Syntheses and Activities of New C10 β-Turn Peptidomimetics

Article abstract of DOI:10.1021/jo034167x

A program to identify small molecules that mimic or disrupt protein-protein interactions led us to design the peptidomimetics 1-3. Solid-phase syntheses of 1-3 were developed. The purities of the crude materials isolated from the resin tend to be highest for the S- and N-compounds 2 and 3 and better than in the corresponding syntheses of peptidomimetics A. The particular dipeptide units incorporated were chosen to correspond with the turn regions of the neurotrophins (e.g., nerve growth factor {NGF} and the neurotrophin factor-3 {NT-3}). Preliminary studies were performed to access the binding of these analogues to Trk receptors and their ability to induce cell survival (just as NGF and NT-3 do). Several active compounds were identified. However, poor water solubilities of some of the other compounds preclude reliable testing. Consequently, solid-phase modifications to the synthetic procedures were investigated to provide access to the derivatives 12-14 in which the aromatic nitro group is replaced by amine, guanidine, or sulfonamide functionalities. The latter are more acceptable pharmacophores than nitro groups and also tend to increase the water solubilities of the peptidomimetics.

Full text of DOI:10.1021/jo034167x

Syn th eses a n d Activities of New C10 â-Tu r n P ep tid om im etics
Hong Boon Lee,† Maria Clara Zaccaro,‡ Mookda Pattarawarapan,† Sudipta Roy,†
H. Uri Saragovi,‡ and Kevin Burgess*,†
Chemistry Department, Texas A & M University, P.O. Box 30012, College Station, Texas 77843-3012,
and Pharmacology and Therapeutics, McGill University, 3655 Drummond no. 1320,
Montreal, Quebec, Canada H3G 1Y6
burgess@tamu.edu
Received February 6, 2003
A program to identify small molecules that mimic or disrupt protein-protein interactions led us to
design the peptidomimetics 1-3. Solid-phase syntheses of 1-3 were developed. The purities of the
crude materials isolated from the resin tend to be highest for the S- and N-compounds 2 and 3 and
better than in the corresponding syntheses of peptidomimetics A. The particular dipeptide units
incorporated were chosen to correspond with the turn regions of the neurotrophins (e.g., nerve
growth factor {NGF} and the neurotrophin factor-3 {NT-3}). Preliminary studies were performed
to access the binding of these analogues to Trk receptors and their ability to induce cell survival
(just as NGF and NT-3 do). Several active compounds were identified. However, poor water
solubilities of some of the other compounds preclude reliable testing. Consequently, solid-phase
modifications to the synthetic procedures were investigated to provide access to the derivatives
12-14 in which the aromatic nitro group is replaced by amine, guanidine, or sulfonamide
functionalities. The latter are more acceptable pharmacophores than nitro groups and also tend to
increase the water solubilities of the peptidomimetics.
In the design and testing of peptidomimetics for
binding target protein receptors, rigidity is a two-edged
sword.1-3 At one extreme, compounds that are too flexible
may pay such high entropic penalties on binding that the
process becomes energetically unfavorable. Conversely,
severe conformational constraints can prevent molecules
from adapting to the binding site at all. A reasonable
approach to finding compounds that bind proteins is
therefore to prepare and test molecules with the ap-
propriate amino acid side chains and moderate confor-
mational constraints and then to increase the conforma-
tional focus in subsequent libraries via incremental
changes that reduce flexibilities. Studies of the conforma-
tions of the peptidomimetics in solution may be used to
guide this process. It is possible that the favored confor-
mation of an active peptidomimetic in solution is not the
bound conformation. However, the likelihood of this
decreases with the rigidity of the mimic, and hence it is
possible to interpret solution-state conformational studies
with increasing levels of certainty as the experiments
hone in on less flexible molecules.
macrocyclization reactions.5-8 It is now evident that
compounds A can disrupt some protein-protein interac-
tions. For instance, a small library of mimics A was
prepared based on the i + 1 and i + 2 residues of the
turn regions of the nerve growth factor (NGF). The NGF
turn regions are thought to be hot spots for the interac-
tion of this protein with its high affinity receptor,
TrkA.9,10 Some members of the library were active in
several pharmacological tests designed to identify this
interaction, including direct and competitive binding to
TrkA, studies based on the ability of NGF (or NGF
mimics) to promote cell survival in media free of growth
hormones, phosphorylation of intracellular tyrosine resi-
dues in the TrkA receptor on activation, upregulation of
choline acetyl transferase, and induction of neuronal cell
growth. Data on one of the lead compounds, D3, has been
described in full,11 and several other interesting leads
have been identified, implying that the combination of
peptidic and nonpeptidic fragments in structures A might
be especially favorable for mimicry of hot spots in
protein-protein interactions that involve â-turn i +1 and
i + 2 regions.
Previous work from these laboratories have focused on
â-turn4 mimetics A, prepared via solid-phase, SNAr
(5) Feng, Y.; Wang, Z.; J in, S.; Burgess, K. J . Am. Chem. Soc. 1998,
120, 10768.
(6) Feng, Y.; Burgess, K. Chem.-Eur. J . 1999, 5, 3261.
(7) Feng, Y.; Burgess, K. Biotechnol. Bioeng. Comb. Chem. 2000,
71, 3.
(8) Park, C.; Burgess, K. J . Comb. Chem. 2001, 3, 257.
(9) LeSauteur, L.; Wei, L.; Gibbs, B.; Saragovi, H. U. J . Biol. Chem.
1995, 270, 6564.
† Texas A & M University.
‡ McGill University.
(1) Searle, M. S.; Williams, D. H. J . Am. Chem. Soc. 1992, 114,
10690.
(2) Gerhard, U.; Searle, M. S.; Williams, D. H. Bioorg. Med. Chem.
Lett. 1993, 3, 803.
(3) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int.
Ed. 1998, 37, 2754.
(4) Rose, G. D.; Gierasch, L. M.; Smith, J . A. Turns in Peptides and
Proteins; Academic Press: New York, 1985; Vol. 37.
(10) Beglova, N.; LeSauteur, L.; Ekiel, I.; Saragovi, H. U.; Gehring,
K. J . Biol. Chem. 1998, 273, 23652.
(11) Maliartchouk, S.; Feng, Y.; Ivanisevic, L.; Debeir, T.; Cuello,
A. C.; Burgess, K.; Saragovi, H. U. Mol. Pharm. 2000, 57, 385.
10.1021/jo034167x CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/09/2004
J . Org. Chem. 2004, 69, 701-713
701

Lee et al.
SCHEME 1. P r ep a r a tion of th e (a ) O-Con ta in in g,
(b) S-Con ta in in g, a n d (c) N-Con ta in in g Tem p la tes
The next step of our work on NGF and related growth
hormones was to produce more rigid turn analogues that
retain the critical structural features of the original
design including the amino acids corresponding to the i
+1 and i + 2 sites in the target turns. To attempt this,
we designed systems of the general type 1-3 shown
above. In these compounds, the a-c bonds are essentially
coplanar and can only move in concert, whereas the
corresponding dihedral angles in A can flex more inde-
pendently. Consequently, mimics 1-3 should be more
rigid than compounds A. The key challenges were to
develop solid-phase syntheses of these materials that
gave crude products with high purities, to explore their
conformations, and to test for biological activities similar
to that shown by compounds A.
Resu lts a n d Discu ssion
Syn th eses of Su p p or ted Tem p la tes. Scheme 1
describes syntheses of templates 4, 5, and 6 that were
pivotal to the solid-phase synthesis of the desired pep-
tidomimetics. The oxygen template 4 was prepared
(Scheme 1a) via hydrolysis of the commercially available
benzyl bromide to give alcohol 7,12 which was then
tritylated to 8.13 This trityl ether does not withstand
reduction of the nitro group with SnCl2 in DMF (protons
are generated in this reaction), but Adams’ catalyst
mediated hydrogenation of the nitro group to the amine
4 without significant hydrogenolysis of the O-protecting
group.14 Amino acid 4 could be activated and coupled to
resins functionalized with the Rink linker.15 The fact that
this reaction is not complicated by reactions at the free
amine group16 simplified the template synthesis but
(14) Hudlicky, M.; Reductions in Organic Chemistry; Wiley: New
York, 1984.
(15) Rink, H. Tetrahedron Lett. 1987, 28, 3787.
(16) Neustadt, B. R.; Smith, E. M.; Nechuta, T.; Zhang, Y. Tetra-
hedron Lett. 1998, 39, 5317.
(12) Berteina, S.; Mesmaeker, A. D. Synlett 1998, 1227.
(13) Mutter, M.; Hersperger, R. Synthesis 1989, 198.
702 J . Org. Chem., Vol. 69, No. 3, 2004

â-Turn Peptidomimetics with Increased Constraints
SCHEME 2. P r ep a r a tion of th e P ep tid om im etics
1-3
implied that conditions to effect subsequent couplings to
the supported arylamine might require some investiga-
tion (see the next section below).
The preferred protecting group for thiols in our solid-
phase synthesis would have been Mmt (monomethoxy-
trityl),17 but the corresponding thiol {(4-MeOC6H4)Ph2-
CSH} is expensive. Consequently, triphenylmethyl thiol
(TrtSH) was used to prepare the sulfur-containing tem-
plate 5 (Scheme 1b). Trityl groups are unmasked from
sulfur using 3% trifluoroacetic acid. These conditions are
a little harsh for the Rink amide linker, so the syntheses
presented here used the Wang ester linker to give
carboxylic acids on cleavage. Syntheses of carboxamides
via Rink resin would simply involve using the more
expensive thiol. In addition to cost factors, however, the
more stable Trt protecting group has the advantage that,
unlike TrtO in the synthesis of the first template, the
TrtS group is stable to on-resin tin(2+) chloride reduction
of the nitro group.18-20 Experimentally, Rink resin has
the advantage that complete coupling of the first amino
acid is facile and is easily monitored using the Kaiser
test.21 For Wang resin, the coupling is not quite as
efficient and cannot be followed as easily. Consequently,
the loading of the sulfur template was checked using a
UV assay monitoring liberation of the trityl group (see
Supporting Information).
The nitrogen-containing template was prepared in Mtt-
protected form {Mtt is 4-methyltrityl, (4-MeC6H4)Ph2C-}
as indicated in Scheme 1c, via a known benzylic amine.22,23
Use of Mtt is compatible with Rink resin. A solution-
phase hydrogenation was used to form the amine 6.
Solid -P h a se Syn th eses of Ma cr ocycles. Coupling
of the first amino acids to the supported templates was
the most difficult step in the macrocycle syntheses
depicted in Scheme 2. The PyBrop reagent (bromo-tris-
pyrrolidino-phosphonium hexafluorophosphate)17,24 with
di-iso-propylethylamine has been used to couple unreac-
tive amines to acids,25 but in the current study this
particular reagent system only gave satisfactory results
for a few amino acids. After optimization of the reaction
conditions (Table 1), it was found that PyBrop/2,6-
lutidine was a superior system; 15 equiv of base was used
here because loss of the trityl-based protecting groups
occurred if less was added. As far as we are aware, this
coupling agent/base combination is relatively unex-
plored.26-28 This set of reagents was superior to any
others that were tested in this work, both with respect
to number of equivalents of the amino acids/PyBrop
required and epimerization. The rest of the steps for the
TABLE 1. Con d ition s for Cou p lin g of F ir st Am in o Acid s
to Su p p or ted Tem p la te 6
PyBrop
(equiv)
base
(equiv)
time puritya
entry
amino acid3
(h)
(%)
1
2
3
Fmoc-Lys(Boc)-OH
Fmoc-Lys(Boc)-OH
Fmoc-Lys(Boc)-OH
3
3
3
iPr2NEt (8)
2,6-lutidine (8)
N-methyl-
8
8
8
57
93
88
morpholine (8)
4
5
6
7
8
9
Fmoc-Asn(Trt)-OH
Fmoc-Asn(Trt)-OH
Fmoc-Asn(Trt)-OH
Fmoc-Asn(Trt)-OHb
Fmoc-Asn(Trt)-OHb
Fmoc-Gly-OH
3
3
3
5
5
3
3
iPr2NEt (8)
2,6-lutidine (15)
2,6-lutidine (15) 20
2,6-lutidine (15)
2,6-lutidine (15) 20
2,6-lutidine (15)
8
8
8
66
79
73
86
95
95
8
8
(17) NovaBiochem Catalog & Peptide Synthesis Handbook; Nova-
Biochem: Darmstadt, 1999.
10 Fmoc-Gly-OH
2,6-lutidine (15) 20
(18) Mayer, J . P.; Zhang, J .; Bjergarde, K.; Lenz, D. M.; Gaudino,
J . J . Tetrahedron Lett. 1996, 37, 8081.
a
b
Three equivalents unless otherwise indicated. Five equiva-
(19) Arumugam, V.; Routledge, A.; Abell, C.; Balasubramanian, S.
Tetrahedron Lett. 1997, 38, 6473.
lents.
(20) Lee, J .; Gauthier, D.; Rivero, R. A. Tetrahedron Lett. 1998, 39,
201.
(21) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.
(22) Rich, D. H.; Gurwara, S. K. Tetrahedron Lett. 1975, 16, 301.
(23) Gelb, M. H.; Abeles, R. H. J . Med. Chem. 1986, 29, 585.
(24) Coste, J .; Frerot, E.; J ouin, P. Tetrahedron Lett. 1991, 32, 1967.
(25) Guarna, A.; Guidi, A.; Machetti, F.; Menchi, G.; Occhiato, E.
G.; Scarpi, D.; Sisi, S.; Trabocchi, A. J . Org. Chem. 1999, 64, 7347.
(26) Humphrey, J . M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243.
(27) Bunin, B. A.; Ellman, J . A. J . Am. Chem. Soc. 1992, 114, 10997.
(28) Wang, H.; Ganesan, A. J . Comb. Chem. 2000, 2, 186.
preparation of the macrocycle are similar to those
outlined previously for the peptidomimetics A. These are
standard amino acid coupling steps, removal of the “P”
protecting group from “X” (1% TFA in CH2Cl2 for OTrt
and NHMtt, and 3% TFA for STrt), base-mediated
cyclization, and simultaneous removal of the side chain
protecting groups and cleavage of the products from the
resin.
J . Org. Chem, Vol. 69, No. 3, 2004 703

Lee et al.
TABLE 2. Yield a n d P u r ity Da ta for Com p ou n d s 1-3a
TABLE 3. Com p a r ison of P u r ities for Com p ou n d s A
a n d 1-3
amino acids
amino acids
purity (%)
1, 2, or 3
compd
i + 1
i + 2
X/Y
puritya (%)
yield (%)
entry
i + 1
i + 2
X
A
1a
1b
1c
1d
1e
1f
1g
1h
1i
Ile
Lys
Lys
Lys
Arg
Gly
Asn
Asn
Gly
Gly
Thr
Asn
Lys
Lys
Lys
Arg
Gly
Asn
Asn
Gly
Gly
Thr
Asn
Lys
Lys
Lys
Arg
Gly
Glu
Ser
Thr
Ser
Thr
Gln
Gln
Thr
Ser
Ala
Asp
O/NH2
O/NH2
O/NH2
O/NH2
O/NH2
O/NH2
O/NH2
O/NH2
O/NH2
O/NH2
O/NH2
S/OH
S/OH
S/OH
S/OH
S/OH
S/OH
S/OH
S/OH
S/OH
96/97
62/81
86/98
78/92
38/49
94/98
88/96
70/87
80
66
43
53
54
67
60
71
74
69
71
47
56
57
50
55
52
67
54
56
52
55
59
44
46
61
61
58
55
64
71
65
82
98
62
94
88
51
Gly
Glu
Ile
Thr
Ile
Asn
Lys
Arg
Lys
Glu
Ile
Gly
Glu
Ile
1
2
3
4
5
6
7
8
Ile
Glu
Ile
Lys
Lys
Asn
Lys
Lys
Lys
Asn
Arg
Lys
Lys
O
O
O
S
S
S
S
S
80
89
61
87
91
85
65
82
86
90
96
86
94
95
97
97
100
87
87
90
Ile
Gly
Glu
Asn
Ile
Ile
Glu
74/92
76/92
56/90
1j
9
10
NH
NH
1k
2a
2b
2c
2d
2e
2f
2g
2h
2i
95/100
97/100
97/100
87/91
94/100
98/100
100/100
99/100
99/100
98/100
85/95
a
Purity assessed by HPLC with UV detection at 254 nm.
for a bioassay). In some cases, the major impurity was
isolated, subjected to MALDI-MS analysis, and shown
to have a molecular mass of twice the value expected for
the product. This apparently corresponds to the dimeric
structure 10 (MS evidence alone). Similar head-to-tail
dimers have been observed in other work from these
laboratories, and they tend to be the major byproducts
when there is some stereoelectronic impediment to the
desired macrocyclization. In this work, however, dimers
such as 10 were only detected for the macrocyclic ethers
1. No dimers were observed in the syntheses of com-
pounds 2 and 3, even at resin loadings as high as 0.6
mmol/g (conditions that accentuate dimer formation
when O-nucleophiles are used in the ring closure step).
This is in contrast to systems A wherein head-to-tail
dimers can form at higher resin loadings even if N- and
S-nucleophiles are used. The macrocyclic thiols and
amines were formed in significantly higher purities
without any appreciable head-to-tail dimer formation.
Compounds 2 had average purities of 98.7% (ELS, over
11 samples), and compounds 3 had average purities of
99.0% (ELS, over 16 samples). For 2 and 3 together, the
minimum purity observed was 91%.
Thr
Ile
Asn
Lys
Arg
Lys
Glu
Ile
Gly
Glu
Ile
Thr
Arg
Glu
Glu
Pro
Lys
Pro
Lys
Gln
Lys
Gln
Thr
2j
S/OH
S/OH
2k
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
NH/NH2
87/94
87/100
90/100
85/93
86/98
71/100
94/100
100/100
97/100
88/100
98/100
100/100
96/100
95/99
97/100
98/100
a
The resin was Wang (1.1 mmol/g) for 1a -k , Rink amide
MBHA (0.55 mmol/g) for 2a -k , Rink (0.6 mmol/g) for 3a -h , and
b
Rapp-HG200RAM (0.62 mmol/g) for 3i-o. Purity as assessed by
HPLC of crude product, monitored UV absorption at 254 nm. and
using an evaporative light scattering detector (Sedex)
Experiments were run in the early stages of this project
to ascertain the effects (if any) of resin on the cyclization
reactions. In the event, the support made relatively little
difference on the outcome of the reaction. These data are
summarized in Supporting Information.
Table 2 summarizes yield and purity data for the
macrocycles produced in this work. Throughout, UV and
evaporative light scattering (ELS) detection were used
to monitor the purities. The percentage purities indicated
via UV and ELS detection can differ by as much as 44%,
but in most cases they are much closer. UV detection
over-represents compounds with larger extinction coef-
ficients at the detection wavelength. ELS has a higher
threshold of detection, so it tends to miss trace impurities.
In this particular study, we believe that ELS, being based
on particle mass, is likely to be more representative of
the actual purity, especially since significant impurities
probably have molecular masses similar to the product.
Table 2 shows that the oxygen compounds 1 were
formed in the lowest purities in the series (49-98% based
on ELS, average 84.1%) where 7 of the 11 compounds
had purities above 85% (a typical quality control “cut-off
value” above which a crude sample might be submitted
The purity trends outlined above are consistent with
those previously observed for compounds A where the
purity of the products is less if oxygen nucleophiles are
involved in the cyclization than for N- or S-nucleophiles.
However, products 1-3 tend to be formed in higher
purities. Table 3 shows data selected from this study for
syntheses of some of the compounds 1-3, presented with
other data from these laboratories involving syntheses
of systems A. Higher purities are obtained for 1-3 in
most cases.
Con for m a tion a l An a lyses of Com p ou n d s 1c, 2c,
a n d 3c. The approach used in this study is the same one
704 J . Org. Chem., Vol. 69, No. 3, 2004

â-Turn Peptidomimetics with Increased Constraints
F IGURE 1. Simulated low energy conformers for compound 1c, 2c, 3c, and 12a -c.
used in these laboratories for conformational analyses
of other peptidomimetics, including compound A.29 J ust
as in the previous work, DMSO was used as the solvent
so that the data obtained could be compared with that
from previous studies and because some of the com-
pounds were insoluble in aqueous media. Details of the
approach used are given in the supplementary. The linear
precursor peptide 11 was isolated and analyzed to check
a feature of the data relating to NH coefficients.
why the CD and molecular simulations experiments
indicate 3c has a turn conformation but the NMR data
does not. An alternative explanation for the atypical
conformational behavior of the amine could be that the
aniline nitrogen is more planar than the corresponding
O or S atoms as a result of conjugation with the nitro
group (we thank a referee for suggesting this). Overall,
we conclude that 1c, 2c, and 3c can adopt conformations
that approximate â-turns, though in the case of the amine
3c this bias is not so pronounced as for the corresponding
ether and thioether.
P r elim in a r y Biologica l Scr een s. Two assays were
used to evaluate the activities of these compounds: a
competitive fluorescence activated cell sorting (FACS)
assay to detect binding to cells that overexpress the Trk
receptors and a cell survival assay to identify compounds
that have neurotrophin-related biological properties.
Early in this work, peptidomimetic 3n was identified as
a very interesting compound. It was tested in these and
several other assays and found to be active. These data
have now been communicated; 3n is the first reported
small molecule mimic of NT-3. It synergizes with this
neurotrophin via binding to the TrkC receptor to induce
intracellular phosphorylation and, subsequently, protects
the cells from apoptotic cell death.30 The subsequent
discussion focuses on the other compounds in this series,
since full details of these activities for 3n have already
been given.
Overall the data obtained from the combined NMR,
CD, and molecular simulations studies of the compounds
are consistent with the following conclusions. The ether
1c and the thioether 2c populate type I-like turn con-
formations (Figure 1). CD (in MeOH/H2O) and QMD data
for compound 3c are indicative of type I-like turn
conformations, but this assertion is not supported by the
NMR data. It is possible that the strong H-bond acceptor
characteristics of DMSO enable it to interact with the
arylamine functionality of 3c in a way that disrupts the
turn conformation and that was not evident in the
calculations since explicit solvent molecules were not
used. Similar interactions would not be so prevalent in
MeOH/H2O during the CD studies. This would explain
The compounds from Table 2 were tested for binding
selectivity toward cells expressing TrkC or TrkA recep-
tors in the FACS assay. Such FACS assays are not
feasible using labeled natural neurotrophins as the
competing ligand. Fortunately, a set of monoclonal
antibodies (mAb’s) that bind to the domain 5 of the
(29) Feng, Y.; Wang, Z.; J in, S.; Burgess, K. Chem.-Eur. J . 1999,
5, 3273.
(30) Pattarawarapan, M.; Zaccaro, M. C.; Saragovi, U.; Burgess, K.
J . Med. Chem 2002, 45, 4387.
J . Org. Chem, Vol. 69, No. 3, 2004 705

Lee et al.
TABLE 4. Com p ou n d s In h ibit th e Bin d in g of m Ab’sa
mAb binding (% maximal)
competitor
none
NTF (0.3 nM)
NTF (1 nM)
2B7‚TrkC
100 ( 0
5C3‚TrkA
100 ( 0
1
2
3
4
5
6
7
8
84 ( 8* (NT-3)
59 ( 7* (NT-3)
32 ( 7* (NT-3)
92 ( 14
92 ( 9 (NGF)
81 ( 7* (NGF)
68 ( 6* (NGF)
73 ( 8*
NTF (2 nM)
2b
2d
3a
3b
3d
3e
3j
97 ( 6
75 ( 11*
83 ( 7*
79 ( 8*
82 ( 7*
92 ( 9
9
84 ( 9
83 ( 3*
10
11
12
13
14
15
94 ( 6
84 ( 5*
115 ( 16
80 ( 4*
83 ( 5*
3l
67 ( 8*
3m
3o
12b
84 ( 15
86 ( 7*
F IGURE 2. Trophic survival assays. The compounds were
tested with cells in the presence of limiting concentrations of
the corresponding neurotrophin (100 pM NT-3 for NIH-TrkC
and 100 pM NGF for NIH-TrkA) that afford ∼25% cell
survival. Data are standardized to maximal trophic survival
afforded by optimal (2 nM) neurotrophins ) 100%. Statistically
reproducible survival of less than ∼25% indicates antagonism,
whereas higher than ∼25% survival indicates potentiation of
agonism. Average ( standard deviation from 3 to 4 indepen-
dent assays, each assay n ) 6. Only the compounds active in
this assay are shown; all others tested were statistically
inactive.
95 ( 11
85 ( 7*
79 ( 11*
84 ( 20
a
The maximal binding of mAb’s 2B7 or 5C3 is shown, relative
to no inhibition (100%). Data ( standard deviation for competitor
compounds (at 50 µM) or for the corresponding neurotrophins
(NTF) with statistically significant inhibition are indicated with
asterisk, n ) 3-5; p e 0.05.
receptors is available from pervious work in these
laboratories. Domain 5 of the Trk receptors is thought
to be crucial for ligand binding and receptor activation.31-33
The binding sites of the anti-TrkA mAb 5C3 and the anti-
TrkC mAb 2B7 partially overlap with the binding sites
of NGF and NT-3, respectively, and the data shown in
Table 4, entries 2-4 is consistent with this. These mAb’s
are therefore useful surrogates for the natural neurotro-
phins. Simultaneous assays using TrkA and TrkC recep-
tors were used to indicate selectivity. Selective competi-
tion of mAb‚receptor interactions by a small molecule
peptidomimetic (added at 50 µM concentration) suggests
that the compound binds to that receptor, likely binding
at the domain 5 of the receptor where the mAb binds.
Only data for the compounds with statistically significant
activities for either receptor are shown in Table 4.
Compounds 3a and 3l competed with both mAb 2B7‚
TrkC and mAb 5C3‚TrkA interactions, suggesting that
they may bind to both receptors. Compound 3l at 100
µM competed with 5C3‚TrkA interactions to the same
degree as 2 nM NGF and also competed 2B7‚TrkC
interactions to the same degree as 0.3 nM NT-3. Because
of solubility issues, higher concentrations of peptidomi-
metics could not be tested to generate a full inhibitory
dose response curve. However, it seems as if peptidomi-
metic 3l has a Ki 4-5 logs higher than those of the
neurotrophins.
be assayed accurately, even though the FACS assay
tolerates higher levels of DMSO than most other in vitro
screens for neurotrophin/Trk interactions. Others may
perhaps have bound the receptor, but not at the Trk-d5
domain where the mAbs used in this assay dock. Con-
versely, the fact that a particular compound binds does
not provide information about biological activity. Conse-
quently, a cell survival assay was used to test the
biological effects of the mimics.
Survival assays feature cells in culture in serum free
media (SFM) that would ordinarily go into apoptotic
death under these conditions due to lack of growth
factors. The cells can be rescued by trophic factors acting
at selective receptors, e.g., NGF‚TrkA or NT-3‚TrkC.
Compounds that enhance the survival afforded by a
trophic factor in these assays have agonist-like proper-
ties, whereas nontoxic compounds that prevent the
survival afforded by a trophic factor are antagonistic.
Only the compounds that are at least partially soluble
in physiological buffers (e.g., DMSO final concentration
less than 5%) could be tested in the cell survival assays.
Unfortunately, solubility difficulties were encountered in
this assay for all of the compounds listed in Table 4
except 3a , and that compound did not elicit a positive
response in cell survival assays.
Of the compounds that were soluble enough to be
tested, 1a and 2c prevented the trophic rescue of NIH-
TrkA cells by NGF, and NIH-TrkC cells by NT-3 (Figure
2). This antagonism was selective for TrkA and TrkC
receptors because compounds 1a and 2c had no effect on
the trophic rescue of these cells by â-fibroblast growth
factor (data not shown). Peptidomimetics 1c, 2a , 2f, 2g,
and 13c (vide infra) antagonized TrkA‚NGF but had no
statistically significant effect on TrkC‚NT-3. Hence their
antagonistic response was TrkA-selective. Like previously
reported compound 3n 30 the compounds shown in Table
2 had no detectable intrinsic biological activity when
tested in the absence of neurotrophins (data not shown).
Peptidomimetics 2b, 2d , 3d , 3e, 3j, and 3o inhibited
mAb 5C3‚TrkA interactions but not mAb 2B7‚TrkC
interactions; these data suggest that those particular
compounds bind selectively to TrkA. Conversely, 3b
(entry 8) appears to have more affinity toward TrkC than
TrkA.
All of the other compounds assayed did not have a
significant effect in this binding competition assay (data
not shown). Some were too insoluble in water/DMSO to
(31) Urfer, R.; Tsoulfas, P.; O’Connell, L.; Shelton, D. L.; Parada,
L. F.; Presta, L. G. EMBO 1995, 14, 2795.
(32) Perez, P.; Coll, P. M.; Hempstead, B. L.; Martin-Zanca, D.; Chao,
M. V. Mol. Cell. Neurosci. 1995, 6, 97.
(33) Urfer, R.; Tsoulfas, P.; O’Connell, L.; Hongo, J .-A.; Zhao, W.;
Presta, L. G. J . Biol. Chem. 1998, 273, 5829.
706 J . Org. Chem., Vol. 69, No. 3, 2004

â-Turn Peptidomimetics with Increased Constraints
TABLE 5. Yield a n d P u r ity Da ta for Com p ou n d s 12-14a
SCHEME 3. Str a tegies To In cr ea se th e Wa ter
Solu bilities of th e P ep tid om im etics
compd
X/Y
Z
purityb (%) yield (%)
12a
12b
12c
13a
13b
13c
14a
14b
14c
O/NH2
O/NH2
O/NH2
S/OH
S/OH
S/OH
NH2
NHC(dNH)NH2
NHSO2Me
NH2
NHC(dNH)NH2
NHSO2Me
93/98
95/100
88/96
74/100
50/66
95/100
90/93
85/88
43/46
56
60
79
64
60
49
55
88
c
NH/NH2 NH2
NH/NH2 NHC(dNH)NH2
NH/NH2 NHSO2Me
a
The resin was Wang (1.1 mmol/g) for 1a -k , Rink amide
MBHA (0.55 mmol/g) for 2a -k , Rink (0.6 mmol/g) for 3a -h , and
b
Rapp-HG200RAM (0.62 mmol/g) for 3i-o. Purity as assessed by
HPLC of crude product, monitored UV absorption at 254 nm, and
b
using an evaporative light scattering detector (Sedex). Yield not
determined because of low purity.
12c-14c. All of these reactions were attempted for three
dipeptide sequences in the starting materials 1-3, and
products were formed with very high purities except in
two cases (Table 5). For the guanidine 13b two unchar-
acterized byproducts formed in significant amounts.
Guanylation of 14a proceeded cleanly even though there
are two N atoms in the substrate that could potentially
react. The corresponding sulfonylation reaction of 14a
was not so straightforward, however, and we were unable
to obtain high crude purities for the desired product.
Qualitatively, it was observed that the solubilities of
compounds 1-3 vary according to the R1 and R2 side
chains. Where these are lypophilic the compounds tend
to be insoluble or sparingly soluble in water, and this is
unacceptable for bioassays. However, derivatization of
these materials as described above gives amine, guani-
dine, or sulfonamide derivatives that are appreciably
more water-soluble.
Conformational analyses of compounds 12a -c were
performed to probe the effects of modification of the nitro
functionality of 1-3. The calculated low energy conform-
ers for 12a -c are shown in Figure 1d-f, and full data
are given in Supporting Information. Salient details from
these QMD studies and NMR analyses of 12a -c reveals
that their conformations are similar to the parent ether
2 (both in DMSO). The CD spectra of the derivatized
compounds (Figure 1b) are similar to the ether 2 in the
190-210 nm region; they only differ in the part of spectra
above 210 nm wherein ellipticities due to the aromatic
rings prevail. These observations imply that substitution
of the nitro group with amine, guanidine, or methane-
sulfonamide functionalities does not significantly perturb
the preferred conformations of these molecules. This
implies that structure-activity relationships (SARs)
within this series of molecules could be studied without
complications arising from conformational differenc-
es.Proper evaluation of 12-14 as neurotrophin analogues
is in progress, but preliminary results are given. Table
Rea ction s To Ad d P h a r m a cop h or es a n d In cr ea se
Wa ter Solu bilities. The data presented above indicates
that compounds in this series can be active in screens
for which no small molecule leads have previously been
reported. However, the testing also revealed that the
water solubilities of the peptidomimetics are generally
borderline and in some cases too low to allow for accurate
assays of activity. In any case, nitroaryl compounds are
not normally regarded as ideal for pharmaceutical de-
velopment. For these reasons, ways to convert the nitro
group to other, more hydrophilic functionalities were
examined.
The nitro functionalities of compounds 1-3 were
reduced to the corresponding arylamines (SnCl2‚2H2O,
DMF) on the resin. The resulting supported amines were
cleaved from the resin to give 12a -14a (Scheme 3) or
derivatized to introduce other potential pharmacophores.
For instance, reactions with guanidinylating agents (O
and N, N,N′-bis-BOC-1-guanylpyrazole {BOCNHC(pyra-
zole)dNBOC}; S, N,N′-bis-BOC-thiourea {BOCNHC(S)-
NHBOC} and N-methyl-2-chloropyridinium chloride)34
gave the guanidines 12b-14b, and reaction with meth-
anesulfonyl chloride gave the corresponding sulfonamides
(34) Yong, Y. F.; Kowalski, J . A.; Lipton, M. A. J . Org. Chem. 1997,
62, 1540.
J . Org. Chem, Vol. 69, No. 3, 2004 707

Lee et al.
have outlined. Their bioavailabilities35-38 may be in-
creased since they have one less natural amino acid than
compounds A (making them more resistant to proteolytic
degradation). Moreover, the extra aromatic ring in mol-
ecules 1-3 is likely to make them more easily crystallized
for X-ray diffraction studies and helps assignment of
chemical shifts due to anisotropy effects.
Finally, we outline a conclusion that may be pertinent
to many studies aimed at design of small molecules to
mimic or disrupt protein-protein interactions. In this
particular area, structure, conformation, activity rela-
tionships (SCARs) may be more appropriate than simpler
correlations between structure and activity (SARs).
TABLE 6. Dose-Resp on se Stu d ies for Com p ou n d 13a
% maximal survival (relative to 2 nM NTF)a
NIH-TrkA cells in SFM
NIH-TrkC cells in SFM
µM 13a untreated + 0.1 nM NGF untreated + 0.1 nM NT-3
1
2
3
4
0
0.4
2
0 ( 0
1 ( 2
25 ( 1
25 ( 2
26 ( 1
25 ( 1
0 ( 0
9 ( 2
10 ( 1
9 ( 1
25 ( 2
32 ( 2*
35 ( 3*
32 ( 1*
0 ( 1
10
-1 ( 1
a
Trophic survival assays of NIH-TrkA and NIH-TrkC in the
absence (SFM) or presence of limiting concentrations of the
corresponding neurotrophin (100 pM NT-3 for NIH-TrkC and 100
pM NGF for NIH-TrkA) that afford ∼25% cell survival. Data are
standardized to maximal trophic survival afforded by optimal (2
nM) neurotrophins ) 100%. Averaged ( standard deviation, n )
6. Bold indicates survival above no compound, untreated cells.
Asterisks indicate survival above suboptimal neurotrophin control.
Exp er im en ta l Section
NMR Con for m a tion Stu d ies. NMR spectra were recorded
on a Varian UnityPlus 500 spectrometer (500 MHz). The
concentrations of the samples were approximately 5 mM in
DMSO-d6 throughout. One-dimensional (1D) 1H NMR spectra
were recorded with a spectral width of 8000 Hz, 16 transients,
and a 3-s acquisition time. Vicinal coupling constants were
measured from 1D spectra at 25 °C. Assignments of 1H NMR
resonances in DMSO were performed using sequential con-
nectivities. Temperature coefficients of the amide protons were
measured via several 1D experiments in the temperature
range 20-50 °C adjusted in 5 °C increments with an equili-
bration time of more than 10 min after successive temperature
steps. Coupling constants are given in hertz.
Two-dimensional (2D) NMR spectra were recorded at 25 °C
with a spectral width of 8000 Hz. Through-bond connectivities
were elucidated by COSY and DQF-COSY spectra, which were
recorded with 512 t1 increments and 16 scans per t1 increment,
with 2 K data points at t2. Through-space interactions were
identified by ROESY spectra. ROESY experiments were
performed using mixing times of 100, 200, 300, and 400 ms.
The intensities of the ROESY cross-peaks were assigned as S
(strong), M (medium), and W (weak) from the magnitude of
their volume integrals.
4 shows that peptidomimetic 12b may have some binding
to TrkA receptors in FACS scans, because it inhibits 5C3‚
TrkA interactions. Figure 2 shows that compound 12b
had no activity in biological assays; but compound 13a
is a partial agonist of TrkC, i.e., at 50 µM it enhances
the trophic activity of NT-3 in cell survival assays from
25% to ∼50% protection. Survival assays of dose-
response curves for compound 13a showed that at 0.4
µM it has some intrinsic trophic activity for NIH-TrkC
cells in the absence of NT-3 (Table 6), and also continues
to potentiate suboptimal concentrations of NT-3. At
concentrations lower than 50 µM, compound 13a had no
antagonism of TrkA‚NGF function.
Con clu sion s
Compounds 1-3 represent a potentially useful pepti-
domimetic design. They can be formed in higher purities
than the first generation compounds A via efficient and
straightforward solid-phase syntheses. Moreover, com-
pared with the routes to the first generation mimics A,
head-to-tail dimers are only a minor impurity (if they are
seen at all), underscoring the assertion that the desired
ring-closure reaction is more facile as a result of the
rigidity of the acyclic precursor to the second generation
systems. Being intrinsically more rigid than structures
A, these second generation turn mimics provide a means
to introduce small structural changes to alter the pre-
ferred conformations of these molecules in solution. The
preferred conformations of 1c and 2c are not ideal turns,
but there is also no guarantee that the ideal conformation
for bioactivity will be a perfect turn either.
Overall, it is evident from these studies that incre-
mental structural changes can have pronounced effects
on conformation. The structural change from A to 1, 2,
or 3 is much smaller than the steps usually taken on the
pathway from peptidomimetic to constrained peptidomi-
metic to small molecule.
Numerous biological assays are being performed on
compounds 1-3. These data and complete correlations
between structures, conformations, and activities will be
reported when those biological investigations are com-
plete. Nevertheless, our preliminary data indicate recep-
tor selectivity and biological activity at TrkA and TrkC
for some of the compounds.
CD Stu d ies. CD measurements were obtained on an Aviv
(model 62 DS) spectrometer. For these experiments the cyclic
peptidomimetics were dissolved in H2O/MeOH (80:20 v/v) (c
) 0.1 mg/mL, 0.1-cm path length). The CD spectra were
recorded at 25 °C.
Molecu la r Sim u la tion s. CHARMm (version 23.2, rev.
96.0501) was used for the molecular simulations performed
in this work. Explicit atom representations were used through-
out the study. The residue topology files (RTF) for all the
peptidomimetics were built using Quanta97 (version 97.0711,
Molecular Simulations Inc.). Quenched molecular dynamics
simulations for the compounds (compounds 1-3) were per-
formed using the CHARMm standard parameters. All mol-
ecules were modeled in a dielectric continuum of 45 (simulat-
ing DMSO). Thus, the starting conformer was minimized using
1000 steps of Steepest Descents (SD) and 3000 steps of the
Adopted-Basis Newton Raphson method (ABNR), respectively,
until an RMS energy derivative of e0.01 kcal mol-1A-1 was
obtained. The minimized structure was then subjected to
heating, equilibration, and dynamics simulation. Throughout,
the equations of motion were integrated using the Verlet
algorithm with a time step 1 fs, and SHAKE was used to
constrain all bond lengths containing polar hydrogens. Each
peptidomimetic was heated to 1000 K over 10 ps and equili-
(35) Navia, M. A.; Chaturvedi, P. R. Drug Discovery Today 1996, 1,
179.
(36) Chan, O. H.; Stewart, B. H. Drug Discovery Today 1996, 1, 461.
(37) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J . Adv.
Drug Delivery Rev. 1997, 23, 3.
(38) Walters, W. P.; Ajay, Murcko, M. A. Curr. Opin. Chem. Biol.
1999, 3, 384.
Several other characteristics of compounds 1-3 are
potentially beneficial with respect to the objectives we
708 J . Org. Chem., Vol. 69, No. 3, 2004

â-Turn Peptidomimetics with Increased Constraints
brated for another 10 ps at 1000 K, and then molecular
dynamics runs were performed for a total time of 600 ps with
trajectories saved every 1 ps. The resulting 600 structures were
thoroughly minimized again, using 1000 steps of SD followed
by 3000 steps of ABNR. After a suitable cutoff (5.0 kcal/mol
for compound 1, 7.9 kcal/mol for compound 2, 3.5 kcal/mol for
compound 3), the lowest energy structures were selected for
further analysis.
to yield 8.7 g (96%) of yellow powder, 5: mp 213-215 °C; Rf
0.67 (1% AcOH in ethyl acetate); υmax 3000 (b), 1702, 1542,
1
1353; H NMR (300 MHz, CDCl3) δ 8.58 (d, J ) 2, 1H), 8.02
(dd, J ) 8 and 2, 1H), 7.44-7.47 (m, 6H), 7.25-7.34 (m, 9H),
6.90 (d, J ) 8, 1H), 3.83 (s, 2H); 13C NMR (75 MHz, CDCl3) δ
173.9, 144.3, 139.6, 133.9, 133.1, 129.9, 128.3, 128.0, 127.2,
126.8, 68.4, 34.1; HRMS (FAB+) calcd for [C27H21NO4SNa]+
478.1089, found 478.10799.
The Quanta97 package was again used to display and to
classify the selected structures into conformational groups. The
best clustering was obtained using the grouping method based
on calculation of RMS deviation for a subset of atoms; in this
study these were the ring backbone atoms. Thus, appropriate
threshold values (0.85 for compound 1, 0.68 for compound 2,
0.82 for compound 3) were selected to obtain families with
reasonable homogeneity. The lowest energy structure from
each family was considered as a favored conformer and thus
selected as a representative of the family as a whole. Inter-
proton distances and dihedral angles from the lowest energy
structures were calculated for comparisons with the ROE and
NMR data.
Mtt-P r otected Am in o Acid 9. A solution of liquid am-
monia (15 mL) in ethanol (15 mL) was added into a stirred
solution of 4-(bromomethyl)-3-nitrobenzoic acid (5.0 g) in
ethanol (10 mL) at -78 °C in a pressure bottle, and the
mixture was stirred for 5 h at 25 °C. After allowing the NH3
to evaporate, the remaining solution was concentrated in
vacuo. The solid was suspended in a small amount of water
and filtered. The product was dried in vacuo over P2O5 to give
4-(aminomethyl)-3-nitrobenzoic acid,22 3.11 g (82%) as an off-
white solid. This material was used in the next step without
further purification.
4-(Aminomethyl)-3-nitrobenzoic acid (4 g, 20.3 mmol) was
added in one portion into a solution of 4-methyltriphenyl-
methyl chloride (12 g, 40 mmol) in CHCl3/DMF (60 mL, 2:1)
and vigorously stirred at 25 °C for 30 min. Then triethylamine
(11 mL, 81 mmol) was added dropwise, and the mixture was
stirred for 1 h. After addition of methanol (50 mL), the reaction
mixture was stirred at 60 °C for 30 min and concentrated in
vacuo. The residue was purified via flash chromatography with
25-50% EtOAc/hexane to give 8.72 g of compound 9 as pale
yellow oil (95%): Rf 0.21 (60% EtOAc/hexane); υmax 3367 (b),
3011 (b), 2916, 1709, 1622, 1535, 1396, 1353; 1H NMR (300
MHz, acetone-d6) δ 8.52 (d, J ) 1.2 Hz, 1H), 8.39-8.41 (m,
2H), 7.58-7.62 (m, 4H), 7.48 (m, 2H), 7.32-7.37 (m, 4H), 7.21
(t, J ) 1.2 Hz, 1H), 7.25 (m, 1H), 7.16 (dd, J ) 20.1, 0.9 Hz,
2H), 3.73 (s, 2H), 2.32 (s, 3H); 13C NMR (75 MHz, acetone-d6)
δ 164.7, 149.0, 146.1, 143.0, 141.6, 136.2, 134.0, 131.4, 128.8,
128.7, 128.1, 125.4, 71.1, 45.4, 20.3; MS (FAB+) calcd for
[C28H24N2O4Na]+ 475, found 475.
4-(Hyd r oxym eth yl)-3-n itr oben zoic Acid 7. A mixture of
4-(bromomethyl)-3-nitrobenzoic acid (3.0 g, 10.95 mmol) and
sodium carbonate (4.6 g, 43.8 mmol) in 50 mL of 1:1 acetone/
water was heated under reflux for 16 h. The mixture was
cooled to room temperature, acidified to pH 2 with 1 M HCl,
extracted with ethyl acetate (50 mL × 3), dried over MgSO4,
and concentrated in vacuo to yield 2.0 g (93%) of brown solid
1
7. The H NMR of the crude material in DMSO-d6 is identical
to that reported.39
Tr it yl E t h er 8. 4-(Hydroxymethyl)-3-nitrobenzoic acid 7
(2.0 g, 10.14 mmol), triphenylmethyl chloride (3.4 g, 12.17
mmol), silver nitrate (2.1 g, 12.17 mmol), N,N-dimethylfor-
mamide (30 mL), and diisopropylethylamine (3.1 mL, 22.3
mmol) were added to a 100-mL round-bottom flask, and the
mixture was stirred at room temperature for 16 h. The solvent
was removed under reduced pressure, and the crude residue
was purified by flash chromatography on silica gel with an
eluent of 30% ethyl acetate and 1% acetic acid in hexane to
yield 3.79 g (85%) of yellow solid 8: mp 217-220 °C; Rf 0.47
(1:2:0.1 ethyl acetate/hexane/AcOH); υmax 3000 (b), 1709, 1542,
Am in o Acid 6. To a solution of 4 g (8.85 mmol) of 9 in
EtOAc (100 mL) was added PtO2 (0.4 g, 0.885 mmol). The
mixture was stirred vigorously under H2 for 5 h. The reaction
mixture was filtered through Celite and concentrated to
dryness. The residue was purified via flash chromatography
with 25-50% EtOAc/hexane to give 3.37 g (90%) of the product
6 as pale yellow oil: Rf 0.39 (60% EtOAc/hexane); υmax 3433
1
1345; H NMR (300 MHz, acetone-d6) δ 8.75 (d, J ) 2, 1H),
8.42 (dd, J ) 6 and 2, 1H), 8.33 (d, J ) 6, 1H), 7.54-7.57 (m,
6H), 7.33-7.41 (m, 9H), 4.77 (s, 2H); 13C NMR (75 MHz,
acetone-d6) δ 165.2, 144.0, 139.9, 134.4, 129.5, 128.8, 128.3,
127.8, 127.6, 127.0, 125.6, 91.6, 63.3; MS (MALDI+) calcd for
[C27H21NO5Na]+ 462, found 462.
1
(b), 3316 (b), 3018 (b), 2916, 1709, 1215; H NMR (300 MHz,
acetone-d6) δ 7.60-7.63 (m, 1H), 7.47 (s, 1H), 7.50 (s, 1H), 7.43
(m, 1H), 7.35-7.38 (m, 5H), 7.33 (m, 2H), 7.25-7.27 (m, 2H),
7.22 (m, 1H), 7.19 (bs, 1H), 7.16 (m, 1H), 3.40 (s, 2H), 2.33 (s,
3H); 13C NMR (75 MHz, acetone-d6) δ 166.8, 146.5, 146.4,
142.5, 136.0, 130.2, 130.0, 128.9, 128.8, 128.7, 128.0, 126.6,
Am in o Acid 4. A solution of 8 (1.87 g, 4.25 mmol) and PtO2
(50 mg, 0.21 mmol) in 3:1 ethyl acetate/methanol (40 mL) was
stirred vigorously under H2 (1 atm) for 16 h. The reaction
mixture was filtered through Celite and concentrated in vacuo
to yield 1.60 g (92%) of yellow solid 4: mp 180-182 °C; Rf 0.43
(30% ethyl acetate in hexane); υmax 3476, 3360, 3000 (b), 1695,
1629; 1H NMR (300 MHz, CDCl3) δ 7.55-7.59 (m, 6H), 7.30-
7.45 (m, 12H), 4.17 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 171.4,
145.7, 143.7, 129.1, 128.5, 128.0, 127.2, 119.7, 116.9, 87.3, 64.7;
HRMS (FAB+) calcd for [C27H23NO3H]+ 410.17562, found
410.17464.
Tr ityl Th ioeth er 5. To triphenylmethanethiol (6.1 g, 22
mmol) and potassium carbonate (5.5 g, 38 mmol) in a 100-mL
round-bottom flask at 0 °C was added 4-(bromomethyl)-3-
nitrobenzoic acid (5.2 g, 20 mmol) in N,N-dimethylformamide
(40 mL), and the mixture was stirred at room temperature
for 16 h. The reaction mixture was acidified to pH 2 using 1
M HCl and extracted with dichloromethane (50 mL × 3). The
combined organic layers were dried over MgSO4 and concen-
trated to yield a yellow oil. The crude residue was purified by
flash chromatography on silica gel with an eluent of gradient
50% ethyl acetate in hexane to 1% acetic acid in ethyl acetate,
118.7, 116.2, 71.1, 45.1, 20.4; HRMS (FAB) calcd for [C28
26N2O2H]+ 423.20725, found 423.20685.
P r oced u r e for Cou p lin g Com p ou n d 5 to P olystyr en e-
-
H
Wa n g Resin . Polystyrene resin functionalized with a 4-hy-
droxybenzyl linker (1.2 g, 1.1 mmol/g, 1.32 mmol), template 5
(842 mg, 1.85 mmol, tetrabutylammonium iodide (244 mg, 0.66
mmol), and N,N-diisopropylethylamine (805 µL, 4.62 µL) in 8
mL of 4:1 dichloromethane/N,N-dimethylformamide were gen-
tly stirred at 4 °C for 24 h. The resin was filtered, washed,
and treated with 10 equiv of SnCl2‚2H2O in DMF for 1 day.
After washing and drying, the loading of the resin was
determined to be 0.5 mmol/g by UV analysis (410 nm) of the
trityl content of a known amount of resin in 10% trifluoroacetic
acid in dichloromethane. Subsequent couplings of amino acids
were performed following the general procedure given for
peptidomimetics 1-3 below.
P r oced u r e for Cou p lin g Com p ou n d s 4 a n d 6 to P oly-
styr en e-Rin k Resin . Rink resin (0.15 g, 0.6 mmol/g) was
swelled in CH2Cl2 (10 mL/g) in a fritted syringe for 30 min.
The FMOC group on the resin was removed by treating the
resin with 20% piperidine in DMF (2 × 1.5 mL, 10 min and
then 15 min). The resin was washed, after which templates 4
(39) Nicolas, E.; Clemente, J .; Ferrer, T.; Albericio, F.; Giralt, E.
Tetrahedron 1997, 53, 3179.
J . Org. Chem, Vol. 69, No. 3, 2004 709

Lee et al.
or 6 (3 equiv), HBTU (3 equiv), HOBt (3 equiv), and DIEA (5
equiv) in DMF (1.5 mL) were added. After 2 h of gentle
shaking, a ninhydrin test on a small sample of beads gave a
negative result. The reaction mixture was drained, and the
resin was subjected to the above washing cycle.
Da ta for 1d : 17 mg, 53%. Analytical HPLC: homogeneous
single peak, tR ) 15.00 min (8-70% B in 30 min); MALDI MS
calcd for C27H34N8O7 (M + H+) 583.26, found 583.27.
Da ta for 1e: 14 mg, 54%. Analytical HPLC: homogeneous
single peak, tR ) 10.63 min (8-70% B in 30 min); MALDI MS
calcd for C21H21N5O8 (M + H+) 494.13, found 494.11.
Gen er a l Exp er im en ta l P r oced u r e for P r ep a r a tion of
t h e Ma cr ocyclic P ep t id om im et ics 1-3. Com p ou n d 3c.
The resin containing template 6 was treated with FMOC-Lys-
(BOC)-OH (4 equiv), PyBrop (4.8 equiv), and 2,6-lutidine (15
equiv) in CH2Cl2 (1.5 mL) for 20 h. After washing and Fmoc
deprotection, the resin was then treated with FMOC-Glu(Ot-
Bu)-OH (3 equiv), DIC (3 equiv), HOBt (3 equiv), and DIEA
(5 equiv) in CH2Cl2/DMF (4:1, 1.5 mL) for 2 h. The washing
cycle and Fmoc deprotection were repeated, and 2-fluoro-5-
nitrobenzoic acid moiety was introduced by treating the resin
with 2-fluoro-5-nitrobenzoyl chloride (3 equiv) and DIEA (3
equiv) in CH2Cl2 (1.5 mL) for 40 min. The side chain protecting
group (Mtt) of the template was removed by treatment with
1% TFA and 5% TIS in CH2Cl2 (7 × 2 min, or until color
disappeared). After the resin was washed and the macrocy-
clization step was carried out by treating the supported peptide
with K2CO3 (10 equiv) in DMF at 25 °C. After gentle shaking
for 2 d, the peptide-resin was washed then dried in vacuo for
4 h. The peptide was cleaved from the resin by treatment with
a mixture of 90% TFA, 5% TIS, and 5% H2O for 2 h. The
cleavage solution was separated from the resin by filtration.
After most of the cleavage cocktail (about 90%) was evaporated
in vacuo, the crude peptide was triturated using anhydrous
ethyl ether, dissolved in H2O, and then lyophilized to give the
crude product. Preparative HPLC (Beckman System, 5-90%
B in 40 min) was carried out to provide a yellowish powder of
3c (18 mg, 46%) obtained as an ammonium salt (in general,
1
Da ta for 1f: 20 mg, 67%; H NMR (300 MHz, DMSO-d6) δ
8.82 (d, J ) 8.0, 1H), 8.75 (s, 1H), 8.33 (dd, J ) 9.0, 3.0, 1H),
8.24 (d, J ) 8.5, 1H), 8.23 (s, J ) 1.5, 1H), 8.22 (d, J ) 3.0,
1H), 8.00 (s, 1H), 7.69 (d, J ) 9.0, 1H), 7.64 (d, J ) 9.0, 1H),
7.63 (dd, J ) 9.0, 1.5, 1H), 7.46 (s, 1H), 7.41 (s, 1H), 6.98 (s,
1H), 5.63 (d, J ) 12.0, 1H), 5.30 (d, J ) 12.0, 1H), 4.79 (m,
1H), 4.13 (m, 1H), 2.69 (dd, J ) 16.0, 6.5, 1H), 2.56 (dd, J )
16.0, 6.5, 1H), 1.80 (m, 1H), 1.52 (m, 1H), 1.18 (m, 1H), 0.85-
0.90 (m, 6H). Analytical HPLC: homogeneous single peak, tR
) 14.95 min (8-70% B in 30 min); MALDI MS calcd for
C
25H28N6O8 (M + H+) 541.20, found 541.19
Da ta for 1g: 18 mg, 60%; 1H NMR (300 MHz, DMSO-d6) δ
9.05 (d, J ) 8.0, 1H), 8.75 (s, 1H), 8.38 (dd, J ) 9.0, 2.5, 1H),
8.34 (d, J ) 8.5, 1H), 8.22 (s, 1H), 8.14 (d, J ) 3.0, 1H), 8.01
(s, 1H), 7.66 (d, J ) 9.0, 1H), 7.59-7.65 (m, 2H), 7.46 (s, 1H),
7.41-7.44 (b, 3H), 7.01 (s, 1H), 6.95 (s, 1H), 5.62 (d, J ) 12.0,
1H), 5.21 (d, J ) 12.0, 1H), 4.70 (m, 1H), 4.59 (m, 1H), 2.53-
2.75 (m, 4H). Analytical HPLC: homogeneous single peak, tR
) 8.92 min (8-70% B in 30 min); MALDI MS calcd for
C
23H23N7O9 (M + Na+) 564.15, found 564.19.
Da ta for 1h : 3.1 mg, 71%; H NMR (300 MHz, DMSO-d6)
1
δ 8.96 (d, J ) 7.0, 1H), 8.60 (d, J ) 9.0, 1H), 8.60 (s, 1H), 8.33
(dd, J ) 9.0, 3.0, 1H), 8.32 (s, J ) 2.0, 1H), 8.22 (d, J ) 3.0,
1H), 8.00 (s, 1H), 7.70 (d, J ) 8.0, 1H), 7.69 (bs, 3H), 7.62 (m,
2H), 7.41 (s, 1H), 5.53 (d, J ) 12.0, 1H), 5.30 (d, J ) 12.0,
1H), 4.33 (m, 1H), 3.93 (m, 1H), 3.71 (m, 1H), 2.81 (m, 2H),
1.72 (m, 2H), 1.55 (m, 2H), 1.38 (m, 2H). Analytical HPLC:
homogeneous single peak, tR ) 9.15 min (8-70% B in 30 min);
MALDI MS calcd for C23H26N6O7 (M + H+) 499.19, found
499.14.
Da ta for 1i: 3.1 mg, 74%; 1H NMR (300 MHz, DMSO-d6) δ
9.10 (b, 1H), 8.79 (b, 1H), 8.64 (s, 1H), 8.35 (dd, J ) 9.0, 3.0,
1H), 8.33 (s, 1H), 8.22 (d, J ) 3.0, 1H), 7.99 (s, 1H), 7.95 (bs,
1H), 7.70 (d, J ) 8.0, 1H), 7.62 (m, 2H), 7.41 (s, 1H), 7.00-
7.40 (m, 3H), 5.53 (d, J ) 12.0, 1H), 5.32 (d, J ) 12.0, 1H),
4.38 (m, 1H), 3.84 (m, 2H), 3.13 (m, 2H), 1.60-1.76 (m, 3H).
Analytical HPLC: homogeneous single peak, tR ) 9.89 min
(8-70% B in 30 min); MALDI MS calcd for C23H26N8O7 (M +
H+) 527.20, found 527.16.
1
all the products were in protonated form.). Da ta for 3c: H
NMR (500 MHz, DMSO-d6) δ 12.1 (bs, 1H), 9.62 (s, 1H), 8.51
(d, J ) 7.0, 1H), 8.26 (m, 2H), 8.05 (m, 2H), 7.94 (s, 1H), 7.66
(m, 5H), 7.57 (d, J ) 7.0, 1H), 7.37 (s, 1H), 6.77 (d, J ) 9.5,
1H), 4.92 (dd, J ) 7.5, 15.7, 1H), 4.45 (dd, J ) 4.5, 16, 1H),
4.31(m, 1H), 4.11 (m, 1H), 2.81 (bm, 2H), 1.96 (m, 1H), 1.88
(m, 1H), 1.78 (m, 1H), 1.63 (m, 4H), 1.33(m, 2 H); 13C NMR
(75 MHz, DMSO-d6) δ 174.6, 173.3, 169.8, 168.0, 151.8, 137.0,
135.9, 134.5, 132.5, 131.9, 128.1, 125.2, 124.5, 124.0, 119.1,
111.5, 60.1, 57.5, 54.6, 45.2, 31.8, 31.1, 28.5, 27.7, 25.8, 23.2.
Analytical HPLC: homogeneous single peak, tR ) 13.1 min
(8-70% B in 30 min); MALDI MS calcd for C26H31N7O8 (M +
H+) 570.23, found 570.12.
Da ta for 1a : 24 mg, 80%; 1H NMR (300 MHz, DMSO-d6) δ
8.70 (s, 1H), 8.65 (d, J ) 7.0, 1H), 8.62 (d, J ) 9.0, 1H), 8.39
(s, J ) 1.5, 1H), 8.33 (dd, J ) 8.5, 2.5, 1H), 8.25 (d, J ) 2.5,
1H), 7.99 (s, 1H), 7.72 (bs, 3H), 7.68 (d, J ) 9.0, 1H), 7.60 (d,
J ) 9.0, 1.5, 1H), 7.40 (s, 1H), 5.64 (d, J ) 12.5, 1H), 5.32 (d,
J ) 12.5, 1H), 4.39 (m, 1H), 4.20 (m, 1H), 2.75 (m, 2H), 1.90
(m, 2H), 1.73 (m, 1H), 1.55 (m, 3H), 1.38 (m, 2H), 1.22 (m,
1H), 0.82-0.94 (m, 6H), Analytical HPLC: homogeneous single
peak, tR ) 14.50 min (8-70% B in 30 min); MALDI MS calcd
for C27H34N6O7 (M + H+) 555.26, found 555.26.
Da ta for 1j: 3.1 mg, 69%; 1H NMR (300 MHz, DMSO-d6) δ
8.98 (d, J ) 7.5, 1H), 8.63 (s, 1H), 8.43 (dd, J ) 3, 9, 1H), 8.40
(d, J ) 3.0, 1H), 8.31 (d, J ) 8.5, 1H), 8.28 (d, J ) 3.0, 1H),
8.02 (s, 1H), 7.59-7.71 (m, 6H), 7.43 (s, 1H), 5.67 (d, J ) 12.0,
1H), 5.16 (d, J ) 12.0, 1H), 4.47 (m, 1H), 4.28 (m, 2H), 2.80
(m, 2H), 1.77 (m, 2H), 1.56 (m, 2H), 1.45 (m, 2H), 1.06 (m,
3H). Analytical HPLC: homogeneous single peak, tR ) 9.36
min (8-70% B in 30 min); MALDI MS calcd for C25H30N6O8
(M + H+) 543.22, found 543.16.
Da ta for 1k : 26 mg, 71%; 1H NMR (300 MHz, DMSO-d6) δ
12.2 (bs, 1H), 8.99 (d, J ) 7.0, 1H), 8.75 (s, 1H), 8.38 (m, 2H),
8.30 (d, J ) 3.0, 1H), 8.21 (s, 1H), 8.01 (s, 1H), 7.59-7.68 (m,
3H), 7.46 (s, 1H), 7.40 (s, 1H), 6.99 (s, 1H), 5.64 (d, J ) 12.0,
1H), 5.17 (d, J ) 12.0, 1H), 4.74 (m, 1H), 4.23 (m, 1H), 2.67
(dd, J ) 16.0, 6.5, 1H), 2.55 (dd, J ) 16.0, 6.5, 1H), 2.40 (m,
2H), 1.89 (m, 2H). Analytical HPLC: homogeneous single
peak, tR ) 10.67 min (8-70% B in 30 min); MALDI MS calcd
for C24H24N6O10 (M + H+) 557.16, found 557.09.
Da ta for 1b: 18 mg, 66%; analytical HPLC: homogeneous
single peak, tR ) 10.08 min (8-70% B in 30 min); MALDI MS
calcd for C23H26N6O7 (M + H+) 499.19, found 499.23.
Da ta for 1c: 46 mg, 43%; 1H NMR (500 MHz, DMSO-d6) δ
12.2 (bs, 1H), 8.73 (d, J ) 7.0, 1H), 8.70 (s, 1H), 8.53 (d, J )
8.5, 1H), 8.41 (dd, J ) 3.0, 9.0, 1H), 8.37 (d, J ) 3.0, 1H), 8.33
(d, J ) 3.0, 1H), 8.00 (s, 1H), 7.68 (d, J ) 9.0, 1H), 7.66 (s,
1H), 7.66 (bs, 3H), 7.64 (d, J ) 3.0, 1H), 7.40 (s, 1H), 5.67 (d,
J ) 12.0, 1H), 5.23 (d, J ) 12.0, 1H), 4.48 (m, 1H), 4.28 (m,
1H), 2.77 (m, 2H), 2.48 (m, 2H), 2.00 (m, 2H), 1.93 (m, 1H),
1.70 (m, 1H), 1.55 (m, 2H), 1.35 (m, 2H); 13C NMR (75 MHz,
DMSO-d6) δ 174.6, 173.0, 171.2, 168.1, 165.8, 160.8, 141.1,
138.0, 136.2, 131.3, 129.7, 127.8, 127.3, 124.7, 124.0, 123.7,
114.4, 69.48, 56.0, 53.2, 31.2, 30.9, 26.9, 26.6, 23.1. Analytical
HPLC: homogeneous single peak, tR ) 15.00 min (8-70% B
in 30 min); MALDI MS calcd for C26H30N6O9 (M + H+) 571.22,
found 571.17.
Da ta for 2a : 5.0 mg, 47%; 1H NMR (300 MHz, DMSO-d6) δ
12.6 (bs, 1H), 9.13 (s, 1H), 8.92 (d, J ) 10.0, 1H), 8.52 (d, J )
8.5, 1H), 8.50 (d, J ) 2.5, 1H), 8.26 (dd, J ) 2.5, 8.5, 1H), 8.10
(d, J ) 2.5, 1H), 8.07 (d, J ) 8.5, 1H), 7.68 (m, 5H), 4.70 (d, J
) 11.5, 1H), 4.48 (d, J ) 11.5, 1H), 4.43 (m, 1H), 4.38 (m, 1H),
2.75 (m, 2H), 1.85 (m, 2H), 1.65 (m, 1H), 1.53 (m, 3H), 1.33
(m, 3H), 0.84-0.93 (m, 6H). Analytical HPLC: homogeneous
single peak, tR ) 17.40 min (8-70% B in 30 min); MALDI MS
calcd for C27H33N5O7S (M + H+) 572.22, found 572.24.
710 J . Org. Chem., Vol. 69, No. 3, 2004

â-Turn Peptidomimetics with Increased Constraints
Da ta for 2b: 5.5 mg, 56%. Analytical HPLC: homogeneous
single peak, tR ) 14.11 min (8-70% B in 30 min); MALDI MS
calcd for C23H25N5O7S (M + H+) 516.16, found 516.16.
Da ta for 2c: 43 mg, 57%; 1H NMR (500 MHz, DMSO-d6) δ
12.6 (bs, 2H), 9.10 (s, 1H), 8.86 (d, J ) 8.0, 1H), 8.54 (d, J )
1.5, 1H), 8.42 (d, J ) 9.0, 1H), 8.33 (dd, J ) 2.5, 8.5, 1H), 8.31
(d, J ) 2.5, 1H), 8.13 (d, J ) 8.5, 1H), 7.71 (dd, J ) 1.5, 8.0,
1H), 7.68 (m, 3H), 7.67 (d, J ) 8.0, 1H), 4.77 (d, J ) 10.5, 1H),
4.53 (m, 1H), 4.46 (d, J ) 10.5, 1H), 4.45 (m, 1H), 2.75 (m,
2H), 2.54 (m, 2H), 2.04 (m, 1H), 1.95 (m, 1H), 1.87 (m, 1H),
1.64 (m, 1H), 1.54 (m, 2H), 1.30 (m, 2H); 13C NMR (75 MHz,
DMSO-d6) δ 173.9, 172.0, 170.0, 166.8, 166.4, 145.3, 143.3,
139.6, 137.0, 132.0, 131.8, 131.0, 130.9, 130.3, 125.5, 124.4,
123.8, 121.2, 54.2, 53.7, 36.2, 31.0, 30.6, 26.9, 26.4, 22.2.
Analytical HPLC: homogeneous single peak, tR ) 14.83 min
(8-70% B in 30 min); MALDI MS calcd for C26H29N5O9S (M +
H+) 588.18, found 588.24.
8.31 (b, 1H), 8.28 (dd, J ) 8.5, 2.5, 1H), 8.24 (d, J ) 2.5, 1H),
8.06 (d, J ) 8.5, 1H), 7.72 (bs, 3H), 7.65 (m, 2H), 4.86 (d, J )
11.0, 1H), 4.47 (m, 1H), 4.39 (m, 1H), 4.30 (m, 1H), 4.21 (d, J
) 11.0, 1H), 2.83 (m, 2H), 1.78 (m, 2H), 1.59 (m, 2H), 1.51 (m,
2H), 1.09 (m, 3H). Analytical HPLC: homogeneous single
peak, tR ) 13.54 min (8-70% B in 30 min); MALDI MS calcd
for C25H29N5O8S (M + H+) 560.18, found 560.15.
1
Da ta for 2k : 1.8 mg, 55%; H NMR (500 MHz, DMSO-d6)
δ 12.6 (bs, 2H), 9.15 (s, 1H), 8.92 (d, J ) 8.5, 1H), 8.38 (s, 1H),
8.29 (d, J ) 9.0, 1H), 8.28 (s, 1H), 8.20 (d, J ) 8.5, 1H), 8.10
(d, J ) 9.0, 1H), 7.67 (dd, J ) 1.5, 7.5, 1H), 7.62 (d, J ) 7.5,
1H), 7.42 (s, 1H), 6.94 (m, 1H), 4.75 (m, 1H), 4.73 (d, J ) 11.0,
1H), 4.40 (m, 1H), 4.38 (d, J ) 11.0, 1H), 2.61 (d, J ) 6.0, 2H),
2.40 (m, 2H), 1.89 (m, 2H). Analytical HPLC: homogeneous
single peak, tR ) 14.28 min (8-70% B in 30 min); MALDI MS
calcd for C24H23N5O10S (M + Na+) 596.11, found 596.07.
Da ta for 3a : 26 mg, 59%; 1H NMR (300 MHz, DMSO-d6) δ
9.70 (s, 1H), 8.45 (d, J ) 8.1, 1H), 8.23 (d, J ) 1.5, 1H), 8.14
(d, J ) 2.7, 1H), 8.05 (dd, J ) 2.7, 9.3, 1H), 7.96 (bs, 1H), 7.90
(bt, 1H), 7.60-7.76 (m, 4H), 7.39 (m, 2H), 7.31 (bm, 1H), 6.77
(d, J ) 9.6, 1H), 4.92 (m, 1H), 4.46 (m, 1H), 4.35 (m, 1H), 3.90
(m, 2H), 2.81 (m, 2H), 1.82 (m, 2H), 1.64 (m, 2H), 1.35 (m,
2H), 0.80-1.25 (m, 8H). Analytical HPLC: homogeneous single
peak, tR ) 16.6 min (8-70% B in 30 min); MALDI MS calcd
for C27H35N7O6 (M + Na+) 576.25, found 576.17.
Da ta for 3b: 13 mg, 44%. Analytical HPLC: homogeneous
single peak, tR ) 12.4 min (8-70% B in 30 min); MALDI MS
calcd for C23H27N7O6 (M + H+) 498.20, found 498.14.
Da ta for 3d : 23 mg, 61%; 1H NMR (300 MHz, DMSO-d6) δ
10.1 (bs, 1H), 8.95 (s, 1H), 8.81 (d, J ) 8.4, 1H), 8.74 (bt, 1H),
8.45-8.60 (m, 3H), 8.08 (m, 1H), 7.54-7.80 (m, 3H), 7.30 (d,
J ) 8.1, 1H), 4.83 (s, 2H), 4.50-4.65 (m, 1H), 4.20-4.35 (m,
3H), 1.17 (d, J ) 6.3, 3H). Analytical HPLC: 86%, tR ) 11.2
min (8-70% B in 30 min); MALDI MS calcd for C21H22N6O7
(M + H+) 471.16, found 471.10.
1
Da ta for 2d : 5.0 mg, 50%; H NMR (300 MHz, DMSO-d6)
δ 12.6 (bs, 1H), 9.18 (s, 1H), 8.84 (d, J ) 9.5, 1H), 8.45 (d, J )
8.5, 1H), 8.46 (s, 1H), 8.25 (dd, J ) 2.5, 9.0, 1H), 8.09 (d, J )
2.5, 1H), 8.07 (d, J ) 8.5, 1H), 7.68 (m, 5H), 7.57 (bs, 1H),
6.90-7.30 (b, 3H), 4.65 (d, J ) 11.5, 1H), 4.50 (d, J ) 11.5,
1H), 4.39 (m, 1H), 4.32 (m, 1H), 3.11 (m, 2H), 1.86 (m, 2H),
1.65 (m, 1H), 1.52 (m, 3H), 1.22 (m, 1H), 0.81-0.94 (m, 6H).
Analytical HPLC: homogeneous single peak, tR ) 18.10 min
(8-70% B in 30 min); MALDI MS calcd for C27H33N7O7S (M +
H+) 600.22, found 600.17.
Da ta for 2e: 5.5 mg, 55%. Analytical HPLC: homogeneous
single peak, tR ) 14.84 min (8-70% B in 30 min); MALDI MS
calcd for C21H20N4O8S (M + H+) 511.09, found 511.09.
Da ta for 2f: 5.0 mg, 52%; 1H NMR (300 MHz, DMSO-d6) δ
12.6 (bs, 1H), 9.12 (s, 1H), 8.81 (d, J ) 9.0, 1H), 8.39 (s, 1H),
8.50 (d, J ) 2.5, 1H), 8.26 (dd, J ) 2.5, 9.0, 1H), 8.21 (d, J )
8.5, 1H), 8.11 (d, J ) 2.5, 1H), 8.09 (d, J ) 9.0, 1H), 7.66 (s,
2H), 7.42 (s, 1H), 6.91 (s, 1H), 4.73 (m, 1H), 4.65 (d, J ) 11.5,
1H), 4.43 (d, J ) 11.5, 1H), 4.28 (m, 1H), 2.64 (dd, J ) 15.5,
6.0, 1H), 2.55 (dd, J ) 15.5, 6.0, 1H), 1.82 (m, 1H), 1.51 (m,
1H), 1.22 (m, 1H), 0.76-0.90 (m, 6H). Analytical HPLC:
homogeneous single peak, tR ) 17.82 min (8-70% B in 30 min);
MALDI MS calcd for C25H27N5O8S (M + Na+) 580.15, found
580.16.
Da ta for 3e: 23 mg, 61%. Analytical HPLC: homogeneous
single peak, tR ) 11.2 min (8-70% B in 30 min); MALDI MS
calcd for C21H22N6O7 (M + Na+) 495.15, found 495.06.
1
Da ta for 3f: 21 mg, 58%; H NMR (300 MHz, DMSO-d6) δ
12.1 (bs, 1H), 9.52 (s, 1H), 8.80 (bs, 1H), 8.22 (b, 1H), 8.13 (m,
2H), 8.00 (dd, J ) 2.7, 9.3, 1H), 7.94 (m, 1H), 7.80 (b, 1H),
7.65 (s, 2H), 7.36 (s, 2H), 7.18 (b, 3H), 6.76 (d, J ) 9.6, 1H),
4.30-4.45 (m, 3H), 4.12 (m, 1H), 3.12 (b, 2H), 2.25 (m, 2H),
1.92 (m, 2H), 1.75 (m, 2H), 1.67 (m, 2H). Analytical HPLC:
71%, tR ) 12.4 min (8-70% B in 30 min); MALDI MS calcd
for C26H31N9O9 (M + H+) 598.24, found 598.18.
Da ta for 2g: 5.5 mg, 67%; 1H NMR (300 MHz, DMSO-d6) δ
12.6 (bs, 1H), 9.15 (s, 1H), 8.90 (d, J ) 8.5, 1H), 8.35 (s, 1H),
8.30 (d, J ) 2.5, 1H), 8.25 (d, J ) 8.4, 1H), 8.16 (s, 1H), 8.11
(d, J ) 2.5, 1H), 7.67 (d, J ) 8.0, 1H), 7.62 (d, J ) 8.0, 1H),
7.46 (s, 1H), 7.39 (s, 1H), 7.00 (s, 1H), 6.89 (s, 1H), 4.70-4.80
(m, 2H), 4.70 (d, J ) 11.0, 1H), 4.39 (d, J ) 11.0, 1H), 2.53-
2.69 (m, 4H). Analytical HPLC: homogeneous single peak, tR
) 13.00 min (8-70% B in 30 min); MALDI MS calcd for
Da ta for 3g: 17 mg, 55%; 1H NMR (300 MHz, DMSO-d6) δ
12.2 (bs, 1H), 9.62 (s, 1H), 8.52 (d, J ) 7.2, 1H), 8.28 (m, 2H),
8.06 (m, 2H), 7.96 (s, 1H), 7.65 (m, H), 7.48 (d, J ) 7.2, 1H),
7.38 (s, 1H), 6.78 (d, J ) 9.6, 1H), 5.00 (m, 2H), 4.36-4.48 (m,
2H), 3.59 (m, 2H), 2.46 (m, 2H), 1.92 (m, 2H); 13C NMR (75
MHz, DMSO-d6) δ 174.5, 173.5, 169.8, 169.2, 168.0, 151.8,
137.1. 135.9, 134.6, 132.4, 131.7, 128.1, 125.5, 124.5, 123.9,
119.1, 111.5, 60.9, 57.5, 56.5, 45.3, 31.1, 25.8. Analytical
HPLC: homogeneous single peak, tR ) 12.9 min (8-70% B in
30 min); MALDI MS calcd for C23H24N6O9 (M + H+) 529.16,
found 529.05.
C
23H22N6O9S (M + Na+) 581.11, found 581.11.
Da ta for 2h : 1.8 mg, 54%; H NMR (300 MHz, DMSO-d6)
1
δ 12.6 (bs, 1H), 9.07 (d, J ) 9.5, 1H), 8.95 (s, 1H), 8.74 (d, J )
8.5, 1H), 8.57 (d, J ) 1.5, 1H), 8.25 (dd, J ) 8.5, 2.5, 1H), 8.21
(d, J ) 2.5, 1H), 8.05 (d, J ) 8.5, 1H), 7.65 (m, 3H), 7.63 (dd,
J ) 8.5, 1.5, 1H), 7.59 (d, J ) 8.5, 1H), 4.64 (d, J ) 11.5, 1H),
4.47 (m, 1H), 4.43 (d, J ) 11.5, 1H), 4.03 (dd, J ) 15.5, 8.0,
1H), 3.74 (dd, J ) 15.5, 6.0, 1H), 2.83 (m, 2H), 1.76 (m, 2H),
1.57 (m, 2H), 1.44 (m, 2H). Analytical HPLC: homogeneous
single peak, tR ) 13.42 min (8-70% B in 30 min); MALDI MS
calcd for C23H25N5O7S (M + H+) 516.16, found 516.16.
Da ta for 2i: 1.9 mg, 56%; 1H NMR (300 MHz, DMSO-d6) δ
12.6 (bs, 1H), 9.25 (b, 1H), 8.98 (s, 1H), 8.86 (b, 1H), 8.56 (s,
1H), 8.23 (d, J ) 8.5, 1H), 8.18 (s, 1H), 8.06 (d, J ) 8.5, 1H),
7.74 (b, 1H), 7.62 (d, J ) 8.5, 1H), 7.59 (d, J ) 8.5, 1H), 7.00-
7.30 (b, 3H), 4.62 (d, J ) 12.0, 1H), 4.48 (d, J ) 12.0, 1H),
4.45 (m, 1H), 4.02 (dd, J ) 15.5, 8.0, 1H), 3.74 (dd, J ) 15.5,
6.0, 1H), 3.15 (m, 2H), 1.71 (m, 3H). Analytical HPLC:
homogeneous single peak, tR ) 13.94 min (8-70% B in 30 min);
MALDI MS calcd for C23H25N7O7S (M + H+) 544.16, found
544.17.
Da ta for 3h : 18 mg, 64%. Analytical HPLC: homogeneous
single peak, tR ) 13.0 min (8-70% B in 30 min); MALDI MS
calcd for C24H26N6O9 (M + H+) 543.17, found 543.08.
1
Da ta for 3i: 24 mg, 80%; H NMR (300 MHz, DMSO-d6) δ
9.43 (s, 1H), 8.57 (s, 1H), 8.19 (d, J ) 2.7, 1H), 8.12 (d, J )
7.2, 1H), 8.00 (dd, J ) 2.4, 9.0, 1H), 7.96 (s, 1H), 7.63 (bs, 2H),
7.44 (d, J ) 4.8, 1H), 7.36 (bs, 1H), 6.52 (d, J ) 9.3, 1H), 4.75
(m, 1H), 4.30-4.54 (m, 3H), 3.70-3.87 (m, 4H), 1.70-2.03 (m,
4H); 13C NMR (75 MHz, DMSO-d6) δ 173.1, 168.5, 168.3, 166.8,
150.2, 137.8, 135.2, 130.5, 129.3, 127.3, 123.8, 123.7, 123.3,
120.1, 111.7, 63.5, 61.1, 59.1, 47.4, 47.2, 32.3, 22.8. Analytical
HPLC: homogeneous single peak, tR ) 11.9 min (8-70% B in
30 min); MALDI MS calcd for C28H24N6O7 (M + H+) 497.17,
found 497.08.
Da ta for 2j: 1.8 mg, 52%; 1H NMR (300 MHz, DMSO-d6) δ
12.6 (bs, 1H), 9.31 (d, J ) 9.5, 1H), 8.78 (s, 1H), 8.66 (s, 1H),
J . Org. Chem, Vol. 69, No. 3, 2004 711

Lee et al.
1
Da ta for 3j: 21 mg, 65%; H NMR (300 MHz, DMSO-d6) δ
DMSO-d6) δ 172.7, 171.8, 169.6, 168.9, 168.1, 151.4, 137.6,
135.9, 134.7, 131.8, 130.8, 127.7, 123.7, 123.1, 120.7, 111.3,
65.7, 64.6, 51.4, 45.2, 35.8, 21.1. Analytical HPLC: homoge-
neous single peak, tR ) 13.2 min (8-70% B in 30 min); MALDI
MS calcd for C23H24N6O9 (M + H+) 529.16, found 529.06.
Gen er a l P r oced u r e for P r ep a r a tion of Der iva tized
Ma cr ocyclic P ep tid om im etics 12a -14a . Resin containing
side chain protected 1c or 2c or 3c was swelled in CH2Cl2 in
a fritted syringe for 30 min and treated with 10 equiv of SnCl2‚
2H2O in DMF for 1 day. After washing, the peptide was cleaved
from the resin by treatment with a mixture of 90% TFA, 5%
TIS, and 5% H2O for 2 h. The cleavage solution was separated
from the resin by filtration. After most of the cleavage cocktail
(about 90%) was evaporated in vacuo, the crude peptide was
triturated using anhydrous ethyl ether, dissolved in H2O, and
then lyophilized to give the crude product.
9.18 (s, 1H), 8.88 (d, J ) 7.8, 1H), 8.58 (d, J ) 2.7, 1H), 8.22
(d, J ) 2.7, 1H), 8.10-8.15 (m, 2H), 7.98 (m, 1H), 7.65-7.81
(m, 5H), 7.40-7.48 (m, 2H), 6.83 (d, J ) 9.3, 1H), 4.78-4.90
(m, H), 4.43 (m, 1H), 4.30 (m, 2H), 4.15-4.22 (m, 2H), 2.84
(m, 2H), 1.78 (m, 2H), 1.61 (m, 4H), 1.10 (d, J ) 6.3, 3H).
Analytical HPLC: homogeneous single peak, tR ) 11.4 min
(8-70% B in 30 min); MALDI MS calcd for C25H31N7O7 (M +
H+) 542.23, found 542.13.
Da ta for 3k : 21 mg, 71%; 1H NMR (300 MHz, DMSO-d6) δ
9.30 (s, 1H), 8.60 (s, 1H), 8.30 (d, J ) 7.8, 1H), 8.15 (d, J )
2.7, 1H), 7.96 (m, 2H), 7.64 (m, 2H), 7.37-7.45 (m, 3H), 6.89
(bs, 1H), 6.51 (d, J ) 9.3, 1H), 4.80 (m, 2H), 4.56 (m, 1H), 4.18
(m, 1H), 3.80 (m, 2H), 1.90-2.20 (m, 8H); 13C NMR (75 MHz,
DMSO-d6) δ 174.5, 173.5, 169.8, 169.2, 168.0, 151.8, 137.1,
135.9, 134.6, 132.4, 131.7, 128.1, 125.5, 124.5, 123.9, 119.1,
111.5, 60.9, 57.5, 56.5, 45.3, 31.1, 25.8. Analytical HPLC:
homogeneous single peak, tR ) 11.8 min (8-70% B in 30 min);
MALDI MS calcd for C25H27N7O7 (M + Na+) 560.19, found
560.07.
Da ta for 12a : 9.5 mg, 41%; 1H NMR (500 MHz, DMSO-d6)
δ 12.2 (bs, 1H), 8.79 (s, 1H), 8.47 (d, J ) 8.5, 1H), 8.44 (d, J )
8.0, 1H), 8.37 (s, 1H), 7.95 (s, 1H), 7.69 (bs, 3H), 7.61 (s, 2H),
7.32 (s, 1H), 7.31 (d, J ) 8.5, 1H), 7.06 (s, 1H), 7.04 (d, J )
8.5, 1H), 5.36 (d, J ) 12.0, 1H), 5.07 (d, J ) 12.0, 1H), 4.40
(m, 1H), 4.29 (m, 1H), 2.76 (m, 2H), 2.36 (m, 2H), 1.96 (m,
2H), 1.91 (m, 1H), 1.67 (m, 1H), 1.52 (m, 2H), 1.31 (m, 2H);
13C NMR (75 MHz, DMSO-d6) δ 174.4, 173.0, 171.0, 168.2,
166.9, 159.5, 159.1, 151.6, 137.7, 135.8, 131.1, 130.3, 127.7,
123.7, 123.1, 120.5, 116.0, 69.3, 55.7, 53.3, 30.9, 26.8, 26.4, 23.1.
Analytical HPLC: homogeneous single peak, tR ) 8.20 min
(2-40% B in 30 min); MALDI MS calcd for C26H32N6O7 (M +
H+) 541.24, found 541.26.
1
Da ta for 3l: 34 mg, 98%; H NMR (300 MHz, DMSO-d6) δ
9.66 (s, 1H), 8.57 (d, J ) 6.9, 1H), 8.24 (d, J ) 1.2, 1H), 8.20
(d, J ) 2.7, 1H), 8.06 (dd, J ) 2.7, 9.3, 1H), 7.98 (bm, 1H),
7.60-7.80 (m, 5H), 7.28-7.50 (m, 4H), 6.77 (m, 2H), 4.90 (m,
1H), 4.30-4.50 (m, 2H), 4.08 (m, 1H), 2.83 (m, 2H), 2.11 (m,
2H), 1.91 (m, 2H), 1.76 (m, 2H), 1.40-1.65 (m, 4H). Analytical
HPLC: homogeneous single peak, tR ) 11.3 min (8-70% B in
30 min); MALDI MS calcd for C26H32N8O7 (M + H+) 569.25,
found 569.15.
1
1
Da ta for 3m : 20 mg, 62%; H NMR (300 MHz, DMSO-d6)
Da ta for 13a : 12.5 mg, 55%; H NMR (300 MHz, DMSO-
δ 9.11 (s, 1H), 8.95 (d, J ) 7.2, 1H), 8.59 (d, J ) 2.7, 1H), 8.27
(d, J ) 2.7, 1H), 8.10-8.15 (m, 2H), 7.97 (bs, 1H), 7.64-7.80
(m, 3H), 7.55 (d, J ) 8.4, 1H), 7.42 (bs, 2H), 6.83 (d, J ) 9.3,
1H), 5.05 (d, J ) 4.8, 1H), 4.84 (m, 1H), 4.43 (m, 1H), 4.20 (m,
1H), 2.34 (m, 2H), 2.00 (m, 2H), 1.10 (d, J ) 6.3, 3H); 13C NMR
(75 MHz, DMSO-d6) δ 174.2, 174.1, 169.9, 169.5, 167.9, 153.6,
151.8, 137.2, 136.6, 134.8, 132.9, 131.6, 128.1, 124.4, 124.2,
112.0, 67.4, 60.0, 57.7, 55.6, 32.3, 26.6, 20.8. Analytical
HPLC: 96%, tR ) 11.7 min (8-70% B in 30 min); MALDI MS
calcd for C24H27N7O9 (M + Na+) 564.18, found 564.07.
d6) δ 12.6 (bs, 2H), 9.01 (s, 1H), 8.50 (d, J ) 1.5, 1H), 8.49 (d,
J ) 8.0, 1H), 8.23 (d, J ) 8.5, 1H), 7.66 (dd, J ) 1.5, 8.0, 1H),
7.66 (m, 3H), 7.54 (d, J ) 8.0, 1H), 7.43 (d, J ) 8.5, 1H), 6.63
(dd, J ) 2.5, 8.5, 1H), 6.60 (d, J ) 2.5, 1H), 4.45 (m, 1H), 4.39
(d, J ) 10.5, 1H), 4.32 (m, 1H), 4.06 (d, J ) 10.5, 1H), 2.76 (m,
2H), 2.38 (m, 2H), 1.97 (m, 2H), 1.90 (m, 1H), 1.65 (m, 1H),
1.53 (m, 2H), 1.33 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ
174.5, 173.3, 170.8, 170.0, 167.5, 149.9, 143.7, 137.2, 137.0,
133.0, 132.1, 131.2, 126.4, 115.7, 115.4, 112.5, 71.1, 55.2, 53.9,
31.2, 31.1, 27.3, 27.2, 23.0. Analytical HPLC: homogeneous
single peak, tR ) 15.51 min (2-40% B in 30 min); MALDI MS
calcd for C26H31N5O7S (M + H+) 558.20, found 558.11.
Da ta for 3n : 30 mg, 94%; 1H NMR (300 MHz, DMSO-d6) δ
9.64 (s, 1H), 8.50 (d, J ) 7.5, 1H), 8.23 (d, J ) 1.2, 1H), 8.17
(d, J ) 2.4 Hz, 1H), 8.06 (dd, J ) 2.7, 9.3, 1H), 7.99 (t, J ) 6.3,
1H), 7.95 (s, 1H), 7.66 (m, 5H), 7.36 (s, 1H). 7.30 (d, J ) 7.2,
1H), 6.77 (d, J ) 9.3, 1H), 4.93-5.02 (m, 2H), 4.34-4.47 (m,
2H), 4.05 (m, 1H), 3.57 (m, 2H), 2.81 (b, 2H), 1.30-1.80 (m,
6H); 13C NMR (75 MHz, DMSO-d6) δ 173.8, 169.9, 169.4, 168.0,
151.7, 137.0, 135.9, 134.6, 132.5, 131.8, 128.2, 125.6, 124.4,
124.0, 119.2, 111.5, 61.0, 57.8, 56.5, 45.2, 29.9, 27.2, 23.4.
Analytical HPLC: homogeneous single peak, tR ) 11.4 min
(8-70% B in 30 min); MALDI MS calcd for C24H29N7O7 (M +
H+) 528.21, found 528.15.
Da ta for 3o: 27 mg, 88%; 1H NMR (500 MHz, DMSO-d6) δ
9.42 (s, 1H), 8.55 (d, J ) 7.0, 1H), 8.42 (dd, J ) 1.5, 1H), 8.22
(d, J ) 3, 1H), 8.06 (dd, J ) 2.5, 9.5, 1H), 7.96 (t, J ) 4.5, 1H),
7.92 (s, 1H), 7.66-7.69 (m, 2H), 7.61 (dd, J ) 1.5, 8.0, 1H),
7.38 (s, 1H), 7.33 (s, 1H), 6.85 (s, 1H), 6.77 (d, J ) 9.5, 1H),
4.93 (m, 1H), 4.45 (m, 1H), 4.31(m, 2H), 4.09 (m, 1H), 2.30 (m,
2H), 1.92 (m, 2H), 1.25 (d, J ) 6.5, 3H); 13C NMR (75 MHz,
DMSO-d6) δ 174.2, 173.1,170.5, 169.5, 168.0, 151.6, 137.4,
136.1, 134.7, 131.8, 131.6, 129.7, 129.0, 127.9, 126.9, 124.1,
124.0, 123.6, 120.0, 111.6, 57.5, 50.3, 45.4, 36.4, 32.2, 26.3, 17.7.
Analytical HPLC: homogeneous single peak, tR ) 12.2 min
(8-70% B in 30 min); MALDI MS calcd for C28H25N7O7 (M +
H+) 512.18, found 512.14.
Da ta for 14a : 8.2 mg, 55%; 1H NMR (500 MHz, DMSO-d6)
δ 12.1 (bs, 1H), 9.60 (bs, 1H), 8.29 (m, 2H), 7.92 (m, 2H), 7.49-
7.82 (m, 5), 7.34 (m, 2H), 7.16 (m, 1H), 6.99 (m, 1H), 6.80 (m,
1H), 4.65 (m, 1H), 4.36 (m, 1H), 4.26 (m, 1H), 4.14 (m, 1H),
2.40 (m, 2H), 1.92 (m, 2H), 1.84 (m, 1H), 1.62 (m, 1H), 1.58
(m, 2H), 1.32 (m, 2H). Analytical HPLC: homogeneous single
peak, tR ) 11.3 min (2-40% B in 30 min); MALDI MS calcd
for C26H33N7O6 (M + H+) 540.25, found 540.30.
Gen er a l P r oced u r e for P r ep a r a tion of Der iva tized
Ma cr ocyclic P ep tid om im etics 12b-14b. Resin containing
side chain protected 12a or 13a or 14a was swelled in CH2Cl2
in a fritted syringe for 30 min and treated with 10 equiv of
N,N′-bis-BOC-1-guanylpyrazole in DMF (for 12a and 14 a ) or
10 equiv of N,N′-bis-BOC-thiourea and 10 equiv of N-methyl-
2-chloropyridinium chloride in CH2Cl2 (for 13a ) for 1 day. After
washing, the peptide was cleaved from the resin by treatment
with a mixture of 90% TFA, 5% TIS, and 5% H2O for 2 h. The
cleavage solution was separated from the resin by filtration.
After most of the cleavage cocktail (about 90%) was evaporated
in vacuo, the crude peptide was triturated using anhydrous
ethyl ether, dissolved in H2O, and then lyophilized to give the
crude product.
Da ta for 12b: 9.6 mg, 40%; 1H NMR (500 MHz, DMSO-d6)
δ 12.2 (bs, 1H), 9.76 (s, 1H), 8.76 (s, 1H), 8.64 (d, J ) 9.5, 1H),
8.59 (d, J ) 7.5, 1H), 8.36 (d, J ) 1.5, 1H), 7.96 (s, 1H), 7.68
(bs, 3H), 7.64 (d, J ) 8.0, 1H), 7.61 (dd, J ) 8.0, 1.5, 1H), 7.46
(d, J ) 9.0, 1H), 7.42 (bs, 4H), 7.38 (s, 1H), 7.35 (d, J ) 3.0,
1H), 7.30 (dd, J ) 9.0, 3.0, 1H), 5.46 (d, J ) 12.0, 1H), 5.14 (d,
J ) 12.0, 1H), 4.41 (m, 1H), 4.32 (m, 1H), 2.76 (m, 2H), 2.37
(m, 2H), 1.97 (m, 2H), 1.92 (m, 1H), 1.68 (m, 1H), 1.52 (m,
Da ta for 3p : 16 mg, 51%; 1H NMR (300 MHz, DMSO-d6) δ
9.59 (s, 1H), 8.53 (d, J ) 1.8, 1H), 8.23 (d, J ) 2.7, 1H), 8.19
(d, J ) 7.8, 1H), 8.04 (dd, J ) 2.7, 9.3, 1H), 7.93 (bs, 2H), 7),
7.81 (d, J ) 7.5, 1H), 7.68 (d, J ) 7.8, 1H), 7.60 (dd, J ) 1.8,
7.8, 1H), 7.35 (bs, 1H), 6.76 (d, J ) 9.6, 1H), 5.23 (d, J ) 4.5,
1H), 4.95-5.04 (m, 1H), 4.06-4.73 (m, 1H), 4.43-4.52 (m, 2H),
3.88-4.00 (m, 3H), 1.10 (d, J ) 6.3, 3H); 13C NMR (75 MHz,
712 J . Org. Chem., Vol. 69, No. 3, 2004

â-Turn Peptidomimetics with Increased Constraints
2H), 1.31 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 174.4, 173.0,
171.0, 168.2, 166.5, 156.6, 153.9, 137.8, 135.9, 131.2, 130.1,
129.0, 127.3, 126.6, 123.8, 123.3, 115.3, 68.7, 55.6, 53.3, 30.9,
30.8, 26.8, 26.5, 23.1. Analytical HPLC: homogeneous single
peak, tR ) 11.37 min (2-40% B in 30 min); MALDI MS calcd
for C27H34N8O7 (M + H+) 583.26, found 583.18.
(2-40% B in 30 min); MALDI MS calcd for C27H33N5O9S2 (M
+ H+) 636.18, found 636.18.
Da ta for 14c: 5 mg, 23%; 1H NMR (500 MHz, DMSO-d6) δ
12.2 (s, 1H), 10.1 (s, 1H), 9.76 (s, 1H), 8.56 (d, J ) 9.0 Hz,
1H), 8.35 (s, 1H), 7.95 (s, 1H), 7.85 (d, J ) 9.0 Hz, 1H), 7.60-
7.73(m, 6H), 7.33-7.38 (m, 4H), 5.15 (d, J ) 18 Hz, 1H), 4.41-
4.46 (m, 2H), 3.83 (d, J ) 17.5 Hz, 1H), 3.07 (s, 3H), 2.80 (m,
2H), 2.37 (m, 2H), 1.95 (m, 2H), 1.72 (m, 1H), 1.57 (m, 2H),
1.50 (m, 1H), 1.32 (m, 2 H). Analytical HPLC: homogeneous
single peak, tR ) 12.4 min (2-40% B in 30 min); MALDI MS
calcd for C27H35N7O8S (M + H+) 618.23, found 618.20.
Cell Lin es. Wild-type NIH3T3 cells do not express any of
the Trk receptors. These cells were stably transfected with
TrkA cDNA (NIH-TrkA cells) or TrkC cDNA (NIH-TrkC cells).
Surface expression of each of the transfected receptors was
routinely controlled in all cells by quantitative FACScan
assays (Becton Dickinson, CA) (data not shown) using anti-
TrkA mAb 5C3 and anti-TrkC mAb 2B7.
Da ta for 13b: 3.2 mg, 22%; 1H NMR (300 MHz, DMSO-d6)
δ 12.6 (bs, 2H), 9.94 (s, 1H), 9.05 (s, 1H), 8.68 (d, J ) 8.5, 1H),
8.48 (d, J ) 1.5, 1H), 8.15 (d, J ) 8.0, 1H), 7.89 (d, J ) 9.0,
1H), 7.68 (dd, J ) 1.5, 8.0, 1H), 7.64 (m, 3H), 7.61 (d, J ) 8.0,
1H), 7.56 (bs, 4H), 7.43 (d, J ) 8.0, 1H), 7.34 (s, 1H), 4.55 (d,
J ) 10.5, 1H), 4.39-4.48 (m, 2H), 4.27 (d, J ) 10.5, 1H), 2.76
(m, 2H), 2.38 (m, 2H), 1.97 (m, 2H), 1.90 (m, 1H), 1.65 (m,
1H), 1.53 (m, 2H), 1.33 (m, 2H). Analytical HPLC: homoge-
neous single peak, tR ) 15.78 min (2-40% B in 30 min);
MALDI MS calcd for C27H33N7O7S (M + H+) 600.22, found
600.16.
1
Da ta for 14b: 14 mg, 88%; H NMR (500 MHz, DMSO-d6)
δ 12.1 (s, 1H), 9.52 (s, 1H), 9.40 (s, 1H), 8.27 (d, J ) 7.5 Hz,
1H), 7.90 (s, 1H), 7.50-7.75 (m, 6H), 7.33(s, 1H), 7.10-7.25
(m, 5H), 7.05 (dd, 1.2, 5.4 Hz, 1H), 6.75 (bs, 1H), 6.73 (d, J )
9.5 Hz, 1H), 4.72-4.76 (m, 1H), 4.25-4.32 (m, 2H), 4.12-4.17
(m, 1H), 2.80 (m, 2H), 2.41 (m, 2H), 1.90 (m, 2H), 1.80 (m,
1H), 1.68 (m, 1H), 1.57 (m, 2H), 1.32 (m, 2 H). Analytical
HPLC: homogeneous single peak, tR ) 12.8 min (2-40% B in
30 min); MALDI MS calcd for C27H35N9O6 (M + H+) 582.27,
found 582.35.
F ACSca n Bin d in g Assa ys. Cells (2 × 105) in FACScan
binding buffer (PBS, 0.5% BSA, and 0.1% NaN3) were immu-
nostained as described30,40,41 using saturating anti-TrkA mAb
5C3, or anti-TrkC mAb 2B7, or control nonbinding IgGs (each
at ∼7 nM). Data were acquired on a FACScan, and mean
channel fluorescence of bell-shaped histograms was analyzed
using the LYSIS II program. Data were averaged relative to
positive control (immunostaining with no competitor), n ) 3
( sd.
Gen er a l P r oced u r e for P r ep a r a tion of Der iva tized
Ma cr ocyclic P ep tid om im etics 12c-14c. Resin containing
side-chain protected 12a or 13a or 14a was swelled in CH2Cl2
in a fritted syringe for 30 min and treated with 10 equiv of
methanesulfonyl chloride, 10 equiv of pyridine, and 1 equiv of
4-(dimethylamino)pyridine in CH2Cl2 for 1 day. After washing,
the peptide was cleaved from the resin by treatment with a
mixture of 90% TFA, 5% TIS, and 5% H2O for 2 h. The cleavage
solution was separated from the resin by filtration. After most
of the cleavage cocktail (about 90%) was evaporated in vacuo,
the crude peptide was triturated using anhydrous ethyl ether,
dissolved in H2O, and then lyophilized to give the crude
product.
Cell Su r viva l Assa ys. Five thousand cells/well in protein-
free media (PFHM-II, GIBCO, Toronto) containing 0.2% bovine
serum albumin (BSA) (Crystalline fraction V, Sigma, St. Louis,
MO) were seeded in 96-well plates (Falcon, Mississauga,
Ontario). The cultures were untreated or treated with the
indicated test or control ligands. Cell viability was quantitated
using the MTT assay after 56-72 h of culture, as described.42
Percent protection was standardized from optical density (OD)
readings relative to optimal neurotrophin (1 nM) ) 100% (the
OD of untreated cells were subtracted).
Ack n ow led gm en t . We thank Dr. J eff Labadie of
Argonaut Technologies, Dr. Wolfgang Rapp of Rapp
Polymere, and Dr. Andrew Bray of Chiron Mimetopes
for generously supplying us with supports for the
studies on the resin effects. Financial support for this
work was provided by National Institutes of Health
(CA82642), The Texas Technology Program, and by The
Robert A. Welch Foundation. We thank The Welcome
Trust for a grant to H.B.L. (059980).
1
Da ta for 12c: 16.9 mg, 45%; H NMR (500 MHz, DMSO-
d6) δ 12.2 (bs, 1H), 9.57 (s, 1H), 8.78 (s, 1H), 8.46 (d, J ) 7.5,
1H), 8.44 (d, J ) 7.5, 1H), 8.36 (d, J ) 1.5, 1H), 7.96 (s, 1H),
7.67 (bs, 3H), 7.62 (d, J ) 8.0, 1H), 7.60 (dd, J ) 8.0, 1.5, 1H),
7.40 (d, J ) 9.0, 1H), 7.39 (s, 1H), 7.27 (d, J ) 2.5, 1H), 7.27
(dd, J ) 9.0, 2.5, 1H), 5.41 (d, J ) 12.0, 1H), 5.12 (d, J ) 12.0,
1H), 4.41 (m, 1H), 4.29 (m, 1H), 2.94 (s, 3H), 2.76 (m, 2H),
2.37 (m, 2H), 1.95 (m, 2H), 1.90 (m, 1H), 1.69 (m, 1H), 1.51
(m, 2H), 1.31 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 174.5,
173.0, 171.0, 168.2, 167.0, 152.2, 137.7, 135.8, 132.1, 131.0,
130.3, 127.5, 124.6, 123.8, 123.2, 122.1, 115.3, 68.9, 55.7, 53.2,
30.9, 30.8, 26.8, 26.4, 23.0. Analytical HPLC: homogeneous
single peak, tR ) 14.66 min (2-40% B in 30 min); MALDI MS
calcd for C27H34N6O9S (M + H+) 619.22, found 619.14.
Su p p or tin g In for m a tion Ava ila ble: ROE connectivities,
tables of chemical shift data, temperature coefficient plots,
calculated molecular parameters from the QMD studies,
tabulated comparisons of ROE cross-peak intensities with
simulated distances, tabulated comparisons of calculated and
actual coupling constants, and ChemBats3D plots of the lowest
energy conformer from each family generated in the QMD
studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
Da ta for 13c: 9.1 mg, 59%; 1H NMR (300 MHz, DMSO-d6)
δ 12.6 (bs, 2H), 10.07 (s, 1H), 9.00 (s, 1H), 8.72 (d, J ) 8.0,
1H), 8.51 (d, J ) 1.5, 1H), 8.36 (d, J ) 8.5, 1H), 7.82 (d, J )
8.5, 1H), 7.68 (dd, J ) 1.5, 8.0, 1H), 7.64 (m, 3H), 7.59 (d, J )
8.0, 1H), 7.30 (dd, J ) 2.0, 8.5, 1H), 7.23 (d, J ) 2.0, 1H), 4.49
(d, J ) 10.5, 1H), 4.48 (m, 1H), 4.37 (m, 1H), 4.29 (d, J ) 10.5,
1H), 3.08 (s, 3H), 2.77 (m, 2H), 2.38 (m, 2H), 1.97 (m, 2H),
1.90 (m, 1H), 1.65 (m, 1H), 1.53 (m, 2H), 1.33 (m, 2H); 13C NMR
(75 MHz, DMSO-d6) δ 173.8, 172.4, 170.0, 168.0, 166.8, 142.0,
138.2, 136.5, 135.3, 131.6, 130.8, 126.1, 125.6, 123.8, 120.3,
117.2, 64.9, 54.3, 53.4, 30.6, 30.4, 26.7, 26.5, 22.2, 15.2.
Analytical HPLC: homogeneous single peak, tR ) 20.42 min
J O034167X
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