Design and synthesis of 3,5-disubstituted benzamide analogues of DNK333 as dual NK1/NK2 receptor probes

Article abstract of DOI:10.1016/j.bmcl.2006.11.064

N-[(R,R)-(E)-(3,4-dichlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide (DNK333, 1b) has been reported to be a potent and balanced dual neurokinin (tachykinin) receptor antagonist. A recent clinical trial using DNK

Full text of DOI:10.1016/j.bmcl.2006.11.064

Bioorganic & Medicinal Chemistry Letters 17 (2007) 890–894
Design and synthesis of 3,5-disubstituted benzamide analogues
of DNK333 as dual NK1/NK2 receptor probes
Venkat Manoj Swarna,a Bradley J. Undemb and Vijaya L. Korliparaa,*
aDepartment of Pharmaceutical Sciences, St. John’s University, 8000 Utopia Parkway, Queens, NY 11439, USA
bJohns Hopkins Asthma and Allergy Center, Baltimore, MD 21224, USA
Received 16 October 2006; revised 16 November 2006; accepted 20 November 2006
Available online 1 December 2006
Abstract—N-[(R,R)-(E)-(3,4-dichlorobenzyl)-3-(2-oxoazepan-3-yl)carbamoyl]allyl-N-methyl-3,5-bis(trifluoromethyl)benzamide
(DNK333, 1b) has been reported to be a potent and balanced dual neurokinin (tachykinin) receptor antagonist. A recent clinical
trial using DNK333 has shown that it blocks the NKA-induced bronchoconstriction in patients with asthma. A series of six ana-
logues 3–8 derived from modification of 3,5-bis(trifluoromethyl)benzamide moiety of DNK333 has been synthesized to serve as the
dual NK1/NK2 receptor probes. The 3,5-dinitro substituted benzamide compound 3 was found to possess potent and balanced dual
NK1/NK2 receptor antagonist activities (pKb = 8.4 for the NK1 receptors, pKb = 7.87 for the NK2 receptors) in the functional assay
using guinea pig trachea. Furthermore, SAR analysis suggests that steric, electronic, and lipophilic characteristics of substituents in
the benzamide region of DNK333 have a crucial effect on both the NK1 and NK2 receptor antagonist activities.
Ó 2006 Elsevier Ltd. All rights reserved.
Neurokinins, also known as tachykinins, are a family of
small peptides that are widely distributed throughout
the central and peripheral nervous system, wherein they
act as neurotransmitters and neuromodulators. The
three tachykinin receptors, NK1, NK2, and NK3, consti-
tute a group of homologous receptors within the family
of G protein-coupled receptors.1,2 The neuropeptides
substance P (SP), neurokinin A (NKA), and neurokinin
B (NKB) act as full agonists on the three neurokinin
receptors, although they exhibit preferential binding to
the NK1, NK2, and NK3 receptors, respectively. These
peptides have been proposed to be involved in a number
of pathological conditions such as pain, arthritis, mi-
graine, emesis, cancer, anxiety, depression, schizophre-
nia, and asthma.3,4 There is mounting evidence that
neurokinins play an important role in airway disease
induction and progression via the activation of NK1
and NK2 receptors.5–8 A number of studies suggest that
neurokinin receptor antagonists, especially dual NK1/
NK2 antagonists, may represent a new treatment option
for asthma and other airway diseases, particularly since
lung tissue from asthma patients has been shown to
overexpress NK1 and NK2 receptors.8,9 Within the
lungs, release of neuropeptides substance P and neurok-
inin A from sensory nerves as well as from inflammatory
cells results in an inflammatory response characterized
by bronchoconstriction, microvascular leakage, and mu-
cus hypersecretion, which are the typical pathological
characteristics of asthma and chronic bronchitis. Sub-
stance P primarily causes microvascular leakage and
mucus hypersecretion, and NKA predominantly pro-
duces hyper-responsiveness. These effects are mediated
through specific receptors, namely NK1 and NK2 recep-
tors. Thus, designed multiple ligand (DML) approach10
has emerged as an attractive strategy for the treatment
of asthma and other airway diseases by simultaneous
and balanced blockade of NK1 and NK2 receptors.11–18
DNK333 1b is an example of a designed multiple
ligand derived from the structural modification of
CGP49823 1a, an NK1 receptor-selective antagonist
(Fig. 1).17,18 DNK333 (IC50 NK1 = 4.8 nM, IC50
NK2 = 5.5 nM in cloned human NK1 and NK2 recep-
tors, respectively) has been shown to inhibit broncho-
constriction induced by NK1 and NK2 receptor
agonists in guinea pigs and squirrel monkeys in preclin-
ical investigations. In patients with mild asthma,
DNK333 has been shown to block NKA-induced bron-
choconstriction, thus further establishing the potential
of dual NK1/NK2 receptor antagonists as anti-asthma
agents.19
Keywords: Neurokinin receptor antagonists; Asthma.
*
Corresponding author. Tel.: +1 718 990 5369; fax: +1 718 990
1877; e-mail: korlipav@stjohns.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.064

V. M. Swarna et al. / Bioorg. Med. Chem. Lett. 17 (2007) 890–894
891
CF3
CH3
O
Me
N
H
N
N
F3C
NH
N
H
O
N
O
Me
O
Cl
Cl
DNK333 1b
CGP49823 1a
IC50 NK1 = 4.8 nM
IC50 NK2 > 5.5 nM
IC50 NK1 = 12 nM
IC50 NK2 > 1000 nM
Figure 1. NK1-selective ligand CGP49823 and dual NK1/NK2 ligand DNK333.
A series of compounds 3–8 derived from DNK333,
which retained the structural characteristics important
for NK1 and NK2 receptor antagonist activities, was de-
signed to further explore the SAR and to obtain dual
NK1/NK2 receptor probes.17,18,20 Previous SAR studies
on DNK333 1b indicate that R,R-stereochemistry is
important. Furthermore, the 3,4-dichlorophenyl ring
and the caprolactam ring were found to be optimal for
balanced dual NK1/NK2 receptor antagonist activity.
No SAR studies investigating modification of the 3,5-
bis(trifluoromethyl) substituted phenyl ring have been
reported. The aim of this project was to investigate mod-
ification of this region of DNK333, with the goal of
obtaining potential electrophilic affinity labels for neu-
rokinin receptors.
derivative 3 with hydrazine monoformate and zinc dust
at room temperature21 which was then reacted with dif-
ferent reagents to provide target compounds 5–8 as
shown in Scheme 2.22 The 3,5-diisothiocyanate 5 was
obtained by reaction of the 3,5-diamine with di-(2-pyr-
idyl) thionocarbonate. Initial efforts to synthesize 3,5-
diisothiocyanate derivative 5 resulted in the formation
of 3,5-dithiocarbamate compound 6. The 3,5-diaceta-
mide 7 was synthesized from the amine by addition of
acetic anhydride at room temperature. The 3,5-dibro-
moacetamide 8 was synthesized from the 3,5-diamine 4
by the controlled addition of bromoacetyl bromide in
THF at 0 °C.
Results of the evaluation of the NK1/NK2 receptor
antagonism and nonreversibility of the target com-
pounds by functional assay using guinea pig trachea
are presented in Table 1. The smooth muscle assays were
conducted on the guinea pig trachea, which was activat-
ed by the NK1-selective agonist ASM-SP (Ac-[Arg6,
Sar9, Met (O2)11]-SP(6–11)) for NK1 receptors and by
the NK2-selective agonist [b-Ala[8]]NKA(4–10) for
NK2 receptors.23 The antagonist potencies of the test
compounds are expressed as mean pKb values.
In the target compounds, the 3,5-bis(trifluoromethyl)
substituents have been replaced by other groups.
3,5-Diisothiocyanate and 3,5-dibromoacetamide ana-
logues (5, 8) were designed to serve as the electrophilic
affinity labels, and to cover a range of reactivities and
chemical selectivities. These ligands have the potential
to label NK1, NK2, or both NK1 and NK2 receptors
depending on the presence of the nucleophilic groups
on these receptors in the region where the benzamide
group binds. Furthermore, presence of two electrophilic
groups in each of these ligands increases the possibility
of covalently labeling these receptors. The synthetic
intermediates, 3,5-dinitro analogue 3 and 3,5-diamino
analogue 4, and the target compound 3,5-diacetamide
analogue 7 were chosen to serve as the nonelectrophilic
controls and to help understand the SAR of the benzam-
ide region of DNK333.
The lead compound, DNK 333 1b, was found to exhibit
potent and reversible NK1 and NK2 receptor antagonist
activities in this assay, with pKb values of 9.2 and 7.43,
respectively. The 3,5-dinitro substituted derivative 3
exhibited a lower NK1 receptor antagonist potency with
a pKb value of 8.4, while the NK2 receptor activity was
increased to a pKb value of 7.87, thus providing a more
balanced NK1/NK2 receptor antagonist activity. Since
the 3,5-dinitro derivative 3 lacks the electrophilic moie-
ties, it exhibited reversible antagonism at both NK1 and
NK2 receptors (Table 1). Overall, 3,5-dinitro substituted
derivative which exhibits similarity in size and shape to
DNK333 (Fig. 2)24 showed a potent and balanced dual
NK1/NK2 receptor antagonist activity. Remaining com-
pounds in the series did not exhibit antagonist activity at
either NK1 or NK2 receptors, when studied at concen-
trations up to 1 lM. The calculated logP and molecular
volumes of the target compounds were compared to
analyze the biological results obtained. The lack of
antagonist activity in 3,5-dithiocarbamate derivative 6
Compound 2 (R, R-isomer) was prepared starting from
commercially available Boc-D-3,4-dichlorophenylala-
nine in five steps according to the procedure reported
by Gerspacher and coworkers.20 Acylation of the
secondary amine with either 3,5-bis(trifluorometh-
yl)benzoyl chloride or 3,5-dinitrobenzoyl chloride led
to the 3,5-bis(trifluoromethyl) derivative 1b (DNK333)
or the 3,5-dinitro derivative 3, respectively (Scheme 1).
The 3,5-diaminobenzamide derivative 4 was prepared in
high yields by reduction of the corresponding 3,5-dinitro

892
V. M. Swarna et al. / Bioorg. Med. Chem. Lett. 17 (2007) 890–894
R
CH3
N
CH3
N
O
O
H
NH
R
NH
N
N
H
H
O
O
O
i or ii
Cl
Cl
Cl
Cl
1b, R=CF3
3, R=NO2
2
Scheme 1. Reagents: (i) 3,5-bis(trifluoromethyl)benzoyl chloride, Et3N, CH2Cl2; (ii) 3,5-dinitrobenzoyl chloride, toluene/satd NaHCO3 solution.
NO2
NCS
CH3
N
CH3
N
O
O
O2N
NH
NH
N
SCN
N
H
H
O
O
O
O
5
3
Cl
Cl
Cl
i
Cl
iii
ii
S
NH
C OCH3
NH2
CH3
N
O
CH3
N
O
H3CO
C
S
HN
NH
N
NH
H
H2N
N
O
O
H
O
O
6
Cl
Cl
Cl
Cl
4
iv
NHCOCH3
v
CH3
N
O
H3COCHN
NH
N
H
O
O
NHCOCH2Br
Cl
CH3
N
O
Cl
7
BrH2COCHN
NH
N
H
O
O
8
Cl
Cl
Scheme 2. Reagents and conditions: (i) Zn/NH2–NH2ÆHCOOH, MeOH, rt; (ii) di-(2-pyridyl)-thionocarbonate, CH2Cl2, rt; (iii) methanol in work up;
(iv) acetic anhydride, CHCl3, rt; (v) bromoacetyl bromide, THF, 0 °C.
and in 3,5-dibromoacetamide 8 might be attributed to
the steric factors (Table 2, Fig. 2)24, whereas relatively
higher hydrophilicity of the 3,5-diamino substituted
benzamide 4 and increased steric hindrance coupled
with reduced hydrophobicity might explain low potency
of 3,5-diacetamide 7 (Table 2). The reason for inactivity
of 3,5-diisothiocyanate substituted benzamide 5 in the
smooth muscle experiments is less clear. Subtle differenc-
es in the shape and electronic character of the isothiocy-
anate group compared to the trifluoromethyl and nitro
groups together with its high lipophilicity might be play-
ing a role.
In conclusion, a series of analogues 3–8 derived from the
modification of 3,5 bis(trifluoromethyl)benzamide moie-
ty of DNK333 1b has been successfully synthesized
starting from commercially available optically active
Boc-D-3,4-dichlorophenylalanine. For comparison and
validation purposes, the lead compound, 1b
(DNK333), was also synthesized. Synthesis of the target

V. M. Swarna et al. / Bioorg. Med. Chem. Lett. 17 (2007) 890–894
893
Table 1. NK1 and NK2 receptor antagonist potencies of substituted DNK333 analogues in guinea pig trachea
R
CH3
O
N
R
NH
N
H
O
O
Cl
Cl
NK1 receptor
Compound
R
NK2 receptor
Maximum response in
a,b
pKb
b,c
pKb
Maximum response in
presence of antagonists
(Control = 97.0 1.2%)
presence of antagonists
(Control = 98.3 0.6%)
DNK333 (1b)
3
CF3
NO2
89.4 2.1
96.1 2.9
9.2 0.4
8.4 0.3
97.0 0.1
96.1 1.3
7.43 0.4
7.87 0.4
a pKb are determined against ASM-SP (Ac-[Arg6, Sar9, Met (O2)11]-SP(6–11)) 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 À 1).
c pKb are determined against [b-Ala8]NKA(4–10) in isolated guinea pig trachea.
compounds consisted of synthesis of the chiral interme-
diate 2 followed by acylation to obtain 3,5-dinitro
substituted benzamide 3. The 3,5-diamino substituted
derivative 4 was prepared by highly efficient reduction
of the 3,5-dinitro derivative 3 using hydrazinium mono-
formate and zinc. The diamine derivative 4 was reacted
with various reagents to provide the target compounds
5–8. The smooth muscle data indicated that 3,5-dinit-
robenzamide 3 exhibited a potent and balanced dual
NK1/NK2 receptor antagonist activity with pKb of 8.4
and 7.87 for the NK1 and NK2 receptors, respectively.
The NK2 antagonist potency of compound 3 was greater
than that of the lead compound, while the NK1 receptor
antagonist potency was reduced. As expected, com-
pound 3 exhibited reversible antagonism of both NK1
and NK2 receptors. Other compounds in the series were
inactive in the smooth muscle assay using guinea pig tra-
chea, suggesting strict steric, electronic, and lipophilic
requirements for substituents in the benzamide region
of DNK333.
References and notes
Figure 2. Superimposition of 3,5-dinitro analogue 3 (yellow) and 3,5-
dibromoacetamide analogue 8 (green) using DNK333 (magenta) as a
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