Design, synthesis and biological evaluation of 3-amino-3-phenylpropionamide derivatives as novel μ opioid receptor ligands

Article abstract of DOI:10.1016/S0960-894X(00)00034-2

3-Amino-3-phenylpropionamide derivatives were produced as small molecule mimics of the cyclic octapeptide octreotide from readily available imine 1. The compounds exhibit high affinity for the μ opioid receptor. (C) 2000 Elsevier Science Ltd. All rights r

Full text of DOI:10.1016/S0960-894X(00)00034-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 523±526
Design, Synthesis and Biological Evaluation of 3-Amino-3-
phenylpropionamide Derivatives as Novel ꢀ Opioid
Receptor Ligands
Martin P. Allen, James F. Blake, Dianne K. Bryce, Mary E. Haggan,
Spiros Liras,* Sta€ord McLean and Barb E. Segelstein
CNS Discovery, Central Research Division, P®zer Inc, Groton, CT 06340, USA
Received 1 October 1999; accepted 4 January 2000
AbstractÐ3-Amino-3-phenylpropionamide derivatives were produced as small molecule mimics of the cyclic octapeptide octreotide
from readily available imine 1. The compounds exhibit high anity for the m opioid receptor. # 2000 Elsevier Science Ltd. All
rights reserved.
The human opioid receptors m, d, and k have been clas-
si®ed as members of the G protein coupled receptor
family.1±3 The opioid receptors are abundantly present
in the CNS and the periphery and mediate signi®cant
physiologic events that range from the perception of
pain, mood and pleasure, to respiratory depression and
regulation of the GI and immune systems.4 The design
and synthesis of potent and selective molecular probes
that will de®ne the biological signi®cance of each
receptor are highly active research areas in con-
temporary medicinal chemistry (for some leading
examples, see refs 5±8). As part of a program that
aimed at providing selective molecular probes for the m
receptor and its subtypes we sought to design and syn-
thesize small molecule mimics of the cyclic octapeptide
octreotide (SMS-201, 995), a selective m ligand. The
targeted molecules are represented by the 3-amino-3-
phenylpropionamide derivatives shown in Figure 1.
relative to the d receptor.9 More recently, interesting
conformationally restricted cyclic peptides have been
discovered as analogues of SMS-201, 995 with good
anity for the m opiate receptor and reduced anity for
somatostatin.10,11 In order to produce small molecule
mimics of these cyclic peptides, our design e€orts
focused on the SAR and spatial relationship of the two
phenylalanine residues in SMS-201, 995. In a similar
approach, small molecule mimics of the d selective cyclic
peptide [(2S, 3R)-TMT1]DPDPE were designed by
appropriately placing the aromatic pharmacophores of
[(2S, 3R)-TMT1]DPDPE on a piperazine sca€old.12
The X-ray structure of SMS-201, 995 contains three
molecules in the asymmetric unit cell and measurement
of the distance between the centroids of the phenylala-
nine aromatic rings for each molecule gives 12.170‹0.11
A. This conformation can be described as relatively
`extended'.13 While this peptide is conformationally
constrained, a more `compact' conformation is also pos-
sible and supported by NMR studies.14 A compact con-
formation can be achieved by modi®cation of w1 of Phe-3
to be 60ꢀ, which decreases the distance between the two
phenylalanine aromatic rings to 9.335‹0.40 A. These
distances provide us with a framework for the selection
of the 3-amino-3-phenylpropionamide as a sca€old with
the desired aromatic ring-hydrophobic group spatial
relationship. For each 3-amino-3-phenylpropionamide
the initial conformations were generated by carrying out
a Monte Carlo conformational search with Macro-
Model v5.5 employing the MMFF force ®eld with the
GB/SA continuum solvation model.15 In each Monte
Results and Discussion
Design
The cyclic octapeptide octreotide, d-Phe-Cys-Phe-d-
Trp-Lys-Thr-Cys-Thr-ol (SMS-201, 995), has been
shown to exhibit anity for the m opioid receptor in
addition to its somatostatin activity and good selectivity
*Corresponding author. Tel.: +1-860-441-1739; fax: +1-860-441-
4111.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00034-2

524
M. P. Allen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 523±526
Figure 1. 2-Dimensional extended representations of octreotide (left) and a 3-amino-3-phenylpropionamide derivative (right).
Carlo search, 2000 possible conformers were generated.
Full geometry optimizations were then carried out with
Gaussian98 (6-31G(d) basis set) on the 10 lowest energy
conformations for each compound within a 5 kcal/mol
window.16 The pharmacophoric elements of each com-
pound (aromatic ring, basic nitrogen, and hydrophobic
group) were then aligned and evaluated against the com-
pact and extended conformations of SMS-201,995. The
overlap of SMS-201, 995 (green) with a 3-amino-3-phe-
nylpropionamide derivative (gold) as shown in Figure 2
illustrates our design strategy.
precursor b-lactam 3 was obtained in high yields after
treatment of imine 1 with titanium tetrachloride and
addition of the commercially available ketene acetal 2
followed by tandem cyclization of an intermediate
amino ester.17 The lactam 3 serves as an internal nitro-
gen-protecting group and sets the stage for ring opening
reactions. Thus, the desired propionamides 4 were pro-
duced in high yields after addition of lithium amides of
primary amines to the lactam 3. The methyl ethers 4d±i
were deprotected in high yields with boron tribromide
in dichloromethane to produce the phenols 5. The ter-
tiary amines 6 were formed by reductive alkylation
under standard conditions.
Synthesis
A convergent approach was designed for the prepara-
tion of the desired opiates (see Scheme 1). Thus, the
The in vitro binding anities for compounds 4, 5 and 6
were determined.19 The binding assays were performed
according to standard protocols. For the d receptor,
radioligand studies were carried out on CHO cells
expressing the human d opioid receptor. Cells were col-
lected and homogenized (50 mM Tris HCl, pH 7.4 and 1
mM EGTA) centrifuged, the pellet washed once and
resuspended in bu€er at 0.8 mg/mL and frozen
( 80 ꢀC). Binding to the d receptor was initiated with
the addition of cell membranes to 96 well plates con-
taining 0.5 nM [3H] naltrindole (Tocris: speci®c activity
50 Ci/mmol) and incubated for 90 min at 37 ꢀC.
Non-speci®c binding was estimated using 1 mM cold
naltrindole.
Rat brain tissue was utilized for the m receptor assay.
Thus, rat brains were homogenized (50 mM Tris HCl
pH 7.4) and a crude membrane fraction was prepared
and kept on ice. Receptor binding was initiated with the
Figure 2. Overlap of SMS-201, 995 with a 3-amino-3-phenylpropio-
namide derivative.
Scheme 1. Preparation of 3-amino-3-phenylpropionamide derivatives.18 (a) TiCl4, CH2Cl2, rt., 10 h; 88%; (b) n-BuLi, R1NH2, THF, 0 ꢀC; 15 min
then add 3; 85±98% yield; (c) BBr3, CH2Cl2, 78 ꢀC to rt; 80±95% yield; (d) RxCHO, NaBH(OAc)3, AcOH, CH2Cl2, rt; 10 h; 65±95% yield.

M. P. Allen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 523±526
525
addition of rat brain membranes to 96-well plates con-
taining 1.5 nM [3H]DAMGO (NEN; speci®c activity 55
Ci/mmol) and incubated for 90 min at 22 ꢀC. Non-spe-
ci®c binding was determined using 1 mM naltrexone.
For the k binding assay membranes (ꢁ5 mg/mL pro-
tein) prepared from HEK 293 cells transfected with the
human k opioid receptor were used. Receptor binding
assays were initiated by the addition of cell membranes
to 96-well plates containing 2 nM [3H]U 69,593 (NEN;
speci®c activity 40 Ci/mmol) and incubated for 1 h at
25 ꢀC. All assays were terminated by rapid ®ltration
using a Skatron harvester onto Whatman GF/C (d
assay), Wallac Filtermat B (m assay) or Wallac Filtermat
B (k assay) and counted in a Beta Plate reader (Wallac).
group (entries 5a±f) delivers compounds with improved
binding anity for the m receptor. Selectivity over the d
receptor is generally maintained with the introduction
of the 3-hydroxy substituent and high binding anity
for the k receptor is generally observed. Biological
results for the methyl ether precursors 4d±i are not
included in Table 1 as the compounds generally show
reduced anity for the receptors (20±50-fold).
Substrates with amide substitutions such as the phen-
ethyl in 5b and 1-naphthylmethyl in 5d exhibit the
highest m receptor binding anities. Compound 5c, with
the larger phenylpropyl amide group, exhibits modestly
lower anity for the m receptor and high anity for the
k receptor. Compound 5e with the hydrophobic cyclo-
hexylmethyl amide group shows high binding anity
for the m and k receptors and good selectivity over the d
receptor. Compound 5f with a smaller hydrophobic
amide group, such as the cyclopropyl methyl group, has
signi®cantly reduced anity for the m receptor and
modest anity for the k receptor. Generally, conversion
of the secondary amines to tertiary amines (entries 6a±b)
via reductive alkylation with small aliphatic aldehydes
delivered compounds with reduced binding anity for
the m receptor.
Results of the biological evaluation of compounds 4, 5
and 6 are included in Table 1. Biological data are
reported in nM. Values reported are the arithmetic
mean‹SEM in parenthesis of a minimum of three
independent receptor binding assays, except when noted.
Compound 4a with a phenethyl amide group represents a
substrate with aromatic ring-hydrophobic group dis-
tance of 11.3‹0.2 A. This value corresponds more clo-
sely to the phenylalanine aromatic group distance
suggested by the X-ray structure of octreotide. Com-
pound 4a exhibits high anity for the m receptor, excel-
lent selectivity over the d receptor and modest anity for
the k receptor. Compound 4b, with a shorter benzyl
amide group exhibits reduced anity for the m receptor
and similar selectivity pro®le. The aromatic ring-hydro-
phobic group distance for 4b was computed to be 9.9 A,
in line with a more compact conformation suggested by
the NMR studies. Interestingly, compound 4c with a
non-aromatic hydrophobic amide substitution such as
the cyclohexylmethyl shows binding anity for the m
receptor similar to the benzamide 4b and increased a-
nity for the k receptor.
In conclusion, the design and synthesis of small mole-
cule mimics of the cyclic octapeptide SMS-201, 995 have
been described herein. The designed propionamide
derivatives were produced from readily available imines.
In vitro biological evaluation revealed compounds with
high anity for the m opioid receptor and excellent
selectivity over the d receptor, consistent with the data
reported for SMS-201, 995 and its analogues. Selectivity
over the k receptor is generally low for substrates
within this class of compounds. Receptor selectivity
optimization, determination of the functional in vitro
characteristics and behavioral in vivo properties of
compounds within this group constitute the subject of
our current investigation. Results will be reported in
due course.
Introduction of a classic opioid-binding moiety, the 3-
hydroxy substituent, to the benzylamino aromatic
Table 1. Binding anities (nM) for compounds 4, 5 and 6 at m, d, and k receptors
Compound
R1
R2
R
m
d
k
4a
4b
4c
5a
5b
5c
5d
5e
5f
PhCH2CH2
PhCH2
c-C6H11±CH2
PhCH2
PhCH2CH2
PhCH2CH2CH2
1-naphthyl-CH2
c-C6H11-CH2
c-C3H5-CH2
PhCH2CH2
PhCH2CH2
H
H
H
H
H
H
H
H
H
H
H
H
13 (20±8.9)
95 (139±65)
85 (110±66)
8.7 (11±6.6)
1.4 (1.7±1.2)
12 (26±5.2)
1.4 (1.6±1.2)
4.4 (5.8±3.3)
364 (408±324)
28 (31±24)
>10,000
>10,000
>10,000
49a (50±48)
467 (754±290)
1.2 (1.5±0.90)
5.0 (9.0±2.7)
6.3 (13±3.1)
3.6 (7.3±1.8)
1.3 (3.1±0.55)
5.7 (14±2.3)
75 (118±47)
2.9 (5.9±1.4)
75 (198±28)
OH
OH
OH
OH
OH
OH
OH
OH
>10,000
176 (320±97)
>10,000
163 (199±134)
881 (972±711)
781 (1000±522)
710 (996±501)
>10,000
6a
6b
c-C3H5±CH2
n-C4H9
26 (30±21)
aValue represents the average of two experiments.

526
M. P. Allen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 523±526
15. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Lis-
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1
azetidin-2-one (3): H NMR (400 MHz, CDCl3) d 7.28 (ap t,
1H), 6.84 (dd, J=8.1, 2.1 Hz, 1H), 6.73 (d, J=7.5 Hz, 1H), 4.25
(s, 1H), 3.80 (s, 3H), 2.85 (s, 3H), 1.40 (s, 3H), 0.78 (s, 3H); 13
C
NMR (100 MHz, CDCl3) d 174.3, 159.9, 137.9, 129.8, 118.9,
113.0, 112.5, 112.4, 68.6, 56.5, 55.2, 27.2, 22.5, 17.7; MS (M+1)
220.0. HRMS calcd for C13H17NO2 +H 239.1495, found
239.1513. 3-(3-Hydroxy-phenyl)-2,2-dimethyl-3-methylamino-
N-phenethyl-propionamide (5b): 1H NMR (400 MHz, CDCl3)
d 7.98 (br, 1H), 7.29±7.04 (comp, 6H), 6.79±6.74 (m, 1H), 6.72±
6.70 (m, 1H), 6.64 (d, J=7.7 Hz, 1H), 3.59±3.48 (comp, 2H),
3.43 (s, 1H), 2.79 (t, J=6.8 Hz, 2H), 2.07 (s, 3H), 1.06 (s, 3H),
0.99 (s, 3 H); 13C NMR (100 MHz, CDCl3) d 178.1, 156.6,
140.8, 139.1, 129.1, 128.1, 128.6, 126.4, 119.8, 115.9, 114.7,
71.0, 45.7, 40.6, 35.4, 34.7, 24.9, 21.0; MS (M+1) 327.3. HRMS
calcd for C20H26N2O2 +H 307.0930, found 307.0930.
19. Naloxone was used as a standard in the binding assays:
mKi=1.4 nM, dKi=41 nM, kKi=1.2 nM.
14. Melacini, G.; Zhu, Q.; Goodman, M. Biochem. 1997, 36,
1233.