968 (Ki 1.0nM)8 as the lead compound in
order to develop electrophilic affinity labels with NK2
receptor selectivity.
acetamide groups were chosen for attachment to the
benzamide group to cover a range of reactivities and
chemical selectivities. ortho, meta and para analogues
were synthesized to map the receptor for the nucleophi-
lic groups. The synthetic intermediates, nitro analogues
4–6, and the acetamide analogues 13–15 were chosen to
serve as the nonelectrophilic controls for the isothiocya-
nate 10–12 and bromoacetamide 16–18 analogues. Due
to the similarity in sizes of fluorine and hydrogen atoms,
para-fluoro analogue 3 was also synthesized.
Since SR 48,968, the S-stereoisomer, was found to exhi-
bit a nearly 2000-fold higher binding affinity for NK2
receptors from rat duodenum membranes than its R-
enantiomer SR 48,965,8 all the target compounds have
been designed to have S-configuration at the chiral cen-
ter. Chimeric receptor studies and site-directed muta-
genesis studies have been utilized in an attempt to
delineate the binding site of SR 48,968 on the NK2 re-
ceptors. The NK1/NK2 chimeric receptor studies suggest
the involvement of transmembrane VI, extracellular
loop three, and part of transmembrane VII in the bind-
ing of this ligand to the NK2 receptors.9 Furthermore,
site-directed mutagenesis studies have provided infor-
mation regarding the important amino acid residues
for high affinity binding of SR 48,968. According to
various studies, His198 plays an important role in the
binding of SR 48,968 to NK2 receptor,10,11 although dis-
crepant results have been reported regarding the role of
His198 by one group.12 Based on the hypothetical mod-
els proposed for SR 48,968 binding mode on the human
NK2 receptor, the benzamide moiety of SR 48,968 is
close to His198 region.13 In addition, other
nucleophilic amino acids such as Tyr197, Tyr206,
Met213, Tyr217, Ser218 are present in the vicinity.13
Therefore, the benzamide moiety of the lead compound
was chosen for substituting the electrophilic groups.
Moreover, previous structure–activity relationship stud-
ies have shown that a wide variety of substituents are
tolerated in the benzamide region of SR 48,968,14 and
of YM-35375, which is a spiro-substituted piperidine
analogue of SR 48,968.15 Isothiocyanate and bromo-
Synthesis of amide 19 was accomplished using asymmet-
ric synthesis according to steps described by Hale and
co-workers.16 Diisobutylaluminum hydride reduction
of amide 19 to the corresponding secondary amine fol-
lowed by acylation with benzoyl chloride, p-fluoro-
benzoyl chloride, or o-, m-, p-nitrobenzoyl chloride led
to the formation of benzamide 20 and substituted benz-
amides 21–24, respectively. Aldehydes 25–29 were ob-
tained by the oxidative cleavage of the olefinic bond of
20–24 using osmium tetroxide and sodium periodate.17
Reductive amination of 25–29 with 4-hydroxy-4-phenyl-
piperidine using sodium cyanoborohydride afforded
compound 2, the S-stereoisomer corresponding to the
lead compound 1b, the fluoro analogue 3, and the nitro
analogues 4–6 as illustrated in Scheme 1.
The synthetic intermediate aminobenzamide derivatives
7–9 were prepared by reduction of the corresponding
nitro derivatives 4–6 with stannous chloride (Scheme
2),15 which were then reacted with different reagents to
provide target compounds 10–18. The isothiocyanates
10–12 were obtained by reacting the amines with di-(2-
pyridyl)thionocarbonate.18 The acetamides 13–15 were
synthesized from the amines by addition of acetic anhy-
O
C
O
O
O
HO
O
N
NH
CH3
N
N
N
R
R
R
iii
ii
CH3
CH3
CH3
i
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
20, R = H
25, R = H
2, R = H
19
21, R = p-F
26, R = p-F
3, R = p-F
22, R = o-NO2
23, R = m-NO2
24, R = p-NO2
27, R = o-NO2
28, R = m-NO2
29, R = p-NO2
4, R = o-NO2
5, R = m-NO2
6, R = p-NO2
Scheme 1. Reagents and conditions: (i) DIBALH, CH2Cl2/toluene, rt, then substituted or unsubstituted benzoyl chloride, toluene/satd NaHCO3
solution; (ii) cat OsO4, N-methylmorpholine N-oxide 2:1:1 v/v/v acetone/t-butanol/water, then NaIO4, 4:1 v/v THF/water; (iii) 1.5equiv 4-hydroxy-4-
phenylpiperidine, Na(CN)BH3, 1:1 v/v THF/methanol.

S.-C. Huang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4779–4782
4781
O
N
HO
N
NO2
CH3
4, R = ortho
5, R = meta
Cl
6, R = para
Cl
i
O
HO
N
N
NCS
CH3
ii
10, R = ortho
11, R = meta
12, R = para
Cl
O
HO
Cl
N
N
CH3
NH2
7, R = ortho
8, R = meta
9, R = para
Cl
Cl
iii
O
HO
N
N
iv
NHCOCH3
CH3
13, R = ortho
14, R = meta
15, R = para
Cl
O
HO
Cl
N
N
NHCOCH2Br
CH3
16, R = ortho
17, R = meta
18, R = para
Cl
Cl
Scheme 2. Reagents and conditions: (i) SnCl2Æ2H2O, ethyl acetate, reflux; (ii) di-2-pyridyl-thionocarbonate, CH2Cl2, rt; (iii) acetic anhydride, CHCl3,
rt; (iv) bromoacetyl bromide, THF, 0°C.
dride at room temperature. The bromoacetamides 16–18
were obtained from the amines by the controlled addi-
tion of bromoacetyl bromide in THF at 0°C.19
pKb of 9.7. Substitution of the N-methylbenzamide moi-
ety with amino group at ortho, meta, or para positions
led to analogues that retained high NK2 receptor antag-
onist potencies. Thus, both electron-withdrawing fluoro
group and the electron-releasing amino group main-
tained high NK2 receptor antagonist potencies.
N-Methylbenzamide substituted analogues derived from
SR 48,968 were synthesized and evaluated for their NK2
receptor antagonist activity using isolated guinea pig
trachea according to experimental protocols described
previously.20 The results are presented in Table 1. The
unsubstituted analogue 2, fluoro substituted analogue
3, and the amino substituted analogues 7–9 were found
to be the most potent compounds in this series of target
compounds with pKb values similar to or greater than
that for the prototypical NK2 receptor antagonist SR
48,968.
Nitro substituted analogues 4 and 6, isothiocyanate ana-
logue 10, acetamide analogue 15, and bromoacetamide
analogue 18 showed somewhat reduced activities with
pKb values in the range of 8.3–8.55. The remaining ana-
logues showed substantially reduced activities with pKb
values less than 8.0. Reduction in potency of nitro (4–
6), isothiocyanate (10–12), acetamide (13–15), and
bromoacetamide (16–18) analogues relative to unsubsti-
tuted analogue 2, fluoro analogue 3, and amino ana-
logues 7–9 may be explained by a relative increase in
the size of these substituents. Since both fluoro and
amino analogues show good NK2 receptor activity, elec-
tronic effect may not be playing a significant role in
the activities of N-methylbenzamide substituted
analogues.
The unsubstituted N-methylbenzamide analogue 2,
which is the S-stereoisomer corresponding to the race-
mic lead compound 1b, exhibited high NK2 receptor
antagonist potency with a pKb value of 9.5. In compari-
son with the previously reported NK2 receptor antago-
nist activity of SR 48,968 (1a) using [b-Ala8]NKA(4-
10) in isolated guinea pig trachea in a similar assay,14
compound 2 was found to be more potent suggesting
that replacement of the 4-acetamide group on the piper-
idine ring of SR 48,968 with a hydroxyl group is a
favorable modification. Additional modifications
focused on substituting the N-methylbenzamide moiety.
Modification of compound 2 by substituting a para
fluoro group on the N-methylbenzamide moiety, which
is sterically similar to hydrogen, led to compound 3,
which is the most potent analogue in this series with a
The irreversibility of the target compounds was evalu-
ated by their ability to inhibit the maximum obtained
contraction of the NK2 agonist [b-Ala8]-NKA(4-10).
The maximum obtainable response of the agonist aver-
aged approximately 96.7% and was not significantly
(P>0.05) inhibited by any of the target compounds.
Therefore, we conclude that antagonist activity of all
the test compounds, including the electrophilic isothiocy-
anate derivatives 10–12 and bromoacetamide derivatives

4782
S.-C. Huang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4779–4782
Table 1. NK2 receptor antagonist potency of substituted N-methyl-
benzamide analogues in guinea pig trachea
by the standard NK2 receptor agonist indicating the
reversible nature of their binding.
O
HO
N
N
Acknowledgements
R
CH3
This work was supported in part by NIH (Grant 1 R15
NS36367-01A1).
Cl
Cl
pKb
a,b
Compd
R
H
Maximum response
in presence of
antagonists
References and notes
(control=96.7 0.3%)
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SR 48,968
2
9.1c
9.5 0.2
9.7 0.2
8.5 0.4
93.3 2.9%
94.7 0.5%
92.7 1.7%
98.6 0.6%
3
p-F
4
o-NO2
m-NO2
p-NO2
o-NH2
m-NH2
p-NH2
o-NCS
m-NCS
p-NCS
5
<8.0
4. Longmore, J.; Hill, R. G.; Hargreaves, R. J. Can. J.
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6
8.55 0.4 98.0 2.0%
7
9.4 0.3
9.4 0.4
9.1 0.5
8.5 0.1
98.2 1.1%
97.4 0.8%
95.9 2.6%
95.5 0.3%
95.8 1.2%
97.6 1.5%
98.3 0.7%
96.3 0.8%
96.6 3.2%
97.1 0.7%
98.0 0.7%
96.0 3.1%
8
9
10
11
12
13
14
15
16
17
18
<8.0
<8.0
<8.0
<8.0
o-NHCOCH3
m-NHCOCH3
p-NHCOCH3
8.4 0.6
o-NHCOCH2Br <8.0
m-NHCOCH2Br <8.0
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p-NHCOCH2Br
8.3 0.4
a pKb are determined against [b-Ala8] NKA(4-10) in isolated guinea pig
trachea; values are means SEM from four experiments.
b pKb is the negative log of Kb where Kb =[antagonist]/(dose ratio-1).
c Data from Ref. 14.
16–18, is surmountable by the NK2 agonist [b-Ala8]-
NKA(4-10). This suggests that the electrophilic isothio-
cyanate and bromoacetamide groups in the ortho, meta,
or para positions of the N-methylbenzamide group in the
target compounds 10–12 and 16–18 are not within the
covalent bonding distance of any nucleophilic group in
the NK2 receptor binding site.
12. Huang, R. R.; Vicario, P. P.; Strader, C. D.; Fong, T. M.
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In conclusion, compounds 2, 3, and 7–9, which are ob-
tained by structural modification of SR 48,968 exhibit
potent NK2 receptor antagonist activity with pKb values
in the range of 9.1–9.7. These ligands are potentially use-
ful as pharmacologic tools when reversible NK2 recep-
tor antagonists are needed. para-Fluoro substituted
analogue 3 was found to be highly potent with a pKb
of 9.7. It is approximately one-half of an order of mag-
nitude more potent than the prototypical NK2 receptor
antagonist SR 48,968. The antagonist activity of the lig-
ands with electrophilic substituents was surmountable
20. Ellis, J. L.; Undem, B. J.; Kays, J. S.; Ghanekar, S. V.;
Barthlow, H. G.; Buckner, C. K. J. Pharmacol. Exp. Ther.
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