New templates for syntheses of ring-fused, C10 β-turn peptidomimetics leading to the first reported small-molecule mimic of neurotrophin-3

Article abstract of DOI:10.1021/jm0255421

β-Turn peptidomimetics 1 were designed to mimic hot spots of neurotrophin-3 (NT-3) and others. Solid-phase syntheses of these were developed, though limitations were encountered with scale-up. Consequently, an alternative design with 2 was investigated. 1 and 2 favored distorted type I β-turn conformations in solution. It was found that peptidomimetic 2b has NT-3-like neurotrophic activity in cell survival assays, selectively binds the NT-3 receptor TrkC, and induces the tyrosine phosphorylation of the TrkC receptor.

Full text of DOI:10.1021/jm0255421

J . Med. Chem. 2002, 45, 4387-4390
4387
Sch em e 1
New Tem p la tes for Syn th eses of
Rin g-F u sed , C10 â-Tu r n P ep tid om im etics
Lea d in g to th e F ir st Rep or ted
Sm a ll-Molecu le Mim ic of Neu r otr op h in -3
Mookda Pattarawarapan,† Maria Clara Zaccaro,‡
Uri H. Saragovi,‡ and Kevin Burgess*,†
Department of Chemistry, Texas A&M University,
P.O. Box 30012, College Station, Texas 77842-3012, and
Departments of Pharmacology and Therapeutics,
Oncology and The Cancer Center, McGill University,
Montre´al, QC, H3G 1Y6, Canada
Received May 20, 2002
Abstr a ct: â-Turn peptidomimetics 1 were designed to mimic
hot spots of neurotrophin-3 (NT-3) and others. Solid-phase
syntheses of these were developed, though limitations were
encountered with scale-up. Consequently, an alternative de-
sign with 2 was investigated. 1 and 2 favored distorted type I
â-turn conformations in solution. It was found that peptido-
mimetic 2b has NT-3-like neurotrophic activity in cell survival
assays, selectively binds the NT-3 receptor TrkC, and induces
the tyrosine phosphorylation of the TrkC receptor.
Our efforts to mimic â-turn hot spots of the nerve
growth factor (NGF)1 led to the design and synthesis of
several ring-fused, C10 â-turn analogues,2 including
compound A.3 That particular mimic has since been
shown to bind the high-affinity NGF receptor TrkA and
to enhance the response obtained from suboptimal
concentrations of NGF.3
The next phase of this research, described here,
involved two goals. The first was to design slightly less
peptidic compounds that hold the key dipeptide frag-
ment in a more rigid (i + 1)/(i + 2) â-turn conformation.
The second was to mimic â-turn hot spots of neurotro-
phin-3 (NT-3), which is a member of the neurotrophin
family and related to NGF.4 The tyrosine kinase recep-
tor for NGF is TrkA, and the tyrosine kinase receptor
for NT-3 is termed TrkC.4 To the best of our knowledge,
there have been no previous reports of a small molecule
that mimics NT-3 by interacting with the TrkC receptor.
Compounds 1 were conceived after consideration of
the issues outlined above. These must be more rigid
than A and related structures because of the constraints
imposed by the extra aromatic ring on the ø1-ø3 vectors.
Consequently, practical solid-phase syntheses of these
materials were required, their conformational biases in
solution needed to be determined, and tests needed to
be performed to determine if compounds of this type
might, like A, elicit a functional response at one of the
Trk receptors.
Scheme 1 shows how compounds 1 were constructed
on Rapp HypoGel 200 RAM resin (Rapp Polymere). The
most challenging, and the most interesting, step in the
sequence is the penultimate one. This SNAr macro-
cyclization is not particularly favorable because of the
poor nucleophilicity of the arylamine. This ring closure
did not occur at room temperature unless copper(+1)
salts were present. Copper(+1)-mediated aryl-to-amine
couplings have been known for decades but tend to
involve elevated temperatures.5 Successful coupling at
room temperature in this study is therefore unusual.
Purities of crude materials isolated from the solid
support are critical in syntheses such as that shown in
* To whom correspondence should be addressed. Phone: 979-845-
4345. Fax: 979-845-8839. E-mail: burgess@tamu.edu.
† Texas A&M University.
‡ McGill University.
10.1021/jm0255421 CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/21/2002

4388 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Letters
Ta ble 1. Purities and Isolated Yields of 1
amino acids
AA1
AA2
UV/sedexa
isolated
compound
(i + 1)
(i + 2)
purities (%)
yields (%)
1a
1b
1c
1d
1e
1f
1g
1h
1i
Glu
Lys
Ile
Gly
Ser
Thr
Arg
Asp
Ile
Lys
Ser
Lys
Lys
Lys
Lys
Ser
Arg
Arg
89/99
86/98
81/91
78/100
83/97
87/100
74/97
81/99
81/98
51
44
40
35
38
36
48
41
37
a
Sedex is evaporative light scattering detection.
Sch em e 2. Synthesis of Compounds 2
F igu r e 1. CD spectra of compounds 1a ,b and 2a ,b in 20%
MeOH/H2O.
Conformational preferences of peptidomimetics 1a ,b
and 2a ,b were studied by experiments that included
measurements of NH temperature coefficients, NH-R
coupling constants, and ROESY connectivities.6 These
experimental data were then compared with virtual
conformations generated via the quenched molecular
dynamics technique.7,8 Molecules such as these must
populate several conformational states, but the infer-
ence from these studies is that all four compounds can
adopt distorted type 1 â-turn conformations (Supporting
Information). This conclusion is supported by the CD
spectra of these compounds (Figure 1), which shows a
minimum in the 208 nm region that is thought to be
characteristic of type 1 turns.9,10
Cell survival screens (MTT assays) were used to
access the potential of these compounds as neurotrophin
analogues.3 The choice of side chains for the compounds
(Table 1) largely reflects the sequences at the NGF and/
or NT-3 turn regions. This assay uses cells stably
transfected with the NT-3 receptor, TrkC, or the NGF
receptor, TrkA. Culture of these cells in serum-deprived
conditions induces their death (0% survival). However,
the cells can be rescued by addition of the appropriate
concentration of neurotrophin (e.g., 2000 pM NT-3;
100% survival). Suboptimal concentrations of neurotro-
phin (e.g., 100 pM NT-3) affords low but significant
survival (26%).
An interesting lead compound, 2b, was identified
using these cell survival assays. Peptidomimetic 2b
enhanced the survival of TrkC-expressing NIH-3T3 cells
exposed to suboptimal NT-3 doses (Table 2; compare
entries 8-11 with entry 3). Compound 2b affords
significant (p < 0.05) enhancement of cell survival when
tested in combination with 100 pM NT-3 (Table 2,
entries 10 and 11; 10 and 50 µM 2b). Lower concentra-
tions of 2b do not afford significant cell survival
enhancement (Table 2, entries 8 and 9; 0.4 and 2 µM
2b). Compound 2b had no effect on the survival of TrkA-
expressing NIH-3T3 cells with or without NGF (Sup-
porting Information), indicating a selective trophic effect
toward TrkC. All the other compounds listed in Table
1 had no significant effect on the survival of TrkC-
expressing cells.
Scheme 1. Table 1 shows that the procedure works well
in this regard for a number of side chain functionalities
R1 and R2.
Methodologies that give other â-turn peptidomimetics
with enhanced rigidities are also being developed.
Compounds 2a and 2b were formed via the route
outlined in Scheme 2. The scope of this approach is still
being explored, but the advantages of avoiding copper
catalysis are clear. Excess copper(+1) iodide, which has
relatively low solubility, seems to interfere with per-
meation of reagents into the resin for the sequence in
Scheme 1 on reasonably large scales. This is not a
problem for the chemistry in Scheme 2.
A direct binding assay was used to access the ability
of peptidomimetic 2b to bind the Trk receptors. For that

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4389
Ta ble 2. Compound 2b Affords Partial NT-3-Like Survival
Activity in Synergy with Suboptimal (100 pM) NT-3
Concentrations
2b
NT-3
concentrated
(pM)
% survival of
TrkC-expressing
NIH-3T3 cells ( SEM
concentrated
(µM)
entry
1
2
3
4
5
6
7
8
9
0
0
0
0.4
2
10
50
0.4
2
0
2000
100
0
0
0
0
100
100
100
100
0 ( 0
100 ( 0
26 ( 3.0
0.2 ( 0.5
0.1 ( 0.5
0.4 ( 0.3
0.2 ( 0.2
32 ( 1.8
33 ( 1.5
38 ( 2.2
36 ( 1.2
10
11
10
50
Ta ble 3. Mean Channel Fluorescence Shifts Induced by
FITC-Labeled Compounds in Cells Expressing the Indicated
Receptor or No Neurotrophin Receptor (Wild-Type Cells),
Demonstrating Direct Binding to Neurotrophin Receptors
F igu r e 2. Western blotting assay showing that the tyrosine
phosphorylation of TrkC receptors caused by 2b at 50 µM is
between that caused by 100 pM and 2 nM NT-3.
mean channel
fluorescence
but this was significant compared to wild-type control
cells that do not express neurotrophin receptors. Indeed,
the fluorescent shift in the presence of saturating NGF
(entry 6) was lower, suggesting specific binding of FITC-
2b to TrkA at a docking site that may overlap with
NGF. In controls, no shift above background was seen
after staining wild-type cells that do not express neu-
rotrophin receptors with FITC-2b or anti-receptor mAbs.
(% of maximal binding)
TrkA TrkC wild-type
entry
1
test agent
cells cells
cells
irrelevant FITC mAb
(background)
10
22
11
11
2
3
4
5
6
7
20 µM FITC-2b
71
100
14
9
FITC-R-TrkC mAb
FITC-R-TrkA mAb
100
9
10
20 µM FITC-2b + NT-3 (2 nM)
20 µM FITC-2b + NGF (2 nM)
20 µM FITC-2b +
42
40
Selective binding to TrkA and TrkC suggests that 2b
behaves like NT-3, which binds both receptors but TrkA
with lower affinity.4 The fact that addition of saturating
concentrations of unlabeled neurotrophin ligands {NT-3
to TrkC-cells or NGF to TrkA-cells} blocked a significant
proportion of the binding of FITC-2b suggests that the
neurotrophins and 2b may bind to overlapping sites on
the receptors. Consistent with this, an anti-TrkC mono-
clonal antibody that binds to the domain of TrkC where
NT-3 binds also blocked the binding of FITC-2b (Table
3, entry 7). Cell survival assays could not be done with
FITC-2b because its poor solubility precludes filter
sterilization (FITC-2b is more water-insoluble than 2b).
More importantly, the presence of the solvent DMSO
would interfere with cell survival assays (testing FITC-
2b in survival assays would expose cells to 2.4% DMSO
for 2-3 days, whereas in FACScan assays they are
exposed to 0.9% DMSO for 1 h). However, we predict
that while FITC-2b and 2b are not chemically the same,
they likely have similar binding and biological proper-
ties.
R-TrkC mAb (20 nM)
purpose, fluoresceinylated 2b (or “FITC-2b”) was pre-
pared via a solid-phase route (Supporting Information).
FITC-2b was tested for binding to transfected TrkC-
expressing or TrkA-expressing cells in a fluorescence
activated cell sorting (FACScan) assay as previously
described.3 Selective FITC-labeled anti-TrkC or anti-
TrkA receptor monoclonal antibodies (mAb) were used
to standardize maximal staining, an FITC-labeled ir-
relevant antibody was used to determine background
fluorescence, and untransfected cells that do not express
any neurotrophin receptor served as a negative control.
In TrkC-expressing cells, a significant shift in the mean
channel fluorescence (7× background) was observed,
indicative of TrkC binding by FITC-2b (compare entry
2 with entry 1 of Table 3). This fluorescence diminished
upon addition of saturating (2 nM) doses of NT-3 (entry
5), suggestive of an overlapping TrkC docking for NT-3
and FITC-2b. In TrkA-expressing cells, there was very
low staining with FITC-2b (entry 2) (2× background),
Tyrosine phosphorylation (pTyr) assays were per-
formed to test if peptidomimetic 2b would affect the
intrinsic kinase activity of TrkC receptors. Such activity
is implied by the enhanced cell survival data described
in Table 2. Treatment with 2b (50 µM) was compared
with treatment with NT-3 (2 nM and 100 pM; positive
control), with saline, and with an irrelevant peptidomi-
metic (negative controls; data not shown). Cells express-
ing TrkC were exposed for 20 min at 37 °C to the
indicated treatments. Detergent extracts prepared from
these cells were studied by Western blotting with anti-
pTyr mAb 4G10 as previously described.11
Compound 2b affords the pTyr of TrkC receptors
(Figure 2). As expected, the pTyr induced by 2b is lower

4390 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Letters
than pTyr induced by 2 nM NT-3, but it is comparable
to pTyr induced by 100 pM NT-3.
Biomolecular NMR Laboratory at Texas A&M Univer-
sity was supported by the National Science Foundation
(Grant DBI-9970232) and the Texas A&M University
System.
It has been reported11 that Trk receptor phosphory-
lation in neuronal cells can activate either a survival
pathway (e.g., cell survival in serum-free media) or a
differentiative pathway (e.g., neurite outgrowth). In-
deed, while NGF-TrkA interactions can activate both
pathways, some ligands of the TrkA receptor activate
one pathway but not the other.11,12 Compound 2b in the
absence of NT-3 induces partial TrkC tyrosine phos-
phorylation but does not afford cell survival; hence, we
propose that it may induce the differentiative pathway.
This hypothesis cannot be tested directly because DMSO
(1.3% final concentration to which the cells are exposed)
is required for solubilizing 2b and this interferes with
the neurite outgrowth assay (but not with the cell
survival assay).
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for the preparation of 1 and 2, tabulated spectroscopic
data, details of the conformational analyses, and experimental
procedure and scheme outlining the synthesis of FITC-2b. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
(1) Maness, L. M.; Kastin, A. J .; Weber, J . T.; Banks, W. A.;
Beckman, B. S.; et al. The Neurotrophins and Their Receptors:
Structure, Function, and Neuropathology. Neurosci. Biobehav.
Rev. 1994, 18, 145-159.
(2) Burgess, K. Solid-Phase Syntheses of â-Turn Analogues To
Mimic or Disrupt Protein-Protein Interactions. Acc. Chem. Res.
2001, 34, 826-835.
Peptidomimetic 2b is a promising lead for develop-
ment of partial agonist TrkC ligands. This NT-3 pepti-
domimetic appears to bind TrkC at a site overlapping
the NT-3 docking region. It induces the pTyr of TrkC
and under certain conditions partially agonizes the
TrkC receptor to afford cell survival under apoptotic
conditions. We believe that it is the first reported small-
molecule mimic of NT-3 that acts on the TrkC receptor.
NT-3 failed clinical trials because of short half-life
(seconds to minutes), so a low efficacy but stable NT-3
peptidomimetic such as 2b may be useful.
(3) Maliartchouk, S.; Feng, Y.; Ivanisevic, L.; Debeir, T.; Cuello, A.
C.; et al. A Designed Peptidomimetic Agonistic Ligand of TrkA
NGF Receptors. Mol. Pharmacol. 2000, 57, 385-391.
(4) Patapoutian, A.; Reichardt, L. F. Trk Receptors: Mediators of
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(6) Feng, Y.; Wang, Z.; J in, S.; Burgess, K. Conformations of
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Membered Ring Systems. Chem.sEur. J . 1999, 5, 3273-3278.
(7) Pettitt, B. M.; Matsunaga, T.; Al-Obeidi, F.; Gehrig, C.; Hruby,
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(9) Perczel, A.; Hollosi, M. Turns. In Circular Dichroism and the
Conformational Analysis of Biomolecules; Fasman, G. D., Ed.;
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(10) Bush, C. A.; Sarkar, S. K.; Kopple, K. D. Circular Dichroism of
â Turns in Peptides and Proteins. Biochemistry 1978, 17, 4951-
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(11) Saragovi, U. H.; Zheng, W.; Maliartchouk, S.; DiGugliemo, G.
M.; Mawal, Y.; et al. A TrkA selective rapidly internalizing NGF-
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Chem. 1998, 273, 34933-34940.
(12) Belliveau, D. J .; Krivko, I.; Kohn, J .; Lachance, C.; Pozniak, C.;
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Ack n ow led gm en t. We thank Dr. Wolfgang Rapp of
Rapp Polymere for a sample of HypoGel 200 RAM resin
and Dr. H. B. Lee for useful discussions. Financial
support for this project was provided by the National
Institutes of Health (Grant CA82642), The Texas Ad-
vanced Technology Program, and the Robert A. Welch
Foundation. U.H.S. is a Scholar of CIHR and is sup-
ported by a grant from CIHR. Dr. Shane Stichy at the
TAMU/LBMS-Applications Laboratory provided mass
spectrometric support, and the Laboratory for Molecular
Simulations (Dr. Lisa Thompson) supported our molec-
ular simulations work. The NMR instrumentation in the
J M0255421