The effects of conformational constraints and steric bulk in the amino acid moiety of philanthotoxins on AMPAR antagonism

Article abstract of DOI:10.1021/jm049906w

Philanthotoxin-343 (PhTX-343), a synthetic analogue of wasp toxin PhTX-433, is a noncompetitive antagonist at ionotropic receptors (e.g., AChR or iGluR). To determine possible effects of variations of the amino acid side chain, a library consisting of seventeen PhTX-343 analogues was prepared. Thus, tyrosine was replaced by either apolar, conformationally constrained, or bulky amino acids, whereas the acyl unit and the polyamine moiety were kept unchanged. Analogues with tertiary amide groups were prepared for the first time. Pentafluorophenyl esters were employed for amide bond formation, establishing general protocols for philanthotoxin solution- and solid-phase synthesis (39-90% and 42-54% overall yields, respectively). The analogues were tested for their ability to antagonize kainate-induced currents of 2-amino-3-(3-hydroxy-5-methyl- 4-isoxazoyl)propanoic acid receptors (AMPAR) expressed in Xenopus oocytes from rat brain mRNA. This showed that steric bulk in the amino acid moiety is well tolerated and suggests that binding to AMPAR does not involve the α-NHCO group as a donor in hydrogen bonding.

Full text of DOI:10.1021/jm049906w

56
J. Med. Chem. 2005, 48, 56-70
The Effects of Conformational Constraints and Steric Bulk in the Amino Acid
Moiety of Philanthotoxins on AMPAR Antagonism
Malene R. Jørgensen,† Christian A. Olsen,† Ian R. Mellor,‡ Peter N. R. Usherwood,‡ Matthias Witt,§
Henrik Franzyk,*,† and Jerzy W. Jaroszewski†
Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2,
DK-2100 Copenhagen, Denmark, Division of Molecular Toxicology, School of Biology, University of Nottingham,
Nottingham NG7 2RD, U.K., and Bruker Daltonik GmbH, Fahrenheitstrasse 4, D-28359 Bremen, Germany
Received February 3, 2004
Philanthotoxin-343 (PhTX-343), a synthetic analogue of wasp toxin PhTX-433, is a noncompeti-
tive antagonist at ionotropic receptors (e.g., AChR or iGluR). To determine possible effects of
variations of the amino acid side chain, a library consisting of seventeen PhTX-343 analogues
was prepared. Thus, tyrosine was replaced by either apolar, conformationally constrained, or
bulky amino acids, whereas the acyl unit and the polyamine moiety were kept unchanged.
Analogues with tertiary amide groups were prepared for the first time. Pentafluorophenyl esters
were employed for amide bond formation, establishing general protocols for philanthotoxin
solution- and solid-phase synthesis (39-90% and 42-54% overall yields, respectively). The
analogues were tested for their ability to antagonize kainate-induced currents of 2-amino-3-
(3-hydroxy-5-methyl-4-isoxazoyl)propanoic acid receptors (AMPAR) expressed in Xenopus
oocytes from rat brain mRNA. This showed that steric bulk in the amino acid moiety is well
tolerated and suggests that binding to AMPAR does not involve the R-NHCO group as a donor
in hydrogen bonding.
Introduction
Polyamine toxins comprise several subtypes of com-
pounds isolated from the venoms of spiders and wasps
that are used for paralysis of prey. Philanthotoxin-433
(PhTX-433, 1) was isolated from the Egyptian digger
wasp Philanthus triangulum (Figure 1).1
PhTX-433 consists of three subunits: a polyamine
moiety containing four amine functionalities separated
by three or four methylene groups (region 1), a butyryl
group (region 2), and a residue of the L-amino acid
tyrosine (region 3). PhTX-433 and its synthetic analogue
PhTX-343 (2) have been shown to be noncompetitive
inhibitors at mammalian ligand-gated cation channels
such as nicotinic acetylcholine receptors (nAChR) and
ionotropic glutamate receptors (iGluR), including 2-
Figure 1. Natural polyamine toxin PhTX-433 (1) and the
amino-3-(3-hydroxy-5-methyl-4-isoxazoyl)propanoic acid
synthetic analogue PhTX-343 (2).
receptors (AMPAR).2-11 In a preliminary model for the
hydrophobicity of the N-acyl group.10,11 The antagonism
receptor binding of these toxins it was envisaged that
on calcium-permeable iGluR4,17 is potentially important
the positively charged polyamine chain interacts elec-
because excessive influx of Ca2+ ions into the neurons
trostatically with ionized carboxylic acid groups of
is associated with various neurodegenerative disorders
and acute brain seizures.18 In addition to serving as
potential drug leads, PhTX-433 and PhTX-343 ana-
logues are of interest as probes in the studies of receptor
structure and function.6,12,19
Recently, philanthotoxin analogues exhibiting varia-
tions in the polyamine chain have been synthesized by
solution- and solid-phase methods,4,5,20-23 and their
antagonistic effects at various subtypes of iGluR and
nAChR were investigated. Although substitution of the
asymmetric polyamine of the natural PhTX-433 with
the symmetric spermine to give PhTX-343 has an
insignificant influence on biological activity,1,8-11 changes
in the number and position of the secondary amino
functionalities can cause pronounced effects in the
aspartic or glutamic acid residues present in the interior
of the ion channel.7,12 More recent models propose
interactions of amino groups with main-chain carbonyl
oxygen atoms that line the narrowest part of the
AMPAR pore.13 Hydrophobic interactions involving the
toxin headgroup (regions 2 and 3) contribute decisively
to the binding, since the biological activities of philan-
thotoxins are qualitatively and quantitatively different
from those of the parent polyamines.14-16 Also, the
potency of philanthotoxins is enhanced with increased
* Corresponding author. Tel: +45 35306255. Fax: +45 35306040.
E-mail: hf@dfuni.dk.
† The Danish University of Pharmaceutical Sciences.
‡ University of Nottingham.
§ Bruker Daltonik GmbH.
10.1021/jm049906w CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/13/2004

Constraints and Steric Bulk in Philanthotoxins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 57
Scheme 1. Synthesis of Spermine Building Blocksa
of the N-trifluoroacetyl group, afforded a mixture of di-,
tri-, and tetra-Boc-protected spermine derivatives (3a,
3b, and 3c, respectively) in the ratio 1:2:1. The sub-
stantial by-product 3a was used for solid-phase synthe-
sis of PhTX-343 analogues (see below). The reaction
conditions were also modified to obtain 3a as the main
product (87% yield) by employing 2.3 equiv of ethyl
trifluoroacetate. The building block for introduction of
the N-acyl moiety, pentafluorophenyl butyrate, was
obtained from butyryl chloride and pentafluorophenol.22
The majority of amino acids used for synthesis of
philanthotoxins 4-20 were commercially available, but
most of these required NR protection and carboxy
activation. The Fmoc group was introduced by conven-
tional treatment25 with fluorenylmethoxycarbonyl chlo-
ride (Fmoc-Cl) in dioxane-10% aqueous Na2CO3 (Scheme
2) generally giving high yields (77-91%), except for
2-[(9H-fluoren-9-ylmethoxycarbonyl)amino]indan-2-car-
boxylic acid (13b), obtained in only 17% yield, probably
due to a steric hindrance of the amino group at the spiro
position. The resulting Fmoc-protected amino acids were
subsequently activated as Pfp esters by base-catalyzed
tandem transesterification with pentafluorophenyl tri-
fluoroacetate in pyridine-DMF.26
a Reagents: (i) CF3COOEt, MeOH, -50 °C; (ii) Boc2O; (iii)
NaOH, H2O.
pharmacological profiles.2-5,20 In contrast to these stud-
ies, only a few analogues representing systematic varia-
tions of the amino acid part of the molecule have been
synthesized and investigated. These comprise PhTX-343
analogues with the p-hydroxy group replaced by a
m-hydroxy group,5 as well as analogues in which ty-
rosine is replaced with phenylalanine or its halogenated,
alkoxylated, or azido-substituted analogues.7-11 The
overall trend in this series is that lack of the phenolic
functionality in PhTX-343 analogues may result in a
decrease or an increase of potency on iGluR depending
on the receptor system studied,9-11 while the antago-
nism of nACh was occasionally enhanced.8,11 Likewise,
replacement of tyrosine in PhTX-343 with nonpolar
aliphatic amino acids (e.g., glycine, alanine, or leucine)
gave rise to comparable potency at some ionotropic
receptors and to decreased potency at other recep-
tors.10,11 The analogues of PhTX-433 and PhTX-343 with
altered amino acid moieties have been tested mostly on
insect ionotropic receptors.8-10 Hence, there is a need
for a more comprehensive exploration of structure-
activity relationships for analogues of PhTX-343 altered
in the amino acid moiety on mammalian ion channels.
Synthesis of these analogues and determination of their
antagonist potencies on rat brain AMPAR is the subject
of the present work. As the three subunits of the PhTX-
343 molecule are interconnected by amide bonds, strate-
gies from peptide synthesis may be employed for se-
quential synthesis of analogues.5 A range of bulky and
conformationally restricted amino acids was investi-
gated as substitutes for tyrosine. Here, the chosen
amino acid protection-activation strategy is based on
the fluorenylmethoxycarbonyl (Fmoc) group and the
pentafluorophenyl (Pfp) ester group, both for synthesis
in solution22,23 and, for the first time, in solid-phase
philanthotoxin synthesis. tert-Butoxycarbonyl (Boc) group
was applied for protection of amino functionalities in
the polyamine. All synthesized analogues contain the
residue of spermine and a butyryl group as in PhTX-
343, to enable a more straightforward interpretation of
the biological results.
The active Pfp esters were chosen due to their ease
of formation, handling, stability, and appropriate reac-
tivity in amide formation with primary amino groups.
Hence, Pfp esters are still a useful and inexpensive
alternative to more recently developed and highly
efficient coupling agents such as PyBOP27 or HATU.28
Moreover, the Pfp ester couplings proceed without any
detectable racemization of the amino acid chiral center
when used in solution-phase as well as in solid-phase
couplings.22,29,30 In general the Pfp esters were obtained
in high yields (76-100%). Only the Pfp ester of NR-
Fmoc-protected 6-hydroxy-1,2,3,4,-tetrahydroisoquino-
line (5c) was isolated in a lower yield (58%), most likely
caused by the lack of phenol protection. The prepared
novel NR-Fmoc-protected amino acid Pfp esters were
characterized by 1H and 13C NMR, and by ion-cyclotron
resonance HRMS. The compounds containing a tertiary
amide group (5, 12, 15, and 17-20) exhibited two sets
1
of H and 13C signals originating from the presence of
rotamers of amide bonds (see Experimental Section).
For this reason, some of the spectra were recorded at
elevated temperatures.
The hydroxyproline-derived building block 19f was
synthesized as depicted in Scheme 3. In the first step,
the carboxy group in NR-benzyloxycarbonyl-4-hydroxy-
proline (19a) was esterified to give methyl ester 19b.31
Subsequently, a Mitsunobu reaction between the partial-
ly protected hydroxyproline derivative 19b and 2-naph-
thol using diethyl azodicarboxylate (DEAD) and Ph3P
in the presence of Et3N afforded the C-4 inverted com-
pound in 74% yield.32 Removal of the Z-group by hydro-
genolysis employing 10% Pd/C as catalyst gave 19d in
86% yield. Alkaline hydrolysis of 19d in MeCN-10%
aqueous Na2CO3 was followed by evaporation of MeCN,
and subsequently the Fmoc group was introduced by
addition of Fmoc-Cl in dioxane. This one-pot procedure
afforded 19e in 68% yield. Finally, the Pfp ester (19f)
was prepared in quantitative yield as described above.
Solution-Phase Synthesis of Philanthotoxins. In
the solution-phase synthetic route (Scheme 4), Fmoc-pro-
Results and Discussion
Synthesis of Building Blocks. The polyamine
building block, tri-Boc-protected spermine (3b), was
synthesized by a modification of a previously reported
procedure (Scheme 1).22,24
Thus, treatment of spermine with 1 equiv of ethyl
trifluoroacetate, followed by Boc protection and removal

58 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Scheme 2. Synthesis of Amino Acid Building Blocksa
Jørgensen et al.
a Reagents: (i) Fmoc-Cl, 10% Na2CO3, dioxane; (ii) CF3COOPfp, pyridine, DMF. Fmoc: fluorenylmethoxycarbonyl. Pfp: pentafluoro-
phenyl. Ph: phenyl. Bn: benzyl.
tected and Pfp ester-activated amino acids were
coupled to the free primary amino group of spermine
derivative 3b.
The resulting intermediates were isolated in high
yields (72-97%) after purification by VLC. Simplifica-
tion of the procedure by omitting the VLC purification
step was not advantageous, as it caused purification of
the subsequent Fmoc-deprotected intermediates to be
more tedious. Removal of the Fmoc group was per-
formed with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
in dry THF with 1-octanethiol as dibenzofulvene scav-
enger,33 giving intermediate NR-deprotected polyamine

Constraints and Steric Bulk in Philanthotoxins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 59
Scheme 5. Solid-Phase Synthesis of Philanthotoxinsa
Scheme 3. Synthesis of the Building Block 19fa
a Reagents: (i) MeI, Et3N, MeCN; (ii) 2-naphthol (1.5 equiv),
DEAD (1.5 equiv), Ph3P (1.5 equiv), dry THF; (iii) H2, 10% Pd/C,
EtOH; (iv) 10% aqueous Na2CO3, MeCN; then removal of MeCN
and addition of Fmoc-Cl in dioxane; (v) CF3COOPfp, pyridine,
DMF. Z: benzyloxycarbonyl.
a Reagents: (i) THF, 50 °C, 4 h, then 10% DIPEA-MeOH; (ii)
Fmoc-NR′(CHR)-OPfp (2.5 equiv), DIPEA (2.5 equiv), HODhbt (1
equiv), DMF, room temperature, overnight; (iii) 20% piperidine-
DMF, room temperature, 2 × 10 min; (iv) C3H7COOPfp (2.5 equiv),
DIPEA (2.5 equiv), HODhbt (1 equiv), DMF, room temperature, 3
h or overnight; (v) CH2Cl2-TFA-triisopropylsilane (78:20:2), room
temperature, 1 h.
Scheme 4. Solution-Phase Synthesis of
Philanthotoxinsa
amino acid moiety were removed by treatment with
trifluoroacetic acid (TFA). The resulting target com-
pounds were isolated by reversed-phase VLC as tris-
(TFA) salts in 85-100% yield.22 By this method (Scheme
4), philanthotoxins 4-8, 12-16, 18, and 20 were
synthesized. Most of the analogues were obtained in
high to excellent overall yield (70-90%). The only
exceptions were the D-4-hydroxyphenylglycine deriva-
tive 4 and the 6-hydroxy-1,2,3,4-tetrahydroisoquinoline
derivative 5, obtained in 45% and 39% overall yield,
respectively. This is probably due to the presence of a
free phenolic functionality in the amino acid side chain,
which may lead to intermolecular esterification giving
phenyl esters. The analogue 17, lacking the N-butyryl
group, was obtained in three steps by conversion of 1H-
indole-2-carboxylic acid (17a) to its Pfp ester 17b,
coupling with 3a, and Boc deprotection, with an overall
yield of 72%.
Solid-Phase Synthesis (SPS) of Philanthotoxins.
A conventional polystyrene-based 2-chlorotrityl chloride
resin was used as solid support. Although this type of
linker is quite acid-labile, its high stability toward bases
makes it ideal for reverse synthesis of N-terminal amino
peptides as well as for acylation of resin-bound amines
using the Fmoc strategy, the product being readily
cleaved from the resin with acid (e.g., TFA-CH2Cl2).
The coupling reactions were followed by using the
Kaiser test.34 The synthetic strategy is outlined in
Scheme 5.
a Reagents: (i) Et3N, CH2Cl2, room temperature, 3 h; (ii) DBU,
1-octanethiol, THF, room temperature, 2 h; (iii) C3H7COOPfp,
CH2Cl2, room temperature, 3 h; (iv) 20% TFA-CH2Cl2, room
temperature, 3 h.
amides in high yields (88-95%). In a second N-acylation
step, the butyryl group was attached using pentafluo-
rophenyl butyrate, to yield Boc-protected philanthotox-
ins in good to excellent yields (69-96%). Finally, the
Boc groups in the polyamine moiety and any other acid-
labile protecting groups present in the side chain of the
In the resin-loading step, di-Boc-protected spermine
(3a) was attached to the 2-chlorotrityl linker using a

60 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Jørgensen et al.
Table 1. IC50 Values (µM) and Yields of Philanthotoxins Tested in the AMPAR Assay (But ) C3H7CO-; Sp )
H2N(CH2)3NH(CH2)4NH(CH2)3NH-)
a Four-step synthetic route in solution. b Four-step solid-phase synthesis. c Two-step synthetic route in solution.
large excess of 3a in order to minimize cross-linking.
The excess 3a used in the reaction was recovered and
employed for loading of subsequent portions of the resin.
The free primary amino group of the resin-bound 3a was
acylated with Fmoc-protected amino acid Pfp esters
using 3-hydroxy-3,4-dihydro-1,2,3-benzotriazin-4-one
(HODhbt) as an additive to enhance coupling efficiency,
while diisopropylethylamine (DIPEA) was employed as
the tertiary amine.35 Removal of the Fmoc group was
performed with 20% piperidine in DMF as in peptide
SPS, followed by N-acylation with pentafluorophenyl
butyrate.22 The product was cleaved from the resin with
CH2Cl2-TFA-triisopropylsilane (78:20:2) with concomi-
tant N-Boc deprotection. The resulting philanthotoxins
9-11 and 19 were isolated as tris(TFA) salts by
reversed-phase VLC in 42-54% overall yield.
rat brain 1-3 days after they were removed from a toad3
expressed high levels of AMPAR 3-5 days later. A two-
electrode voltage clamp was used to measure the effect
of the philanthotoxins on currents evoked by application
of 10-4 M kainic acid to these oocytes. All measurements
were performed at a holding potential of -80 mV. The
oocytes were successively exposed to five concentrations
of each toxin, and the resulting reductions in the
amplitude of the kainate-induced current were used to
derive IC50 values. In Table 1 the IC50 values (µM) of
the novel philanthotoxins 4-6 and 8-20 synthesized
in this work are listed together with the values obtained
for PhTX-343 (2) and its phenylalanine analogue 7.
Two of the synthesized PhTX-343 analogues, 4 and
5, still contain a phenolic functionality as in the parent
compound. Shortening of the distance between the
amino acid R-carbon and the aromatic ring (compound
4) improved the potency by a factor of 2, whereas the
Pharmacology. For AMPAR assays, toad (Xenopus
laevis) oocytes injected with mRNA isolated from whole

Constraints and Steric Bulk in Philanthotoxins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 61
constrained analogue 5 was 2-fold less active. Com-
pound 4 has the opposite absolute configuration com-
pared to compound 2, whereas compound 5 is racemic
and has the hydroxy group shifted from para to meta
position with respect to the amino acid side chain. The
observed potencies for analogues 4 and 5 are in ac-
cordance with previous studies which showed that the
enantiomers of PhTX-343 (2) have similar potencies at
several types of ionotropic receptors.36 However, the
change to a meta-hydroxylated benzene ring only led
to a moderate decrease in activity, as compared to the
complete loss of antagonistic activity at iGluR, which
was seen for the m-hydroxy analogue of compound 2 in
the nAChR assay.5 Moreover, due to the minor differ-
ences observed between 2 and 5, other conformationally
constrained analogues of PhTX-343, including other
hydroxylated 1,2,3,4-tetrahydroisoquinoline derivatives,
may be of interest for future studies.
Removal of the hydroxy group in PhTX-343 (2) to give
the phenylalanine analogue 7 represents a known
modification,8-11 which resulted in a 3-fold decrease on
AMPAR (Table 1). Next, an array of nonphenolic
philanthotoxins (i.e. 6-20) representing variations of
the basic structure 7 was considered. These compounds
may be subdivided into three groups: (i) simple homo-
logues 6-8; (ii) compounds containing two benzene rings
9-11; and (iii) cyclic analogues 12-15 and 17-20. The
selection of amino acids in the latter series followed
their commercial availability.
of the amino acid nitrogen from the R-position to the
γ-position is tolerated, supporting the conclusion that
the R-nitrogen is not involved in the receptor binding.
Moreover, the present results demonstrate that cyclic
aliphatic substructures in the amino acid (compounds
5, 12-14, 18, 19, and in particular 20) are compatible
with AMPAR antagonism.
Conclusion
The present work has demonstrated the following: (i)
The Fmoc-Pfp methodology22 is equally applicable in the
solution-phase and the solid-phase synthesis of philan-
thotoxins containing a range of amino acids represent-
ing a high structural diversity. The protocol is compat-
ible with the presence of unprotected phenol groups,
although at a penalty of somewhat diminished yield. (ii)
Simple purification by reversed-phase VLC22 proved
extendable to all synthesized analogues. Reversed-phase
VLC is therefore an attractive, rapid, and broad-scope
alternative to reversed-phase HPLC4,5,20,21,36 for large-
scale purification of philanthotoxins. (iii) All 18 philan-
thotoxins (except 17) investigated (Table 1) proved to
have potencies roughly within 1 order of magnitude, in
spite of strongly dissimilar amino acid residues. Thus,
this relatively modest effect of the amino acid structure
on the antagonistic potency on AMPAR seems to suggest
that the headgroups of philanthotoxins occupy a non-
polar binding site with no strongly direction-dependent
binding interactions, resulting in a broad-scope tolera-
tion of the headgroup structure. (iv) The presence of
cyclized amino acid structures appears to be of minor
importance for the activity. The relative insensitivity
of the antagonism of philanthotoxins on AMPAR to
amino acid structure is similar to the results previously
obtained with a limited number of analogues tested on
insect iGluR sensitive to quisqualate (qGluR)10 and
contrasts the pronounced dependency on the structure
of the polyamine chain.2-5,20 (v) The vast majority of
philanthotoxins so far described in the literature contain
a benzene ring. However, the present results together
with an earlier report10 showing a leucine analogue to
be equipotent with PhTX-343 at qGluR and indicate
that philanthotoxins containing bulky aliphatic amino
acid side chains are attractive targets for future studies.
(vi) Finally, the present observation that hydrogen-bond
donor capacity of the amino acid R-nitrogen appears to
be unessential for the activity is an important guidance
for future SAR investigations.
Homologues 6 and 7 displayed similar activity with
IC50 values 0.32 µM and 0.43 µM, respectively, whereas
the phenethyl analogue 8 is 2-fold more potent (IC50
)
0.17 µM) and essentially equivalent to PhTX-343 (2).
Replacement of the benzene ring of 7 with an indole ring
(compound 16) resulted in a practically equipotent
derivative. For philanthotoxins containing two aromatic
rings, the linearly extended aromatic structures 9 and
10 gave rise to a higher potency than the branched
analogue 11, which is equipotent with phenylalanine
derivative 7. Introduction of steric constraints by cy-
clization involving the aromatic ring and either the
R-carbon (compounds 13 and 14) or the R-nitrogen (5,
12, and 15) of the amino acid moiety resulted in
potencies comparable to that of 7. In addition, the
absence of a phenolic functionality resulted in slightly
lowered activity while a more hydrophobic but still
relatively flexible aromatic amino acid side chain is
favorable.
Compounds 5, 12, 15, and 17-20 are to our knowl-
edge the first tertiary amide philanthotoxins prepared.
While the loss of activity of 17 is readily explainable by
the lack of the N-butanoyl group, the data for the
remaining compounds support that a secondary amide
(NHCO) is not required for antagonist activity at rat
brain AMPAR, since tertiary amides 5 and 12 have
activities equivalent to that of the secondary amide 7.
Thus, it can be concluded that the R-NHCO portion of
the amino acid moiety does not participate as a hydrogen-
bond donor upon interaction with AMPAR.
Of the two analogues (18 and 19) based on an
L-hydroxyproline scaffold but differing in stereochem-
istry at C-4, the benzyl derivative 18 was somewhat
more active than the naphthyl derivative 19. The
retained activity of derivative 20 shows that the shift
Experimental Section
General Procedures. Unless otherwise stated, starting
materials and solvents were purchased from commercial
suppliers (amino acid derivatives from Bachem or Novabio-
chem, reagents and solvents from Sigma-Aldrich, Fluka, or
Lancaster, resins for solid-phase synthesis from Novabiochem)
and used as received. CH2Cl2 was distilled from P2O5 and
stored over 4 Å molecular sieves. THF was distilled from Na/
benzophenone immediately before use. MeCN was dried over
3 Å molecular sieves. Dried DMF for solid-phase synthesis was
obtained from Fluka. 1H NMR and 13C NMR spectra were
recorded at 400.14 and 100.62 MHz, respectively, on a Bruker
AMX 400 spectrometer, or at 300.06 and 75.45 MHz, respec-
tively, on a Varian Mercury Plus spectrometer, using CDCl3
or CD3OD as solvents and tetramethylsilane (TMS) as internal
standard. Coupling constants (J values) are given in Hertz
(Hz), and multiplicities of 1H NMR signals are reported as
follows: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet,

62 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Jørgensen et al.
sx, sextet; m, multiplet; br, broad. Assignments are based on
2D NMR experiments (COSY, HMBC, and HSQC). High-
resolution mass spectrometry (HRMS) measurements were
performed on a Bruker APEX III Q Fourier transform mass
spectrometer equipped with a 7 T superconducting magnet and
an external electrospray ion source (Apollo source). The spectra
were calibrated with a capillary skimmer dissociation spec-
trum of LHRH (luteinizing hormone-releasing hormone). The
samples were introduced into the elctrospray ion source using
a 250 µL syringe with a syringe pump flow of 2 µL/min.
Vacuum liquid chromatography (VLC) was performed using
Merck silica gel 60H (particle size < 45 µm) or Merck
Lichroprep RP-18 (40-63 µm) for normal-phase and reversed-
phase VLC, respectively. Analytical RP-HPLC was performed
on a Shimadzu system consisting of an SCL-10A VP controller,
an SIL-10AD VP autosampler, an LC-10AT VP pump, an SPD-
M10A VP diode array detector, and a CTO-10AC VP column
oven, using a 150 × 4.6 mm i.d. Phenomenex Luna C18(2) 3µ
column. The HPLC system was controlled by Class VP 6
software; elution was performed with two different solvent
systems (total flow of 0.8 mL/min). System I: solvent A )
MeCN-H2O-TFA 10:90:0.1 and solvent B ) MeCN-H2O-
TFA 90:10:0.1; t ) 0-5 min 0% B, t ) 5-30 min 0-40% B, t
) 30-35 min 40-100% B). System II: solvent A ) MeOH-
H2O-TFA 10:90:0.1 and solvent B ) MeOH-H2O-TFA 90:
10:0.1; t ) 0-5 min 0% B, t ) 5-35 min 0-100% B. The
purities of target compounds 2 and 4-20 were determined
(from UV absorption intergration at λ ) 215 nm) in both
system I and II, showing purities within the ranges 97.0-
99.8% and 95.9-99.5%, respectively.
General Procedure A: Fmoc Protection of Amino
Acids. A solution of Fmoc-Cl (1 equiv) in dioxane (2.6 mL/
mmol) was added to a suspension of the amino acid in dioxane
(1.3 mL/mmol) and 10% aqueous Na2CO3 (2.6 mL/mmol) at 0
°C. The mixture was stirred for 1 h at 0 °C and then for 1 h at
room temperature. The reaction mixture was poured into
water and washed with Et2O. The aqueous phase was acidified
with concentrated aqueous HCl, and the precipitated product
was isolated by filtration and dried in vacuo.
General Procedure B: Fmoc Protection of Amino
Acids. General procedure B was the same as general proce-
dure A, except that upon acidification with concentrated HCl
the reaction mixture was extracted with EtOAc (3 times). The
combined EtOAc phases were dried (Na2SO4), filtered, and
concentrated in vacuo. The product was either used without
further purification or purified by VLC (hexanes-EtOAc 2:1
to 1:4 with 0.1% AcOH added).
General Procedure C: Pfp Ester Formation. The Fmoc-
protected amino acid was dissolved in dry DMF (2 mL/mmol).
Pyridine (1.1 equiv) and pentafluorophenyl trifluoroacetate (2.0
equiv) were added. The mixture was stirred for 1 h at room
temperature under N2, when it was diluted with EtOAc, and
then washed with 0.1 M aqueous HCl, saturated aqueous
NaHCO3, and water. The organic phase was dried (Na2SO4),
filtered, and concentrated. The product was either used
without further purification or purified by VLC (hexanes-
EtOAc 20:1 to 5:1).
General Procedure D: Coupling of Pfp Esters with
3b. The amino acid Pfp ester (1.0 equiv) and 3b (1.0 equiv)
were dissolved in dry CH2Cl2 (1 mL/100 mg of Pfp ester), Et3N
(1.0 equiv) was added, and the mixture was stirred under N2
for 3 h at room temperature. The reaction mixture was
concentrated, and the product was isolated by VLC (hexanes-
EtOAc 3:1 to 1:2).
General Procedure E: Fmoc Deprotection. The Fmoc-
protected compound was dissolved in dry THF (1 mL/100 mg),
and 1-octanethiol (10 equiv) and DBU (0.2 equiv) were added.
The mixture was stirred under N2 for 2 h at room temperature,
when it was concentrated in vacuo. The product was isolated by
VLC (CH2Cl2-MeOH-32% aqueous NH3 400:4:1 to 400:40:1).
General Procedure F: Introduction of N-Butyryl
Group. The partially protected spermine amino acid conjugate
and pentafluorophenyl butyrate (1.1 equiv) were dissolved in
dry CH2Cl2 (1 mL/100 mg of conjugate), and Et3N (1.0 equiv)
was then added. The mixture was stirred under N2 at room
temperature for 3 h. The reaction mixture was concentrated,
and the crude product was purified by VLC (hexanes-EtOAc
2:1 to 1:4).
General Procedure G: Boc Deprotection. The tri-Boc-
protected philanthotoxin was treated with 10% TFA (100
equiv) in dry CH2Cl2 for 3 h at room temperature. The reaction
mixture was concentrated in vacuo, and the resulting philan-
thotoxin was isolated by reversed-phase VLC (0.1% TFA in
H2O to MeCN-H2O-TFA 300:700:1).
General Procedure H: Solid-Phase Synthesis of Philan-
thotoxins. 2-Chlorotrityl resin preloaded with 3a36 was
swelled in dry DMF (12 mL/mmol of resin) for 30 min. The
solvent was removed by suction, and the resin was treated with
a Pfp-activated Fmoc-protected amino acid (2.0 equiv), HODh-
bt (1.0 equiv), and DIPEA (2.0 equiv) in dry DMF (12 mL/
mmol of resin). The mixture was left overnight under N2 at a
shaking table. The solvent was removed by suction, and the
resin was washed with DMF (3 × 12 mL/mmol of resin),
CH2Cl2 (3 × 12 mL/mmol of resin), and DMF (3 × 12 mL/mmol
of resin). The resin was treated with 20% piperidine in DMF
(12 mL/mmol of resin) for 2 × 10 min at room temperature
and was then washed with DMF (3 × 12 mL/mmol of resin).
The resulting resin was treated with pentafluorophenyl bu-
tyrate (2.5 equiv), HODhbt (1.0 equiv), and DIPEA (2.5 equiv)
in dry DMF (12 mL/mmol of resin) under N2 for 3 h at room
temperature. The solvent was removed by suction, and the
resin was washed successively with DMF (3 × 12 mL/mmol of
resin), CH2Cl2 (3 × 12 mL/mmol of resin), and MeOH (3 × 12
mL/mmol of resin) and then dried overnight in vacuo. The
dried resin was treated with CH2Cl2-TFA-triisopropylsilane
(78:20:2, 12 mL/mmol of resin) for 1 h at room temperature.
The filtrate was collected by suction, and the resin was treated
with 20% TFA in CH2Cl2 (12 mL/mmol of resin) and then with
CH2Cl2 (12 mL/mmol of resin). The combined filtrates were
concentrated, and the product was isolated by reversed-phase
VLC (0.1% TFA in H2O to MeCN-H2O-TFA 300:700:1).
4,9-Diaza-4,9-di-(tert-butoxycarbonyl)dodecane-1,12-
diamine (3a). Spermine (5.19 g, 0.026 mol) was dissolved in
MeOH (350 mL) under N2. Ethyl trifluoroacetate (7.0 mL,
0.059 mol) was added during 0.5 h at -50 °C. An additional
amount of MeOH (100 mL) was added to dissolve the precipi-
tate formed, and the mixture was stirred under N2 for an
additional 0.5 h at 0 °C. Di(tert-butyl) pyrocarbonate (16.78 g,
0.077 mol) in MeOH (30 mL) was added during 10 min, and
the mixture was stirred overnight at room temperature. The
reaction mixture was treated with 2 M aqueous NaOH (115
mL) for 4 h at room temperature and then overnight at 5 °C.
MeOH was removed in vacuo, and the residue was partitioned
between CH2Cl2 (350 mL) and water (200 mL). The CH2Cl2
phase was washed with brine (100 mL), whereas the aqueous
phase was extracted with CH2Cl2 (5 × 100 mL). The combined
organic phases were dried (Na2SO4) and filtered, and the
solvent was evaporated. Purification by VLC (CH2Cl2-MeOH
100:1 to CH2Cl2-MeOH-32% aqueous NH3 40:10:1) gave 3a
(9.02 g, 87%) as a yellow syrup. TLC: Rf 0.14 (CH2Cl2-
MeOH-32% aqueous NH3 50:10:1). 1H NMR and 13C NMR
spectra were as earlier reported.36
Loading of 2-Chlorotrityl Chloride Resin with 3a.
Compound 3a (4.25 g, 10.56 mmol) was dissolved in dry THF
(15 mL), and 2-chlorotrityl chloride resin (0.892 g, loading 1.14
mmol/g, 1.02 mmol) was added in 4 portions during 1 h at 50
°C. The mixture was stirred for 3 h at 50 °C and, upon cooling,
was transferred to a syringe fitted with a Teflon filter and a
Teflon stopcock. The solvent was removed by suction, and the
resin was treated with 10% DIPEA in MeOH (15 mL) for 10
min to trap remaining 2-chlorotrityl chloride groups. The resin
was washed with DMF (3 × 10 mL), CH2Cl2 (3 × 10 mL), and
MeOH (3 × 10 mL) and then dried in vacuo to give the resin
loaded with 3a (1.123 g, loading approximately 0.80 mmol/g
based on the increase in weight).
Pentafluorophenyl D-r-[(9H-fluoren-9-ylmethoxycar-
bonyl)amino]-4-hydroxybenzeneethanoate (4c). The Fmoc
group was introduced to D-(4-hydroxyphenyl)glycine (4a, 506

Constraints and Steric Bulk in Philanthotoxins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 63
mg, 3.027 mmol) according to general procedure B. The
resulting 4b (1.211 g, 79%) was used without further purifica-
tion. The carboxylic acid 4b (730 mg, 1.875 mmol) was
subjected to general procedure C, which gave the Pfp ester 4c
(786 mg, 76%) as a white foam. TLC: Rf 0.11 (hexanes-EtOAc
according to general procedure D to give the conjugate (90 mg,
89%) as a foam. TLC: Rf 0.32 (hexanes-EtOAc 1:2). Fmoc
deprotection of the conjugate (80 mg, 0.089 mmol) was
performed by general procedure E to give the amine interme-
diate (52 mg, 87%) as a syrup. TLC: Rf 0.21 (CH2Cl2-MeOH-
32% aqueous NH3 150:10:1). The N-butyryl group was intro-
duced to the amine intermediate (44 mg, 0.065 mmol) by using
general procedure F to give the Boc-protected 5 (25 mg, 51%)
as a syrup. Deprotection of the latter (25 mg, 0.033 mmol) was
performed according to general procedure G to give 5 (26 mg,
100%, overall yield: 39%) as a syrup. 1H NMR (300 MHz,
CD3OD): δ 7.09 (1H, d, J ) 8 Hz, H-b), 6.72-6.63 (2H, m, H-c
and H-e), 4.56 (1H, t, J ) 5.0 Hz, H-R), 4.65 (1H, d, J ) 14.6
Hz, HA-γ, major rotamer), 4.64 (1H, d, J ) 16.0 Hz, HA-γ, minor
rotamer), 4.57 (1H, d, J ) 14.6 Hz, HB-γ, major rotamer), 4.47
(1H, d, J ) 16.0 Hz, HB-γ, minor rotamer), 3.32-3.22 (1H, m,
HA-1), 3.20-3.00 (9H, m, H-â, HB-1, H-4, H-8, and H-10), 2.86
(2H, t, J ) 7.4 Hz, H-7), 2.78-2.18 (4H, m, H-2′ and H-3),
2.27-2.04 (2H, m, H-9), 1.88-1.62 (8H, m, H-2, H-5, H-6, and
H-3′), 1.03 (3H, t, J ) 7.4 Hz, H-4′, major rotamer), 0.99 (3H,
t, J ) 7.4 Hz, H-4′, minor rotamer). 13C NMR (75 MHz, CD3-
OD): δ 175.8 (C-1′), 174.8 (CdO, amino acid), 157.8 (C-d),
136.4 (C-f), 128.4 (C-b), 125.4 (C-a), 115.3, 114.8 (C-c and C-e),
56.2 (C-R), 48.3, 48.1 (C-4 and C-7), 46.9 (C-γ), 45.9 (C-8), 45.7
(C-3), 37.8 (2C, C-10 and C-2′), 36.4 (C-1), 33.2 (C-â), 27.5
(C-2), 25.5 (C-9), 24.3 (2C, C-5 and C-6), 19.3 (C-3′), 14.3 (C-
4′); only the signals corresponding to the major rotamer are
given. HRMS: calcd for C24H42N5O3 [M + H]+ 448.32822, found
448.32767.
Pentafluorophenyl L-r-[(9H-Fluoren-9-ylmethoxycar-
bonyl)amino]-r-benzeneethanoate (6b). Carboxylic acid 6a
(1.08 g, 2.89 mmol) was esterified by using general procedure
C to give 6b (1.38 g, 89%) as a white foam. TLC: Rf 0.60
(petroleum ether-EtOAc 2:1). [R] 2D5: +36.6° (c 0.56, CHCl3).
1H NMR (300 MHz, CDCl3): δ 7.73 (2H, d, J ) 7.7 Hz, H-y),
7.56 (2H, d, J ) 7.3 Hz, H-v), 7.45-7.24 (9H, m, Ph-H, H-w,
and H-x), 5.54 (1H, s, H-R), 4.50-4.40 (2H, m, Fmoc-CH2), 4.21
(1H, t, J ) 7.0 Hz, Fmoc-CH). 13C NMR (75 MHz, CDCl3):
168.3 (CdO, amino acid), 155.2 (CdO, Fmoc), 143.6, 143.4 (2C,
C-u), 141.2 (2C, C-z), 140.9 (2C, C-2′/C-6′, Pfp), 139.3 (C-4′,
Pfp), 137.7 (2C, C-3′/C-5′, Pfp), 134.4 (C-a), 129.4 (5C, Ph-C),
127.7 (2C, C-w or C-x), 127.5 (Ph-C), 127.1 (2C, C-w or C-x),
124.9 (2C, C-v), 124.8 (C-1′, Pfp), 120.0 (2C, C-y), 67.5 (Fmoc-
CH2), 58.2 (C-R), 47.2 (Fmoc-CH). Anal. Calcd for C29H18F5-
NO4: C, 64.57; H, 3.36; F, 17.61; N, 2.60. Found: C, 64.55; H,
3.61; F, 17.61; N, 2.51.
4:1). [R] 2D5
:
-49.2° (c 0.52, CHCl3). 1H NMR (300 MHz,
CDCl3): δ 7.74 (2H, d, J ) 7.3 Hz, H-y), 7.56 (2H, d, J ) 6.6
Hz, H-v), 7.38 (2H, t, J ) 7.3 Hz, H-x), 7.32-7.26 (4H, m, H-d
and H-w), 6.85 (2H, d, J ) 7.3 Hz, H-c), 5.63 (1H, s, H-R), 4.39-
4.50 (2H, m, Fmoc-CH2), 4.21 (1H, t, J ) 7.3 Hz, Fmoc-CH).
13C NMR (75 MHz, CDCl3): δ 167.5 (CdO, amino acid), 156.5
(2C, CdO Fmoc and C-d), 143.4 (C-u), 141.2 (2C, C-z), 140.5
(2C, C-2′/C-6′, Pfp), 139.0 (C-4′, Pfp), 137.5 (2C, C-3′/C-5′, Pfp),
129.0 (2C, C-b), 127.8, 127.1 (each 2C, C-w and C-x), 125.0
(2C, C-v), 124.9 (C-1′, Pfp), 120.0 (2C, C-y), 116.3 (2C, C-c),
67.9 (Fmoc-CH2), 57.9 (C-R), 47.3 (Fmoc-CH). HRMS: calcd
for C29H18F5NO5Na [M + Na]+ 578.09973, found 578.09926.
D-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
4-hydroxy-r-[(1-oxobutyl)amino]benzeneethanamide Tris-
(trifluoroacetate) (4). Compounds 4c (97 mg, 0.175 mmol)
and 3b (88 mg, 0.175 mmol) were coupled by using general
procedure D to give the conjugate as a foam (110 mg, 72%).
TLC: Rf 0.45 (hexanes-EtOAc 1:2). Fmoc deprotection of the
conjugate (422 mg, 0.483 mmol) was performed according to
general procedure E to give the amine intermediate (284 mg,
90%) as a syrup. TLC: Rf 0.2 (CH2Cl2-MeOH-32% aqueous
NH3 50:10:1). The N-butyryl group was introduced to the
amine intermediate (209 mg, 0.321 mmol) according to general
procedure F to give the protected analogue (160 mg, 69%) as
an oil. TLC: Rf 0.44 (hexanes-EtOAc 1:9). Deprotection of the
latter (110 mg, 0.152 mmol) was performed according to
general procedure G to give 4d (116 mg, 100%; overall yield:
45%) as a syrup. 1H NMR (300 MHz, CD3OD): δ 7.24 (2H, dt,
J ) 8.8 and 2.5 Hz, H-b), 6.77 (2H, dt, J ) 8.8 and 2.5 Hz,
H-c), 5.12 (1H, s, H-R), 3.40-3.20 (2H, m, H-1), 3.18-2.90
(10H, m, H-3, H-4, H-7, H-8, and H-10), 2.25 (2H, t, J ) 7.5
Hz, H-2′), 2.09 (2H, m, H-9), 1.91-1.80 (2H, p, J ) 6.8 Hz,
H-2), 1.83-1.73 (4H, m, H-5 and H-6), 1.63 (2H, sx, J ) 7.5
Hz, H-3′), 0.95 (3H, t, J ) 7.5 Hz, H-4′). 13C NMR (75 MHz,
CD3OD): δ 175.8 (C-1′), 174.3 (CdO, amino acid), 158.7
(C-d), 130.0 (C-b), 128.2 (C-a), 116.4 (C-c), 59.4 (C-R), 48.2, 48.1
(C-4 and C-7), 46.1 (C-3), 45.8 (C-8), 38.4 (C-2′), 37.8 (C-10),
36.7 (C-1), 27.6 (C-2), 25.4 (C-9), 24.3 (2C, C-5 and C-6), 20.3
(C-3′), 14.1 (C-4′). HRMS: calcd for C22H40N5O3 [M + H]+
422.31257, found: 422.31218.
Pentafluorophenyl DL-2-(9H-Fluoren-9-ylmethyl)-6-hy-
droxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (5c).
6-Hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (5a,
250 mg, 1.18 mmol) was Fmoc protected as described in
general procedure A to give 5b (394 mg, 80%). Compound 5b
(200 mg, 0.481 mmol) was esterified by using general proce-
dure C to give 5c (162 mg, 58%) as a white foam. 1H NMR
(300 MHz, CDCl3): δ 7.77 (2H, d, J ) 7.9 Hz, H-y), 7.65-7.42
(2H, m, H-v), 7.45-7.35 (2H, m, H-x), 7.34-7.24 (2H, m, H-w),
7.00 (1H, d, J ) 7.9 Hz, H-b), 6.74-6.69 (2H, m, H-c, H-e),
5.42 (1H, dd, J ) 3.8 and 5.5 Hz, H-R), 5.17 (1H, br s, OH),
4.74-4.42 (4H, m, Fmoc-CH2, H-γ), 4.34*, 4.28* (1H, t, J )
6.6 Hz, Fmoc-CH), 3.37-3.20 (2H, m, H-â). 13C NMR (75 MHz,
CDCl3): δ 167.7*, 167.6* (CdO, amino acid), 156.1*, 155.3*,
155.0*, 154.9* (2C, CdO Fmoc, C-d), 144.0*, 143.9*, 143.7*,
143.5* (2C, C-u), 141.5*, 141.4* (2C, C-z), 140.0 (2C, C-2′/C-
6′, Pfp), 139.0 (C-4′, Pfp), 137.5 (2C, C-3′/C-5′, Pfp), 132.7*,
132.5* (C-f), 128.0*, 127.9*, 127.8* (2C, C-x and C-b), 127.2
(2C, C-w), 125.2*, 125.1*, 125.1*, 125.0*, (2C, C-v), 124.9
(C-1′ Pfp), 124.4 (C-a), 120.2 (2C, C-y), 115.0, 114.8 (C-c, and
C-e), 68.6*, 68.4* (Fmoc-CH2), 54.0*, 53.6* (C-R), 47.4*, 47.3*
(Fmoc-CH), 44.4*, 44.3* (C-γ), 32.2*, 31.7* (C-â); (*) denotes
double signals originating from two amide rotamers present.
HRMS: calcd for C31H21F5NO5 [M + H]+ 582.13344, found
582.13318.
L-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
r-[(1-oxobutyl)amino]benzeneethanamide Tris(trifluo-
roacetate) (6). Pfp ester 6b (242 mg, 0.448 mmol) and
compound 3b (198 mg, 0.395 mmol) were dissolved in dry CH2-
Cl2 (2.0 mL), Et3N (55 µL, 0.40 mmol) was added, and the
mixture was stirred for 2 h at room temperature. The reaction
mixture was diluted with CH2Cl2 (20 mL) and washed with
water (2 × 15 mL). The organic phase was dried (Na2SO4) and
filtered, and the solvent was evaporated. The crude conjugate
(0.395 mmol) was dissolved in dry THF (3.5 mL), and DBU
(79 µL, 0.079 mmol) and 1-octanethiol (10 equiv) were added.
After 2 h at room temperature the reaction mixture was
concentrated, and the product was purified by VLC to give
quantitatively the Fmoc-deprotected product (240 mg, 0.377
mmol). TLC: Rf 0.16 (CH2Cl2-MeOH-32% aqueous NH3 200:
10:1). Coupling with pentafluorophenyl butyrate according to
general procedure F gave the Boc-protected intermediate (249
mg, 94%). Removal of the Boc groups from the latter (211 mg,
0.300 mmol) was performed as described in general procedure
1
G to give 6 (215 mg, 92%; overall yield: 86%). H NMR (400
MHz, CD3OD): δ 7.46-7.32 (5H, m, H-b, H-c and H-d), 5.26
(1H, s, H-R), 3.41-3.22 (2H, m, H-1), 3.18-2.91 (10H, m, H-3,
H-4, H-7, H-8, and H-10), 2.27 (2H, t, J ) 7.5 Hz, H-2′), 2.09
(2H, p, J ) 7.8 Hz, H-9), 1.87 (2H, p, J ) 6.7 Hz, H-2), 1.82-
1.74 (4H, m, H-5 and H-6), 1.64 (2H, sx, J ) 7.5 Hz, H-3′),
0.94 (3H, t, J ) 7.5 Hz, H-4′). 13C NMR (100 MHz, CD3OD):
δ 176.2 (C-1′), 174.2 (CdO, amino acid), 138.0 (C-a), 129.9 and
116.4 (2C, C-b and C-c), 129.6 (C-d), 59.8 (C-R), 48.2 and 48.1
DL-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]pro-
pyl]-6-hydroxy-2-(1-oxobutyl)-1,2,3,4-tetrahydroisoquin-
oline-3-carboxylamide Tris(trifluoroacetate) (5). Pfp ester
5c (65 mg, 0.11 mmol) and 3b (61 mg, 0.12 mmol) were coupled

64 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Jørgensen et al.
(C-4 and C-7), 46.1 and 45.8 (C-3 and C-8), 38.4 (C-2′), 37.8
(C-10), 36.8 (C-1), 27.5 (C-2), 25.4 (C-9), 24.2 (2C, C-5 and C-6),
20.2 (C-3′), 14.0 (C-4′). HRMS: calcd for C22H40N5O2 [M + H]+
406.31765, found 406.31724.
(C-R), 48.2 and 48.1 (C-4 and C-7), 46.2 (C-3), 45.8 (C-8), 38.6
(C-2′), 37.8 (C-10), 36.8 (C-1), 34.6 (C-â), 33.2 (C-γ), 27.5
(C-2), 25.4 (C-9), 24.3 (2C, C-5 and C-6), 20.4 (C-3′), 14.2
(C-4′). HRMS: calcd for C24H44N5O2 [M + H]+ 434.34895, found
434.34848.
L-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
4-r-[(1-oxobutyl)amino]benzenepropanamide Tris(tri-
fluoroacetate) (7). Compounds 7a (200 mg, 0.361 mmol) and
3b (181 mg, 0.361 mmol) were coupled by using general
procedure D to yield the conjugate (296 mg, 94%) as a foam.
TLC: Rf 0.22 (hexanes-EtOAc 1:1). Fmoc deprotection of the
conjugate (275 mg, 0.315 mmol) was performed according to
general procedure E to give the product (106 mg, 100%) as a
colorless syrup. TLC: Rf 0.10 (CH2Cl2-MeOH-32% aqueous
NH3 200:10:1). The N-butyryl group was introduced to the
latter (178 mg, 0.274 mmol) using general procedure F to give
the Boc-protected intermediate (191 mg, 97%) as a foam.
TLC: Rf 0.30 (hexanes-EtOAc 1:4). Deprotection of the latter
(161 mg, 0.226 mmol) was performed according to general
procedure G to give 7 (161 mg, 94%; overall yield: 86%) as a
syrup.1H NMR (400 MHz, CD3OD): δ 7.33-7.18 (5H, m, ArH),
4.47 (1H, dd, J ) 6.8 Hz and J ) 8.7 Hz, H-R), 3.33-2.87 (14H,
m, H-1, H-3, H-4, H-7, H-8, H-10, and H-â), 2.16 (2H, t, J )
7.4 Hz, H-2′), 2.14-2.03 (2H, m, H-9), 1.86-1.76 (6H, m, H-2,
H-5, and H-6), 1.53 (2H, sx, J ) 7.4 Hz, H-3′), 0.83 (3H, t, J )
7.4 Hz, H-4′). 13C NMR (100 MHz, CD3OD): δ 176.2 (C-1′),
175.0 (CdO, amino acid), 138.4 (C-a), 130.2 and 129.5 (each
2C, C-b and C-c), 127.9 (C-d), 56.7 (C-R), 48.4 and 48.1 (C-4
and C-7), 46.2 and 45.8 (C-3 and C-8), 38.6 (2C, C-2′ and C-â),
37.8 (C-10), 36.8 (C-1), 27.4 (C-2), 25.4 (C-9), 24.3 and 24.2
(C-5 and C-6), 20.2 (C-3′), 13.9 (C-4′). HRMS: calcd for
C23H42N5O2 [M + H]+ 420.33330, found 420.33290.
Pentafluorophenyl L-r-[(9H-Fluoren-9-ylmethoxycar-
bonyl)amino]-4-biphenylpropanoate (9b). Compound 9a
(500 mg, 1.079 mmol) was esterified using general procedure
C to give 9b (493 mg, 73%) as a white foam. TLC: Rf 0.29
(hexanes-EtOAc 4:1). [R] 2D5: -8.0° (c 0.64, CHCl3). H NMR
1
(300 MHz, CDCl3): δ 7.74 (2H, d, J ) 7.4 Hz, H-y), 7.57-7.50
(6H, m, H-v, H-b, and H-c′), 7.45-7.32 (5H, m, H-d′, H-w, and
H-x), 7.32-7.22 (4H, m, H-c and H-b′), 5.25 (1H, d, J ) 8.3
Hz, NH), 5.04 (1H, q, J ) 8.3 Hz, H-R), 4.52-4.36 (2H, m,
Fmoc-CH2), 4.21 (1H, t, J ) 6.9 Hz, Fmoc-CH), 3.36 (1H, dd,
J ) 14.0 and 8.3 Hz, HA-â), 3.27 (1H, dd, J ) 14.0 and 8.3 Hz,
HB-â). 13C NMR (75 MHz, CDCl3): δ 167.9 (CdO, amino acid),
155.4 (CdO, Fmoc), 143.6 and 143.5 (2C, C-u), 141.3 (2 C, C-z),
141.0 (2C, C-2′/C-6′, Pfp), 140.5 and 140.4 (C-d, and C-a′), 139.5
(C-4′, Pfp), 137.8 (2C, C-3′/C-5′, Pfp), 133.5 (C-a), 129.7†, 128.8†,
127.6†, 127.4, 127.1†, 127.0†, 125.1, 125.0 (13C, C-b, C-c, C-b,
C-c′, C-d′, C-v, C-w, and C-x), 124.9 (C-1′, Pfp), 120.0 (2C, C-y),
67.4 (Fmoc-CH2), 54.7 (C-R), 47.3 (Fmoc-CH), 37.7 (C-â); (†)
denotes two-carbon signals. HRMS: calcd for C36H24F5NO4-
Na [M + Na]+ 652.15177, found 652.15176.
L-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
r-[(1-oxobutyl)amino]-4-biphenylpropenamide Tris(tri-
fluoroacetate) (9). Using general procedure H, resin loaded
with 3a (250 mg, loading approximately0.80 mmol/g, 0.20
mmol) was elongated successively with 9b and pentafluo-
rophenyl butyrate to give, after deprotection and cleavage,
1
philanthotoxin 9 (91 mg, 54%). H NMR (300 MHz, CD3OD):
Pentafluorophenyl L-r-[(9H-Fluoren-9-ylmethoxycar-
bonyl)amino]benzenebutanoate (8b). Carboxylic acid 8a
(500 mg, 1.25 mmol) was esterified by using general procedure
C to give 8b (679 mg, 96%) as a white foam. TLC: Rf 0.50
δ 7.50-7.48 (4H, m, H-b′ and H-c), 7.39-7.33 (2H, ddt, J )
7.7, 7.1, and 1.1 Hz, H-c′), 7.29-7.20 (3H, m, H-b and H-d′),
4.45 (1H, dd, J ) 8.5 and 6.9 Hz, H-R), 3.30-2.80 (14H, m,
H-1, H-8, H-â, H-10, H-3, H-4, and H-7), 2.14 (2H, t, 7.4 Hz,
H-2′), 2.14-1.98 (2H, m, H-9), 1.84-1.68 (6H, m, H-2, H-5,
and H-6), 1.51 (2H, sx, J ) 7.4 Hz, H-3′), 0.80 (3H, t, J ) 7.4
Hz, H-4′). 13C NMR (75 MHz, CD3OD): δ 175.9 (C-1′), 174.6
(CdO, amino acid), 141.7 and 140.8 (C-d and C-a′), 137.2
(C-a), 130.5 (2C, C-b), 129.7 (2C, C-c′), 128.1 (C-d′), 127.8, 127.6
(C-c and C-b′), 56.7 (C-R), 48.2 (C-4), 48.1 (C-7), 46.2 (C-3),
45.9 (C-8), 38.6 (C-2′), 38.3 (C-â), 37.8 (C-10), 36.8 (C-1), 27.5
(C-2), 25.5 (C-9), 24.4 and 24.3 (C-5 and C-6), 20.3 (C-3′), 14.0
(C-4′). HRMS: calcd for C29H46N5O2 [M + H]+ 496.36460, found
496.36401.
(hexanes - EtOAc 7:1). [R] 2D5: +2.4° (c 3.2, CHCl3). H NMR
1
(300 MHz, CDCl3): δ 7.74 (2H, d, J ) 8.5 Hz, H-y), 7.56 (2H,
d, J ) 7.5 Hz, H-v), 7.42-7.14 (9H, m, Ph-H, H-w, and H-x),
5.28 (1H, d, J ) 8.8 Hz, NH), 4.75 (1H, m, H-R), 4.46 (2H, d,
J ) 7.0 Hz, Fmoc-CH2), 4.22 (1H, t, J ) 7.0 Hz, Fmoc-CH),
2.76 (2H, t, J ) 7.9 Hz, H-γ), 2.42-2.25, 2.25-2.10 (each 1H,
m, H-â). 13C NMR (75 MHz, CDCl3): δ 168.7 (CdO, amino
acid), 155.6 (CdO, Fmoc), 143.6 and 143.5 (2C, C-u), 141.3 (2C,
C-z), 140.7 (2C, C-2′/C-6′, Pfp), 139.8 (C-a), 139.5 (C-4′, Pfp),
137.8 (2C, C-3′/C-5′, Pfp), 128.7, 128.4 (4C, C-b and C-c), 128.3
(C-1′, Pfp), 127.7 (2C, C-x), 127.1 (2C, C-w), 126.5 (C-d), 125.0
(2C, C-v), 120.0 (2C, C-y), 67.3 (Fmoc-CH2), 53.7 (C-R), 47.3
(Fmoc-CH), 34.3 (C-â), 31.7 (C-γ). Anal. Calcd for C31H22F5-
NO4: C, 65.61; H, 3.91; F, 16.71; N, 2.47, found: C, 65.24; H,
4.04; F, 17.69; N, 2.39.
Pentaflouorophenyl D-r-[(9H-Fluoren-9-ylmethoxy-
carbonyl)amino]-2-naphthalenepropanoate (10b). Com-
pound 10a (500 mg, 1.14 mmol) was esterified using general
procedure C give 10b (686 mg, 100%) as a white solid. TLC:
Rf 0.47 (hexanes-EtOAc 3:1). [R] 25: +10.3° (c 1.2, CHCl3).
L-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
r-[(1-oxobutyl)amino]benzenebutanamide Tris(trifluo-
roacetate) (8). Pfp ester 8b (400 mg, 0.705 mmol) and
compound 3b (354 mg, 0.705 mmol) were coupled by using
general procedure D to yield the conjugate (83 mg, 93%).
TLC: Rf 0.22 (hexanes-EtOAc 1:1). Fmoc deprotection of the
conjugate (403 mg, 0.455 mmol) was performed by using
general procedure E to give the amine intermediate (287 mg,
95%) as a syrup. TLC: Rf 0.18 (CH2Cl2-MeOH-32% aqueous
NH3 200:10:1). The N-butyryl group was introduced to the
amine intermediate (219 mg, 0.330 mmol) using general
procedure F to give the Boc-protected intermediate (222 mg,
92%). TLC: Rf 0.08 (hexanes - EtOAc 1:2). Deprotection of
the latter (183 mg, 0.249 mmol) was performed according to
general procedure G to give 8 (191 mg, 99%; overall yield: 80%)
as a syrup. 1H NMR (300 MHz, CD3OD): δ 7.28-7.12 (5H, m,
Ph-H), 4.14 (1H, dd, J ) 9.1 and 5.2 Hz, H-R), 3.40-3.20 (2H,
m, H-1), 3.17-2.94 (10H, m, H-3, H-4, H-7, H-8, and H-10),
2.80-2.60 (2H, m, H-γ), 2.35-2.19 (2H, m, H-2′), 2.14-2.02
(2H, m, H-9), 2.10-1.90 (2H, m, H-â), 1.87 (2H, p, J ) 6.8 Hz,
H-2), 1.84-1.74 (4H, m, H-5 and H-6), 1.66 (2H, sx, J ) 7.5
Hz, H-3′), 0.98 (3H, t, J ) 7.5 Hz, H-4′). 13C NMR (75 MHz,
CD3OD): δ 176.2 (C-1′), 175.5 (CdO, amino acid), 141.8
(C-a), 129.3 and 129.2 (each 2C, C-b and C-c), 126.9 (C-d), 55.1
D
1H NMR (400 MHz, CDCl3): δ 7.84-7.70 (6H, m, H-y, H-c,
H-e, H-h, and H-j), 7.53-7.48 (4H, m, H-v, H-f, and H-g), 7.40-
7.24 (5H, m, H-b, H-w, and H-x), 5.24 (1H, d, J ) 8.4 Hz, NH),
5.14 (1H, m, H-R), 4.52-4.36 (2H, m, Fmoc-CH2), 4.21 (1H, t,
J ) 6.9 Hz, Fmoc-CH), 3.50 (1H, dd, J ) 14.1 and 5.8 Hz, HA-
â), 3.44 (1H, dd, J ) 14.1 and 6.5 Hz, HB-â). 13C NMR (100
MHz, CDCl3): δ 168.2 (CdO, amino acid), 155.7 (CdO, Fmoc),
143.8 and 143.7 (C-u), 141.5 (2C, C-z), 141.0 (2C, C-2′/C-6′,
Pfp), 139.5 (C-4′, Pfp), 137.8 (2C, C-3′/C-5′, Pfp), 133.6, 132.8,
132.2 (C-a, C-d, and C-i), 128.9, 128.4, 128.0†, 127.9† (6C, C-c,
C-e, C-h, C-j, and C-x,), 127.2 (C-w), 127.1 (C-b), 126.6 and
126.3 (C-f and C-g), 125.1 (2C, C-v), 124.8 (C-1′, Pfp), 120.1
(2C, C-y), 67.5 (Fmoc-CH2), 54.8 (C-R), 47.2 (Fmoc-CH), 38.2
(C-â); (†) denotes two-carbon signals. HRMS: calcd for C34H22F5-
NO4Na [M + Na]+ 626.13612, found 626.13627.
D-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
r-[(1-oxobutyl)amino]-2-naphthalenepropanamide Tris-
(trifluoroacetate) (10). Using general procedure H, resin
loaded with 3a (250 mg, loading approximately 0.80 mmol/g,
0.20 mmol) was elongated successively with 10b and pen-
tafluorophenyl butyrate to give, after deprotection and cleav-
age, philanthotoxin 10 (71 mg, 44%) as a syrup. 1H NMR (300
MHz, CD3OD): δ 7.84-7.75 (3H, m, H-c, H-e, and H-h), 7.70

Constraints and Steric Bulk in Philanthotoxins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 65
(1H, s, H-j), 7.49-7.36 (3H, m, H-b, H-f, and H-g), 4.59 (1H,
dd, J ) 8.4 and 7.0 Hz, H-R), 3.33-3.01 (10H, m, H-1, H-3,
H-8, H-10, and H-â), 2.90-2.75 (4H, m, H-4 and H-7), 2.17
(2H, t, J ) 7.4 Hz, H-2′), 2.09 (2H, m, H-9), 1.84-1.69 (6H, m,
H-2, H-5, and H-6), 1.51 (2H, sx, J ) 7.4 Hz, H-3′), 0.80 (3H,
t, J ) 7.4 Hz, H-4′). 13C NMR (75 MHz, CD3OD): δ 176.2
(C-1′), 174.8 (CdO, amino acid), 135.9, 134.9, 133.9 (C-a, C-d,
and C-i), 129.2, 128.9, 128.7 and 128.6 (C-c, C-e, C-h, and C-j),
128.4 (C-b), 127.2 and 126.8 (C-f and C-g), 56.9 (C-R), 48.5 and
48.3 (C-4 and C-7), 46.4 (C-3), 46.1 (C-8), 39.1 (C-2′), 38.9
(C-10), 38.1 (C-1), 37.1 (C-â), 27.7 (C-2), 25.7 (C-9), 24.6 (2C,
C-5 and C-6), 20.6 (C-3′), 14.2 (C-4′). HRMS: calcd for
C27H44N5O2 [M + H]+ 470.34895, found 470.34851.
Pentafluorophenyl DL-2-[(9H-Fluoren-9-ylmethoxycar-
bonyl)amino]-3,3-diphenylpropanoate (11c). The Fmoc
group was attached to DL-â,â-diphenylalanine (15a, 500 mg,
2.07 mmol) using general procedure B, which afforded 11b (937
mg, 98%) as a foam. TLC: Rf 0.28 (hexanes-EtOAc-AcOH
20:10:1). The latter compound (779 mg, 1.68 mmol) was
esterified using general procedure C to give 11c (855 mg, 81%).
TLC: Rf 0.18 (hexanes-EtOAc 7:1). 1H NMR (300 MHz,
CDCl3): δ 7.68 (2H, d, J ) 7.2 Hz, H-y), 7.41 (2H, d, J ) 7.2
Hz, H-v), 7.35-7.15 (14H, m, H-w, H-x, and 2 × Ph), 5.47 (1H,
t, J ) 8.5 Hz, NH), 5.10 (1H, d, J ) 8.5 Hz, H-R), 4.53 (1H, d,
J ) 8.5 Hz, H-â), 4.52-4.36 (2H, m, Fmoc-CH2), 4.21 (1H, t, J
) 6.9 Hz, Fmoc-CH). 13C NMR (100 MHz, CDCl3): δ 168.0
(CdO, amino acid), 155.7 (CdO, Fmoc), 143.7 (2C, C-u), 141.5
(2C, C-z), 141.0 (2C, C-2′/C-6′, Pfp), 139.5 (C-4′, Pfp), 139.3
and 138.8 (2C, C-a), 137.5 (2C, C-3′/C-5′, Pfp), 129.3 and 129.1
(each 4C, C-b and C-c), 128.6 and 128.2 (2C, C-x), 127.9 and
127.8 (2C, C-d), 127.2 (2C, C-w), 125.2 (2C, C-v), 125.1 (C-1′
Pfp), 120.1 (2C, C-y), 67.7 (Fmoc-CH2), 57.2 (C-R), 53.2 (C-â),
47.1 (Fmoc-CH). HRMS: calcd for C36H25F5NO4Na [M + Na]+
652.15177, found 652.15103.
DL-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]pro-
pyl]-3,3-diphenyl-2-[(1-oxobutyl)amino]propanamide Tris-
(trifluoroacetate) (11). Using general procedure H, resin
loaded with 3a (250 mg, loading approximately 0.80 mmol/g,
0.20 mmol) was elongated successively with 11c and with
pentafluorophenyl butyrate to give, after deprotection and
cleavage, philanthotoxin 11 (71 mg, 42%) as a syrup. 1H NMR
(300 MHz, CD3OD): δ 7.39-7.12 (10H, m, Ph-H), 5.24 (1H,
d, J ) 12.0 Hz, H-R), 4.37 (1H, d, J ) 12.0 Hz, H-â), 3.20-
2.96 (8H, m, H-1, H-7, H-8, and H-10), 2.87-2.73 (2H, m, H-4),
2.90-2.75 (1H, m, HA-3), 2.37-2.25 (1H, m, HB-3), 2.10 (2H,
p, J ) 7.7 Hz, H-9), 2.01 (2H, t, J ) 7.3 Hz, H-2′), 1.87-1.67
(4H, m, H-5 and H-6), 1.67-1.52 (2H, m, H-2), 1.45-132 (2H,
sx, J ) 7.3 Hz, H-3′), 0.70 (3H, t, J ) 7.3 Hz, H-4′). 13C NMR
(75 MHz, CD3OD): δ 175.3 (C-1′), 173.6 (CdO, amino acid),
142.3 and 141.7 (2C, C-a), 129.5, 129.3 and 129.0 (8C, C-b and
C-c), 127.9 (2C, C-d), 57.6 (C-R), 54.5 (C-â), 48.2 and 47.1 (C-7
and C-4), 45.8 (2C, C-3 and C-8), 39.6 (C-2′), 37.8 (C-10), 36.5
(C-1), 27.2 (C-2), 25.4 (C-9), 24.3 (2 C, C-5 and C-6), 20.3
(C-3′), 13.8 (C-4′). HRMS: calcd for C29H46N5O2 [M + H]+
496.36460, found 496.36428.
65.84; H, 3.56; F, 16.80; N, 2.51. Found: C, 65.91; H, 3.44; F,
16.93; N, 2.39.
L-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
2-(1-oxobutyl)isoquinoline-3-carboxylamide Tris(trifluo-
roacetate) (12). Pfp ester 12b (346 mg, 0.435 mmol) and
compound 3b (199 mg, 0.386 mmol) were dissolved in dry CH2-
Cl2 (2.0 mL), Et3N (55 µL, 0.40 mmol) was added, and the
mixture was stirred for 2 h at room temperature, when it was
diluted with CH2Cl2 (20 mL) and washed with water (2 × 15
mL). The organic phase was dried (Na2SO4), filtered, and
concentrated. The crude product (0.398 mmol) was dissolved
in dry THF (3.5 mL), DBU (79 µL, 0.079 mmol) and 1-oc-
tanethiol (10 equiv) were added, and then the mixture was
stirred for 2 h at room temperature. The reaction mixture was
concentrated, and the Fmoc-deprotected compound was iso-
lated in quantitative yield by VLC. TLC: Rf 0.20 (CH2Cl2-
MeOH-32% aqueous NH3 200:10:1). The N-butyryl group was
attached to the latter product (255 mg, 0.386 mmol) using
general procedure F to yield the Boc-protected intermediate
(267 mg, 95%). Deprotection of the latter (221 mg, 0.302 mmol)
was performed according to general procedure G to give 12
1
(221 mg, 95%; overall yield: 90%) as a syrup. H NMR (400
MHz, CD3OD, T ) 328 K): δ 7.29-7.18 (4H, m, 4 Ar-H), 4.56
(1H, t, J ) 5.5 Hz, H-R), 4.72 (2H, br s, H-γ), 3.30-3.13 (6H,
m, H-1, H-3, and H-8), 3.09 (4H, t, J ) 7.5 Hz, H-4 and H-7),
2.88 (2H, t, J ) 7.5 Hz, H-10), 2.75 (2H, m, H-â), 2.78-2.18
(2H, t, J ) 7.4 Hz, H-2′), 2.27-2.04 (2H, p, J ) 7.5 Hz, H-9),
1.88-1.63 (8H, m, H-2, H-5, H-6, and H-3′), 1.06-0.95 (3H, t,
J ) 7.4 Hz, H-4′). 13C NMR (100 MHz, CD3OD): δ 176.1
(C-1′), 175.4 (CdO, amino acid), 135.2-127.4 (6C, Ph-C, one
major and several minor rotamers), 56.4 (C-R), 48.3 and 48.2
(C-4 and C-7), 47.4 (C-γ), 45.9 and 45.8 (C-3 and C-8), 37.8
(C-10), 36.7 (C-2′), 36.4 (C-1), 32.9 (C-â), 27.5 (C-2), 25.4
(C-9), 24.3 and 24.2 (C-5 and C-6), 19.3 (C-3′), 14.2 (C-4′); only
signals for the major rotamer have been assigned. HRMS:
calcd for C23H42N5O2 [M + H]+ 432.33330, found 432.33308.
Pentafluorophenyl 2-[(9H-Fluoren-9-ylmethoxycar-
bonyl)amino]indan-2-carboxylate (13c). The Fmoc group
was introduced to 13a (hydrochloride; 255 mg, 1.19 mmol)
using general procedure B to give 13b (82 mg, 17%). TLC: Rf
0.09 (hexanes-EtOAc-AcOH 20:10:1). The carboxylic acid 13b
(131 mg, 0.33 mmol) was esterified using general procedure
C to give 13c (147 mg, 79%). TLC: Rf 0.64 (hexanes-EtOAc
2:1). 1H NMR (300 MHz, CDCl3): δ 7.74 (2H, br d, J ) 7.4 Hz,
H-y), 7.56 (2H, br d, J ) 7.4 Hz, H-v), 7.38 (2H, br t, J ) 7.4
Hz, H-x), 7.29 (2H, br t, J ) 7.4 Hz, H-w), 7.26-7.24 (4H, m,
H-b and H-c), 5.49 (1H, s, NH), 4.60 (2H, d, J ) 6.3 Hz, Fmoc-
CH2) 4.21 (1H, t, J ) 6.3 Hz, Fmoc-CH), 3.85 (2H, d, J ) 16.5
Hz, HA-â), 3.40 (2H, d, J ) 16.5 Hz, HB-â). 13C NMR (75 MHz,
CDCl3): δ 169.1 (CdO, amino acid), 155.4 (CdO, Fmoc), 143.5
(2C, C-u), 141.2 (2C, C-z), 140.7 (2C, C-2′/C-6′, Pfp), 139.5
(C-4′, Pfp), 138.7 (2C, C-a), 137.8 (2C, C-3′/C-5′, Pfp), 127.7
and 127.0 (2C, C-x and C-w), 127.4 (2C, C-b), 124.9 (2C, C-v),
124.7 (2C, C-c), 124.5 (C-1′, Pfp), 120.0 (2C, C-y), 67.4 (Fmoc-
CH2), 66.3 (C-R), 47.3 (Fmoc-CH), 44.1 (2C, C-â). HRMS: calcd
for C31H20F5NO4Na [M + Na]+ 588.12047, found 588.12006.
Pentafluorophenyl L-2-(9H-Fluoren-9-ylmethoxycar-
bonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (12).
Compound 6a (507 mg, 1.27 mmol) was esterified using
general procedure C to give 6b (544 mg, 76%) as a white foam.
TLC: Rf 0.56 (hexanes-EtOAc 2:1). [R] 25: 0° (c 0.68, CHCl3).
1H NMR (400 MHz, CDCl3): δ 7.77*, 7.7D6 (each 2H, d, J ) 7.5
Hz, H-y), 7.62, 7.57* (each 2H, t, J ) 7.8 Hz, H-v), 7.44-7.37
(2H, m, H-x), 7.34-7.12 (6H, m, H-w, H-b, H-c, H-d, and H-e),
5.42 (1H, dd, J ) 5.8 and 3.6 Hz, H-R), 5.22* (1H, t, J ) 4.9
Hz, H-R), 4.82-4.45 (4H, m, Fmoc-CH2, H-γ), 4.35, 4.28* (each
1H, t, J ) 6.8 Hz, Fmoc-CH), 3.43-3.29 (2H, m, H-â). 13C NMR
(100 MHz, CDCl3): δ 168.3 (CdO, amino acid), 143.8 (2C, C-u),
141.5 (2C, C-z), 133.0, 132.4, 131.2, 128.6, 128.1, 127.9, 127.7,
127.5, 127.3, 126.7, 126.5, 125.1 and 120.2 (C-v, C-w, C-x, C-y,
C-b. C-c, C-d, C-e, mixture of rotamers), 68.5* and 68.3 (Fmoc-
CH2), 54.1* and 53.7 (C-R), 47.3 (Fmoc-CH), 44.8* and 44.7
(C-γ), 32.5* and 31.7 (C-â); (*) denotes signals arising from a
minor amide bond rotamer. Anal. Calcd for C31H20F5NO4: C,
DL-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]pro-
pyl]-2-[(1-oxobutyl)amino]indan-2-carboxylamide Tris-
(trifluoroacetate) (13). Pfp ester 13c (100 mg, 0.177 mmol)
and compound 3b (89 mg, 0.177 mmol) were coupled using
general procedure D to yield the conjugate (159 mg, 100%) as
a foam. TLC: Rf 0.13 (hexanes-EtOAc 1:1). Fmoc deprotection
of the conjugate (127 mg, 0.141 mmol) was performed using
general procedure E to give the amine intermediate (92 mg,
96%) as an oil. TLC: Rf 0.21 (CH2Cl2-MeOH-32% aqueous
NH3 200:10:1). The N-butyryl group was introduced to the
latter (76 mg, 0.112 mmol) according to general procedure F
to give the Boc-protected product (62 mg, 44%) as an oil. TLC:
Rf 0.25 (hexanes-EtOAc 1:4). Deprotection of the latter (36
mg, 0.048 mmol) as described in general procedure G yielded
13 (40 mg, 100%; overall yield: 71%) as a syrup. 1H NMR (300
MHz, CDCl3): δ 7.74 (2H, d, J ) 7.3 Hz, H-y), 7.55 (2H, d, J
) 7.3 Hz, H-v), 7.38 (2H, t, J ) 7.3 Hz, H-x), 7.29 (2H, t, J )
7.3 Hz, H-w), 7.24-7.18 (4H, m, H-b and H-c), 5.36 (1H, s,

66 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Jørgensen et al.
NH), 4.60 (2H, d, J ) 6.0 Hz, Fmoc-CH2), 4.20 (1H, t, J ) 6.0
Hz, Fmoc-CH), 3.85 (2H, d, J ) 16.3 Hz, HA-â), 3.40 (2H, d, J
) 16.3 Hz, HB-â). 13C NMR (75 MHz, CDCl3): δ 177.3 (C-1′),
176.7 (CdO, amino acid), 142.2 (2C, C-a), 128.0 (2C, C-b), 125.6
(2C, C-c), 68.1 (C-R), 48.3 and 48.2 (C-4 and C-7), 46.0 and
45.8 (C-3 and C-8), 44.2 (2C, C-â), 38.6 (C-2′), 37.8 (C-10), 36.8
(C-1), 27.8 (C-2), 25.4 (C-9), 24.3 and 24.2 (C-5 and C-6), 20.1
(C-3′), 14.0 (C-4′). HRMS: calcd for C24H42N5O2 [M + H]+
432.33330, found 432.33318.
Pentafluorophenyl DL-2-[(9H-Fluoren-9-ylmethoxycar-
bonyl)amino]-1,2,3,4-tetrahydronaphthalene-2-carboxy-
late (14b). Compound 14a (150 mg, 0.363 mmol) was esterified
using general procedure C to give 14b (202 mg, 96%) as a white
foam. TLC: Rf 0.38 (hexanes-EtOAc 7:1). 1H NMR (300 MHz,
CDCl3): δ 7.72 (2H, br d, J ) 7.5 Hz, H-y), 7.52 (2H, br d, J )
7.5 Hz, H-v), 7.35 (2H, br t, J ) 7.5 Hz, H-x), 7.24 (2H, br t, J
) 7.5 Hz, H-w), 7.20-7.06 (4H, br m, side chain Ar-H), 5.49
(1H, s, NH), 4.45 (2H, br d, J ) 6.5 Hz, Fmoc-CH2), 4.21 (1H,
t, J ) 6.5 Hz, Fmoc-CH), 3.59 (1H, br d, J ) 17.0 Hz, HA-â′),
3.17 (1H, br d, J ) 17.0 Hz, HB-â′), 3.00-2.75 (2H, m, H-γ),
2.75-2.59 (1H, m, HA-â), 2.36-2.20 (1H, m, HB-â). 13C NMR
(75 MHz, CDCl3): δ 169.5 (CdO, amino acid), 155.2 (CdO,
Fmoc), 143.5 (2C, C-u), 141.3 (2C, C-z), 140.9 (2C, C-2′/C-6′,
Pfp), 139.5 (C-4′, Pfp), 138.0 (2C, C-3′/C-5′, Pfp), 134.4 and
130.9 (C-a and C-f), 129.4 and 128.9 (C-b and C-e), 127.7 (2C,
C-x), 127.0 (2C, C-w), 126.9 and 126.4 (4C, C-c and C-d), 124.9
(2C, C-v), 124.8 (C-1′, Pfp), 120.0 (2C, C-y), 67.1 (Fmoc-CH2),
58.7 (C-R), 47.3 (Fmoc-CH), 38.1 (C-â′), 28.6 (C-â), 25.1 (C-γ).
HRMS: calcd for C32H22F5NO4Na [M + Na]+ 602.13612, found
602.13541.
DL-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]pro-
pyl]-2-[(1-oxobutyl)amino]-1,2,3,4-tetrahydronaphthalene-
2-carboxylamide Tris(trifluoroacetate) (14). Pfp ester 14b
(175 mg, 0.302 mmol) and compound 3b (153 mg, 0.302 mmol)
were coupled using general procedure D to yield the conjugate
(243 mg, 90%) as a foam. TLC: Rf 0.17 (hexanes-EtOAc 1:1).
Fmoc deprotection of the conjugate (206 mg, 0.229 mmol) was
performed as described in general procedure E to give the
amine intermediate (150 mg, 97%) as an oil. TLC: Rf 0.70
(CH2Cl2-MeOH-32% aqueous NH3 200:10:1). The N-butyryl
group was introduced to the amine intermediate (109 mg,
0.161 mmol) according to general procedure F but with the
reaction time prolonged to 7 h. An additional amount of
pentafluorophenyl butyrate (45 mg, 0.177 mmol) in dry CH2-
Cl2 (0.5 mL) was added, and the mixture was stirred overnight.
Dry THF (2 mL) was added, and the mixture was stirred
overnight at 40 °C. DBU (5 µL, 0.032 mmol) was added, and
then the mixture was stirred at 40 °C for 5 days, when the
solvents were removed in vacuo. Purification of the residue
by VLC gave unreacted starting material (18 mg, 16%) and
the Boc-protected product (98 mg, 82%). TLC: Rf 0.21 (hex-
anes-EtOAc 1:4). Deprotection of the latter (70 mg, 0.094
mmol) as described in general procedure G yielded 14 (75 mg,
100%; overall yield: 72%) as a syrup. 1H NMR (300 MHz, CD3-
OD): δ 7.10-6.90 (4H, m, Ar-H), 3.32-3.26 (2H, m, H-1), 3.19
(1H, d, J ) 17.0 Hz, HA-â′), 3.11-2.94 (10H, m, H-3, H-4, H-7,
H-8, and H-10), 2.92 (1H, d, J ) 17.0 Hz, HB-â′), 2.75 (2H, dd,
J ) 8.1 and 5.1 Hz, H-γ), 2.43-2.32 (1H, m, J ) 13.2 and 5.1
Hz, HA-â), 2.11 (2H, t, 7.3 Hz, H-2′), 2.08-1.95 (3H, m, H-9
and HB-â), 1.86-1.70 (6H, m, H-2, H-5, and H-6), 1.50 (2H,
sx, J ) 7.3 Hz, H-3′), 0.82 (3H, t, J ) 7.3 Hz, H-4′). 13C NMR
(75 MHz, CD3OD): δ 177.8 (C-1′), 176.1 (CdO, amino acid),
135.8 and 133.4 (C-a and C-f), 130.1 and 129.1 (C-b and C-e),
127.0 and 126.9 (C-c and C-d), 124.8 (C-1′, Pfp), 59.6 (C-R),
48.3 and 48.1 (C-4 and C-7), 46.0 and 45.8 (C-3 and C-8), 38.8
(C-â′), 38.7 (C-2′), 37.8 (C-10), 36.8 (C-1), 29.4 (C-â), 27.8
(C-2), 26.2 (C-γ), 25.4 (C-9), 24.4 and 24.3 (C-5 and C-6), 20.4
(C-3′), 14.2 (C-4′). HRMS: calcd for C25H44N5O2 [M + H]+
446.34895, found 446.34849.
saturated aqueous NaHCO3 (2 × 80 mL). The organic phase
was dried (Na2SO4) and filtered, and the solvent was evapo-
rated. Purification by VLC (petroleum ether to petroleum
ether-EtOAc 4:1) yielded methyl indole-2-carboxylate (0.984
g, 91%) as a powder. TLC: Rf 0.28 (petroleum ether-EtOAc
4:1). 1H NMR data: as earlier reported.38 13C NMR (100 MHz,
CDCl3): δ 162.8 (CdO), 137.1, 127.6, 127.2, 125.5, 122.7 and
120.9 (6C, Ar-C), 52.2 (OCH3). The methyl ester (395 mg, 2.25
mmol) was suspended in MeOH (12 mL), Mg (225 mg, 9.0
mmol) was added, and the mixture was stirred under N2 at
room temperature overnight. The reaction mixture was poured
into cold 2 M aqueous HCl (12 mL), and then the mixture was
stirred until it became clear, when pH was adjusted to 8-9
with 2 M aqueous NH3. MeOH was removed in vacuo, the
residue was suspended in water (50 mL), and the solution was
extracted with EtOAc (3 × 100 mL). The combined EtOAc
phases were dried (Na2SO4) and filtered, and the solvent was
evaporated. Purification by VLC (hexanes-EtOAc 20:1 to 10:
1) afforded methyl indoline-2-carboxylate39 (295 mg, 74%) as
a yellowish solid. TLC: Rf 0.34 (hexanes-EtOAc 4:1). 1H NMR
(400 MHz, CDCl3): δ 7.07 (2H, m, Ar-H), 6.70 (2H, m, Ar-
H), 4.39 (1H, dd, J ) 11.0 and 5.5 Hz, H-R), 4.74 (3H, s, CH3O),
3.39 (1H, dd, J ) 18.0 and 11.0 Hz, H-2), 3.31 (1H, dd, J )
18.0 and 5.5 Hz). The methyl ester (295 mg, 1.66 mmol) was
suspended in water (12.5 mL), a solution of KOH (95 mg, 1
equiv) in MeOH (0.5 mL) was added, and the mixture was
stirred at room temperature overnight. The reaction mixture
was concentrated, and then the residue was suspended in
water (50 mL), acidified with 4 M aqueous HCl, and extracted
with EtOAc (3 × 50 mL). The combined EtOAc phases were
dried (Na2SO4) and filtered, and then the solvent was removed
to give crude 15a (237 mg, 87%), which was used in the next
step without further purification. TLC: Rf 0.36 (hexanes-
EtOAc 4:1). NMR data: essentially as earlier reported.40
Pentafluorophenyl DL-1-(9H-Fluoren-9-ylmethoxycar-
bonyl)indoline-2-carboxylate (15c). Compound 15a (233
mg, 1.43 mmol) was converted to the Fmoc derivative using
general procedure B to give 15b (422 mg, 77%) as a foam.
TLC: Rf 0.18 (hexanes-EtOAc 2:1). Compound 15b (407 mg,
1.06 mmol) was esterified according to general procedure C to
give 15c (542 mg, 93%) as a white foam. TLC: Rf 0.37
(hexanes-EtOAc 7:1). 1H NMR (400 MHz, CDCl3): δ 7.77 (2H,
d, J ) 7.3 Hz, H-y), 7.61 (2H, d, J ) 7.3 Hz, H-v), 7.41 (2H, t,
J ) 7.3 Hz, H-x), 7.33-7.29 (2H, m, H-w), 7.29-6.80 (4H, m,
indoline-ArH), 5.30-5.20 (1H, br m, H-R), 4.83 and 4.58 (each
1H, br m, Fmoc-CH2), 4.40-4.28 (1H, m, Fmoc-CH), 3.88-3.68
(1H, br m, H-â′), 3.49-3.25 (1H, br m, H-â′). 13C NMR (100
MHz, CDCl3): δ 167.7*, 167.6* (CdO, amino acid), 153.6*,
152.1* (CdO, Fmoc), 144.0-136.0 (Pfp C-F, C-u, C-z, C-a, C-f),
128.5-127.2 (6C, C-x, C-w, C-b, C-d), 125.0-123.3 (C-v, C-c,
C-1′ Pfp), 120.2 (C-y), 115.1 and 114.9 (C-e), 68.4 (Fmoc-CH2),
59.9* and 59.8* (C-R), 47.2 (Fmoc-CH), 33.3* and 32.2* (C-â);
(*) denotes doubling of signals originating from two amide
rotamers present. HRMS: calcd for C30H19F5NO4 [M + H]+
552.12288, found 552.12269.
DL-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]pro-
pyl]-1-(1-oxobutyl)indoline-2-carboxylamide Tris(triflu-
oroacetate) (15). Pfp ester 15c (199 mg, 0.360 mmol) and
compound 3b (181 mg, 0.360 mmol) were coupled according
to general procedure D to give the conjugate (297 mg, 95%) as
a foam. TLC: Rf 0.26 (hexanes-EtOAc 4:1). Fmoc deprotection
of the conjugate (200 mg, 0.230 mmol) according to general
procedure E afforded the amine intermediate (142 mg, 95%)
as a syrup. TLC: Rf 0.38 (CH2Cl2-MeOH-32% aqueous NH3
200:10:1). The amine intermediate (111 mg, 0.171 mmol) was
dissolved in dry CH2Cl2 (1 mL) and a solution of pentafluo-
rophenyl butyrate (48 mg, 0.188 mmol) in dry CH2Cl2 (1 mL)
and Et3N (24 µL, 0.171 mmol) were added. The mixture was
stirred for 3 h at room temperature, when it was concentrated
in vacuo. The residue was dissolved in dry CH2Cl2 (1 mL),
additional amounts of pentafluorophenyl butyrate (48 mg,
0.188 mmol) and Et3N (24 µL, 0.171 mmol) were added, and
the mixture was stirred at room temperature overnight.
Butyryl chloride (35.5 µL, 0.342 mmol) and Et3N (120 µL, 0.855
DL-Indoline-2-carboxylic Acid (15a). A solution of indole-
2-carboxylic acid (1.00 g, 6.21 mmol) and concentrated H2SO4
(0.56 mL) in MeOH (34 mL) was heated under reflux for 6
h.37 The reaction mixture was concentrated in vacuo, and the
residue was dissolved in CH2Cl2 (200 mL) and washed with

Constraints and Steric Bulk in Philanthotoxins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 67
mmol) were added, and the stirring was continued for 2 h at
room temperature. The solvent was evaporated and the residue
was purified by VLC to give the Boc-protected product (112
mg, 91%) as a syrup. TLC: Rf 0.28 (hexanes-EtOAc 1:2).
Deprotection of the latter (71 mg, 0.10 mmol) was performed
according to general procedure G to give 15 (64 mg, 85%;
overall yield: 70%) as a syrup. 1H NMR (400 MHz, CD3OD, T
) 328 K): δ 7.23-7.21 (3H, m, H-b, H-c, and H-d), 7.04 (1H,
t, J ) 7.9 Hz, H-e), 5.04 (1H, dd, J ) 10.9 and 3.6 Hz, H-R),
3.32-3.31 (2H, m, H-1), 3.16-2.99 (12H, m, H-â, H-3, H-4,
H-7, H-8, and H-10), 2.15-2.04 (2H, p, J ) 7.4 Hz, H-2′), 1.90
(2H, p, J ) 6.9 Hz, H-9), 1.85-1.67 (8H, m, H-2, H-5, H-6,
and H-3′), 1.01 (3H, t, J ) 7.4 Hz, H-4′). 13C NMR (75 MHz,
CD3OD, T ) 328 K): δ 174.2, (CdO, amino acid), 128.6 (Ar-
C), 125.1 (Ar-C), 63.2 (C-R), 48.3 and 48.2 (C-4 and C-7), 46.4
(C-8), 45.9 (C-3), 37.9, 37.8, and 36.4 (C-2′, C-1, and C-10), 27.5
(C-2), 25.3 (C-9), 24.2 (2C, C-5 and C-6), 19.1 (C-3′), 14.0
(C-4′); several signals are missing due to overlapping and
extensively broadened lines. HRMS: calcd for C23H40N5O2 [M
+ H]+ 418.31765, found 418.31718.
L-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
r-[(1-oxobutyl)amino]-1H-indole-3-propanamide Tris-
(trifluoroacetate) (16). Compound 16a (402 mg, 0.581 mmol)
and compound 3b (292 mg, 0.581 mmol) were coupled using
general procedure D to give the conjugate (572 mg, 97%) as a
foam. TLC: Rf 0.47 (hexanes-EtOAc 1:2). Fmoc deprotection
of the conjugate (382 mg, 0.378 mmol) was performed using
general procedure E to give the amine intermediate (279 mg,
94%) as an oil. TLC: Rf 0.11 (CH2Cl2-MeOH-32% aqueous
NH3 200:10:1). The N-butyryl group was introduced to the
amine intermediate (382 mg, 0.378 mmol) according to general
procedure F to give the Boc-protected product (194 mg, 70%).
TLC: Rf 0.13 (hexanes-EtOAc 1:2). Deprotection as described
in general procedure G yielded 16 (141 mg, 85%; overall
yield: 54%). 1H NMR (300 MHz, CD3OD): δ 7.56 (1H, br d, J
) 8.0 Hz, H-g), 7.33 (1H, br d, J ) 8.0 Hz, H-d), 7.13 (1H, s,
H-b), 7.09 and 7.01 (each 1H, dt, J ) 2 × 8.0 and 1.1 Hz, H-e
and H-f), 4.50 (1H, t, J ) 7.6 Hz, H-R), 3.30-3.00 (10H, m,
H-1, H-4, H-8, H-10, and H-â), 2.90-2.75 (4H, m, H-3 and H-7),
2.17 (2H, t, J ) 7.4 Hz, H-2′), 2.09 (2H, p, H-9), 1.84-1.69
(6H, m, H-2, H-5, and H-6), 1.51 (2H, sx, J ) 7.4 Hz, H-3′),
0.80 (3H, t, J ) 7.4 Hz, H-4′). 13C NMR (75 MHz, CD3OD): δ
175.9 (C-1′), 175.3 (CdO, amino acid), 137.7 (C-c), 128.4
(C-h), 124.3 (C-b), 122.3 and 119.6 (C-e and C-f), 119.1 (C-g),
112.2 (C-d), 110.6 (C-a), 56.3 (C-R), 48.2 and 48.0 (C-4 and C-7),
45.8 (2C, C-3 and C-8), 38.6 (C-2′), 37.8 (C-10), 36.6 (C-1), 28.7
(C-â), 27.4 (C-2), 25.5 (C-9), 24.3 (2C, C-5 and C-6), 20.3
(C-3′), 14.0 (C-4′). HRMS: calcd for C25H43N6O2 [M + H]+
459.34420, found 459.34377.
Pentafluorophenyl 1H-Indole-2-carboxylate (17b). In-
dole-2-carboxylic acid (17a, 100 mg, 0.624 mmol) was esterified
according to general procedure C to give 17b (195 mg, 96%)
as a white foam. TLC: Rf 0.61 (hexanes-EtOAc 5:1). 1H NMR
(400 MHz, CDCl3): δ 8.74 (1H, s, NH), 7.50 (1H, d, J ) 8.1
Hz, H-7), 7.30 (1H, s, H-3), 7.18 (2H, m, H-5 and H-6), 6.97
(1H, m, H-4). 13C NMR (100 MHz, CDCl3): δ 167.5 (CdO),
141.0 (2C, C-2′/C-6′, Pfp), 139.3 (C-4′, Pfp), 137.8 (2C, C-3′/
C-5′, Pfp), 138.1, 127.4, 127.1, 124.8, 123.3†, 121.8 (C-R, C-1′
pfp, C-a, C-b, C-c, C-d, and C-f), 112.8 and 112.2 (C-â and C-e);
(†) denotes two-carbon signal. HRMS: calcd for C15H6F5NO2
[M + H]+ 328.03915, found 328.03949.
N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]propyl]-
r-[(1-oxobutyl)amino]-1H-indole-3-carboxylamide Tris-
(trifluoroacetate) (17). Pfp ester 17b (79 mg, 0.241 mol) and
compound 3b (121 mg, 0.241 mmol) were coupled using general
procedure D to give the conjugate (124 mg, 79%) as a foam.
TLC: Rf 0.25 (petroleum ether-EtOAc 4:1). Deprotection was
performed according to general procedure G to give 17 (36 mg,
92%; overall yield: 72%) as a syrup. 1H NMR (400 MHz, CD3-
OD): δ 7.60 (1H, br d, J ) 8.2 Hz, H-b), 7.45 (1H, br d, J )
8.2 Hz, H-e), 7.23 (1H, br t, J ) 8.2 Hz, H-c), 7.09 (1H, s, H-â),
7.07 (1H, br t, 8.2 Hz, H-d), 3.53 (2H, t, J ) 6.5 Hz, H-1), 3.15-
3.03 (10H, m, H-3, H-4, H-7, H-8, and H-10), 2.11-2.00 (4H,
m, H-2 and H-9), 1.85-1.80 (4H, m, H-5 and H-6). 13C NMR
(100 MHz, CD3OD): δ 165.2 (CdO), 136.4, 131.6, and 129.0
(C-f, C-a, and C-R), 125.3, 122.8, and 121.3 (C-b, C-c, and C-d),
113.1 and 104.8 (C-â and C-e), 48.3 and 48.1 (C-4 and C-7),
46.4 and 45.8 (C-8 and C-3), 37.8 (C-10), 37.0 (C-1), 27.9
(C-2), 25.4 (C-9), 24.3 and 24.2 (C-5 and C-6). HRMS: calcd
for C19H32N5O [M + H]+ 346.26014, found 346.25988.
Pentafluorophenyl (2R,4S)-1-(9H-Fluoren-9-ylmethoxy-
carbonyl)-4-phenylmethyl-pyrrolidine-2-carboxylate(18b).
Compound 18a (200 mg, 0.45 mmol) was esterified using
general procedure C to give 18b (274 mg, 100%) as a white
foam. TLC: Rf 0.39 (hexanes-EtOAc 4:1). [R] 2D5: -42.6° (c
0.65, CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.72 (2H, dd, J )
7.3 and 3.3 Hz, H-y), 7.56 (2H, m, H-v), 7.37-7.21 (9H, m,
H-x, H-w, and Ph), 4.82 (1H, dt, J ) 2 × 7.7 and 4.0 Hz, H-R),
4.60-4.48 (2H, m, PhCH2-O), 4.46-4.34 (2H, m, Fmoc-CH2),
4.33-4.19 (2H, m, Fmoc-CH and H-γ), 3.92 (1H, br d, J ) 12.1
Hz, HA-δ minor rotamer), 3.78-3.65 (2H, m, HA-δ and HB-δ
major rotamer, and HB-δ minor rotamer), 2.75-2.57 (1H, m,
HA-â), 2.39-2.20 (1H, m, HB-â). 13C NMR (75 MHz, CDCl3): δ
168.9* and 168.5* (CdO, amino acid), 154.9* and 154.2* (CdO,
Fmoc), 144.0*, 143.7*, 143.6*, and 143.2* (2C, C-u), 141.2 and
141.1 (2C, C-z), 141.0 (2C, C-2′/C-6′, Pfp), 139.3 (C-4′, Pfp),
137.8 (2C, C-3′/C-5′, Pfp), 137.2* and 137.2* (C-a), 128.5, 128.0,
127.7, and 127.6 (6C, C-b, C-c, C-d, and C-x), 127.0 and 126.9
(2 × C-w), 125.1*, 125.0*, 124.9*, and 124.8* (3C, C-v, C-1′
Pfp), 120.0 (2C, C-y), 76.7* and 75.7* (CH-γ), 71.4 (PhCH2-
O), 68.5* and 67.9* (Fmoc-CH2), 57.9* and 57.7* (C-R), 52.2*
and 51.9* (C-δ), 47.2 (Fmoc-CH), 37.5* and 36.0* (C-â); (*)
denotes double signals originating from two amide rotamers
present. HRMS: calcd for C33H25F5NO5 [M + H]+ 610.16475,
found 610.16451.
(2R,4S)-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]-
propyl]-4-phenylmethyl-1-(1-oxobutyl)pyrrolidine-2-car-
boxylamide Tris(trifluoroacetate) (18). Pfp ester 18b (111
mg, 0.182 mmol) and compound 3b (92 mg, 0.182 mmol) were
dissolved in dry CH2Cl2 (1 mL), Et3N (25 µL, 0.182 mmol) was
added, and the mixture was stirred at room temperature for
1.5 h, when the solvent was removed in vacuo. The crude
conjugate (0.182 mmol) was dissolved in dry THF (1.5 mL),
and 1-octanethiol (320 µL, 1.82 mmol) and DBU (12 µL) were
added. After stirring of the mixture for 3 h at room temper-
ature, TLC showed incomplete conversion. The mixture was
then concentrated and redissolved in THF (1.5 mL), additional
amounts of 1-octanethiol (320 µL, 1.82 mmol) and DBU (18
µL) were added, and the mixture was stirred for 0.5 h at room
temperature. Purification by VLC yielded the amine interme-
diate (113 mg, 98%) as a syrup. TLC: Rf 0.22 (CH2Cl2-
MeOH-32% aqueous NH3 400:20:1). The amine intermediate
(103 mg, 0.162 mmol) was coupled with pentafluorophenyl
butyrate according to general procedure F to yield the Boc-
protected product (99 mg, 87%) as an oil. Deprotection of the
latter (94 mg, 0.135 mmol) was performed as described in
general procedure G to give 18 (103 mg, 94%; overall yield:
1
83%) as a syrup. H NMR (300 MHz, CD3OD): δ 7.26-7.13
(5H, Ph), 4.49 (1H, d, J ) 12.0 Hz, PhCH2-O), 4.43 (1H, d, J
) 12.0 Hz, PhCH2-O), 4.25 (1H, dd, J ) 9.2 and 7.7 Hz, H-R),
4.22-4.17 (1H, m, H-γ), 3.67 (1H, br d, J ) 11.5 Hz, HA-δ),
3.07 (1H, dd, J ) 11.5 and 3.7 Hz, HB-δ), 3.41-3.30 (2H, m,
H-1), 3.16-2.87 (10H, m, H-3, H-4, H-7, H-8, and H-10), 2.49-
2.38 (2H, m, HA-â), 2.38-2.22 (2H, m, HB-â), 2.19 (2H, t, J )
7.3 Hz, H-2′), 2.04-1.90 (2H, m, H-9), 1.86-1.73 (2H, m, H-2),
1.73-1.64 (4H, m, H-5 and H-6), 1.51 (2H, sx, J ) 7.3 Hz, H-3′),
0.86 (3H, t, J ) 7.3 Hz, H-4′). 13C NMR (100 MHz, CD3OD):
δ 175.8, 174.8 (C-1′ and CdO, amino acid), 139.5 (C-a), 129.5
and 128.7 (5C, C-b, C-c, and C-d), 78.7 (PhCH2-O), 71.9
(C-γ), 60.7 (C-R), 54.2 (C-δ), 48.2 and 48.1 (C-4 and C-7), 46.2
and 45.8 (C-3 and C-8), 37.8 (C-10), 37.3 (C-2′), 36.7 (C-1), 36.5
(C-â), 27.5 (C-2), 25.3 (C-9), 24.2 (2C, C-5 and C-6), 19.3
(C-3′), 14.1 (C-4′). HRMS: calcd for C26H46N5O3 [M + H]+
476.35958, found 476.35891.
Methyl (2S,4R)-4-Hydroxy-1-[(phenylmethoxy)carbon-
yl]pyrrolidine-2-carboxylate (19b). Compound 19a (2.00 g,
7.54 mmol) and Et3N (1.6 mL, 11.3 mmol) were dissolved in
MeCN (19.0 mL), and MeI (0.9 mL, 15.08 mmol) in MeCN (4.0

68 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Jørgensen et al.
mL) were added at 0 °C. The mixture was stirred at 55 °C
overnight, when additional amounts of MeI (0.9 mL, 15.08
mmol) and Et3N (3.3 mL, 22.62 mmol) were added, and then
the mixture was stirred at 55 °C for 4 h. The reaction mixture
was distributed between Et2O (100 mL) and 0.1 M aqueous
HCl (100 mL), and the aqueous phase was extracted with Et2O
(2 × 100 mL). The combined organic phases were washed with
0.1 M aqueous NaOH (100 mL), dried (Na2SO4), and filtered,
and the solvent was removed to give crude 19b (1.35 g, 64%),
which was used without further purification. TLC: Rf 0.18
(hexanes-EtOAc 1:1). NMR data: as earlier reported.41
(Ar-C, naphthyl), 127.8, 127.7, 127.1, 127.0, 126.7, 126.6,
126.5, 125.0, 124.9, 124.8, 124.2, and 124.1 (C-x, C-w, C-v, 8
× Ar-C naphthyl, C-1′ Pfp), 120.0 (2C, C-y), 119.3 and 119.2*
(C-b), 108.1 and 108.0* (C-j), 75.4 and 74.4* (C-γ), 68.3 and
68.0* (Fmoc-CH2), 58.0 and 57.8* (C-R), 52.6* and 52.1 (C-δ),
47.4* and 47.3 (Fmoc-CH), 37.1* and 35.9 (C-â); (*) denotes
additional signals originating from a minor amide rotamer.
HRMS: calcd for C36H25F5NO5 [M + H]+ 646.16474, found
646.16439.
(2S,4S)-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]-
propyl]-4-(2-naphthyloxy)-1-(1-oxobutyl)pyrrolidine-2-
carboxylamide Tris(trifluoroacetate) (19). Using general
procedure H, resin loaded with 3a (250 mg, loading ap-
proximately 0.80 mmol/g, 0.20 mmol) was elongated succes-
sively with 19f and pentafluorophenyl butyrate to give, after
deprotection and cleavage, 19 (89 mg, 52%) as a syrup. 1H
NMR (300 MHz, CD3OD): δ 7.76 (3H, m, H-c, H-e and H-h),
7.43 (1H, t, J ) 7.4 Hz, H-g), 7.33 (1H, t, J ) 7.4 Hz, H-f),
7.22 (1H, s, H-j), 7.04 (1H, dd, J ) 8.8 and 2.2 Hz, H-b), 5.22
(1H, m, H-γ), 4.56 (1H, d, J ) 8.0 Hz, H-R), 4.08-3.82 (2H, m,
H-δ), 3.46-3.24 (2H, m, H-1), 3.15-2.80 (10H, m, H-3, H-4,
H-7, H-8 and H-10), 2.70-2.56 (1H, m, HA-â), 2.51-2.40 (3H,
m, H-2′ and HB-â), 2.08 (2H, p, J ) 7.4 Hz, H-9), 1.98-1.82
(2H, m, H-2), 1.84-1.69 (6H, m, H-5, H-6 and H-3′), 1.00 (3H,
t, J ) 7.2 Hz, H-4′). 13C NMR (75 MHz, CD3OD): δ 175.3 and
175.2 (C-1′ and CdO, amino acid), 155.5 (C-a), 135.6 (C-i),
130.6, 128.4, and 127.7 (3C, C-c, C-e and C-h), 130.4 (C-d),
127.4 (C-g), 124.9 (C-f), 119.8 (C-b), 109.2 (C-j), 77.1 (C-γ), 60.8
(C-R), 54.2 (C-δ), 48.2 and 48.1 (C-4 and C-7), 46.1 (C-3), 45.8
(C-8), 37.8 (C-10), 37.3 (C-2′), 36.8 (C-1), 35.8 (C-â), 27.6
(C-2), 25.4 (C-9), 24.3 (2C, C-5 and C-6), 18.9 (C-3′), 14.3 (C-
4′). HRMS: calcd for C29H46N5O3 [M + H]+ 512.35952, found
512.35990.
Pentafluorophenyl (2S,4S)-1-(9H-Fluoren-9-ylmethoxy-
carbonyl)-4-(2-naphthyloxy)pyrrolidine-2-carboxylate
(19f). Alcohol 19b (123 mg, 0.44 mmol), 2-naphthol (95 mg,
0.66 mmol), and Ph3P (173 mg, 0.66 mol) were dissolved in
dry THF (1.5 mL), and then a solution of DEAD (104 µL, 0.66
mmol) in dry THF (0.5 mL) was added dropwise. The mixture
was stirred at room temperature under N2 overnight, and then
the solvent was removed in vacuo. Purification by VLC
(hexanes to hexanes-acetone 5:1) afforded 19c (133 mg, 74%)
as a yellowish solid contaminated with a small amount of
hydrazine-N,N′-dicarboxylic acid diethyl ester according to 1H
NMR. TLC: Rf 0.33 (petroleum ether-EtOAc 4:1). A solution
of 19c (552 mg, 1.36 mmol) in absolute EtOH (10 mL) and
glacial HOAc (0.3 mL) was hydrogenated in the presence of
10% Pd/C (75 mg) with vigorous stirring at room temperature
overnight. The reaction mixture was filtered through a bed of
Celite and concentrated, and the residue was dissolved in CH2-
Cl2 (50 mL) and washed with 1 M aqueous NaOH (20 mL) and
brine (20 mL). The organic phase was dried (Na2SO4), filtered,
and concentrated and the residue subjected to purification by
VLC (CH2Cl2-MeOH-32% aqueous NH3, 400:4:1 to 400:10:
1) to give 19d (317 mg, 86%) as a syrup. TLC: Rf 0.29 (CH2-
Cl2-MeOH-32% aqueous NH3 400:10:1). 1H NMR (400 MHz,
CDCl3): δ 7.77-7.69 (3H, m, H-c, H-e and H-h), 7.41 and 7.35
(each 1H, br t, J ) 8.2 Hz, H-f and H-g), 7.06-7.03, (2H, m,
H-b, H-j), 4.96 (1H, m, H-R), 3.88 (1H, dd, J ) 9.3 and 4.2 Hz,
H-γ), 3.50 (3H, s, CH3-O), 3.40 (1H, dt, J ) 12.4 and 1.4 Hz,
HA-δ), 3.10 (1H, dd, J ) 12.4 and 4.2 Hz, HB-δ), 2.50 (1H, m,
HA-â), 2.40 (1H, m, HB-â). 13C NMR (100 MHz, CDCl3): δ 174.9
(CdO), 155.0 (C-a), 134.5 and 129.2 (C-d and C-i), 129.8 (C-c),
127.8, 126.9 and 126.5 (3C, C-e, C-g and C-h), 119.5 (C-b),
108.3 (C-j), 77.1 (C-γ), 59.5 (C-R), 53.0 and 52.4 (CH3-O and
C-δ), 36.8 (C-â).
Compound 19d (197 mg, 0.726 mmol) was suspended in a
mixture of MeCN (10 mL) and 10% aqueous Na2CO3 (15 mL),
and the mixture was stirred overnight at room temperature,
followed by stirring for 16 h at 40 °C. MeCN was removed in
vacuo, and dioxane (20 mL) and Fmoc-Cl (188 mg, 0.726 mmol)
in dioxane (3 mL) were added successively at 0 °C. The mixture
was stirred at 0 °C for 1 h and was then poured into water
(100 mL). The resulting mixture was washed with hexane (50
mL), and the aqueous phase was acidified with 4 M aqueous
HCl and extracted with EtOAc (4 × 50 mL). The combined
EtOAc phases were dried (Na2SO4), filtered, and concentrated.
Purification by VLC (hexanes-EtOAc 2:1 to hexanes-EtOAc-
HOAc 1000:1000:1) afforded 19e (237 mg, 68%) as a syrup.
TLC: Rf 0.20 (hexanes-EtOAc-HOAc 1000:1000:1). Com-
pound 19e (286 mg, 0.596 mmol) was esterified according to
general procedure C to give 19f (392 mg, 100%). TLC: Rf 0.40
(hexanes-EtOAc 2:1). [R] 2D5: -52.2° (c 0.55, CHCl3). 1H NMR
(300 MHz, CDCl3): δ 7.81-7.64 (5H, m, H-y and 3 × Ar-H,
naphthyl), 7.58-7.50 (2H, m, H-v), 7.49-7.20 (5H, m, H-x,
H-w, and Ar-H, naphthyl), 7.17-7.09 (1H, dt, J ) 8.0 and
1.1 Hz, Ar-H, naphthyl), 7.08-6.98 (2H, m, H-b and H-j), 5.16
and 5.12* (1H, m, H-R), 4.97 and 4.87* (2H, dd, J ) 9.5 and
1.8 Hz, H-γ), 4.59-4.49 and 4.42-4.31* (each 1H, m, Fmoc-
CH2), 4.29-4.19 (1H, m, Fmoc-CH), 4.00-3.80 (2H, m, H-δ),
2.92-2.79 (1H, m, HA-â), 2.79-2.62 (1H, m, HB-â). 13C NMR
(75 MHz, CDCl3): 167.4* and 167.1 (CdO, amino acid), 154.5*
and 154.1 (CdO, Fmoc), 154.2 (C-a), 144.1*, 143.8, 143.5, and
143.3* (2C, C-u), 141.3 and 141.1* (2C, C-z), 134.1, (2C, C-i),
141.0 (2C, C-2′/C-6′, Pfp), 139.3 (C-4′, Pfp), 137.8 (2C, C-3′/C-
5′, Pfp), 130.0 and 129.9* (Ar-C, naphthyl), 129.4 and 129.3*
Pentafluorophenyl DL-1-(9H-Fluoren-9-ylmethoxycar-
bonyl)-4-phenylpiperidine-4-carboxylate (20c). Com-
pound 20a (p-toluenesulfonate; 500 mg, 1.33 mmol) was Fmoc
protected using general procedure A to give 20b (517 mg, 91%)
as a white solid. The latter compound (510 mg, 1.19 mmol)
was esterified according to general procedure C to give 20c
1
(613 mg, 87%) as a white foam. H NMR (300 MHz, CDCl3):
δ 7.73 (2H, d, J ) 7.4 Hz, H-y), 7.56 (2H, br d, J ) 7.4 Hz,
H-v), 7.43-7.25 (9H, m, 4 × Ar-H, and Ph), 4.49 (2H, br d, J
) 6.5 Hz, Fmoc-CH2), 4.23 (1H, t, J ) 6.5 Hz, Fmoc-CH), 4.17
(1H, br d, J ) 11.6 Hz, Heq-R), 3.96 (1H, br d, J ) 11.6 Hz,
Heq-R′), 3.08 (2H, br t, J ) 11.6 Hz, Hax-R and Hax-R′), 2.60
(2H, m, Heq-â and Heq-â′), 1.99 (1H, m, Hax-â), 1.83 (1H, m,
Hax-â′). 13C NMR (75 MHz, CDCl3): δ 170.4 (CdO, amino acid),
155.0 (CdO, Fmoc), 143.8 (2C, C-u), 141.3 (2C, C-z), 141.0 (2C,
C-2′/C-6′, Pfp), 140.1 (C-a), 139.3 (C-4′, Pfp), 137.8 (2C, C-3′/
C-5′, Pfp), 129.0 and 125.7 (2C, C-b and C-c), 128.1 (C-d), 127.7
(2C, C-x), 127.0 (2C, C-w), 124.9 (2C, C-v), 124.8 (C-1′, Pfp),
120.0 (2C, C-y), 67.3 (Fmoc-CH2), 50.4 (C-γ), 47.7 (Fmoc-CH),
41.7 (2C, C-R/C-R′), 33.8/33.5 (C-â/C-â′). Anal. Calcd for
C33H24F5NO4: C, 66.78; H, 4.08; F, 16.00; N, 2.36. Found: C,
66.57; H, 3.82; F, 16.13; N, 2.28.
DL-N-[3-[[4-[(3-Aminopropyl)amino]butyl]amino]pro-
pyl]-1-(1-oxobutyl)-4-phenyl-piperidine-4-carboxyl-
amide Tris(trifluoroacetate (20). Pfp ester 20c (400 mg,
0.674 mmol) and compound 3b (339 mg, 0.674 mmol) were
coupled using general procedure D to yield the conjugate (546
mg, 89%) as a foam. TLC: Rf 0.17 (hexanes-EtOAc 1:1). Fmoc
deprotection of the conjugate (403 mg, 0.442 mmol) was
performed using general procedure E to give the amine
intermediate (294 mg, 96%) as a syrup. TLC: Rf 0.23 (CH2-
Cl2-MeOH-32% aqueous NH3 200:20:1). The N-butyryl group
was introduced to the latter (217 mg, 0.315 mmol) using
general procedure F to give the Boc-protected product (208 mg,
87%) as a syrup. TLC: Rf 0.20 (hexanes-EtOAc; 1:2). Depro-
tection of the Boc-protected product (158 mg, 0.208 mmol) was
performed according to general procedure G to give 20 (164
1
mg, 98%; overall yield: 80%) as a syrup. H NMR (300 MHz,
CD3OD): δ 7.46-7.32 (4H, m, H-b and H-c), 7.30-7.22 (1H,
m, H-d), 4.08 (1H, dt, J ) 14.0 and 4.1 Hz, Heq-R), 3.76 (1H,
dt, J ) 14.0 and 4.1 Hz, Heq-R′), 3.44 (1H, m, Hax-R′), 3.20 (1H,

Constraints and Steric Bulk in Philanthotoxins
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 69
(9) Karst, H.; Piek, T. Structure-Activity Relationship of Philan-
thotoxins. II. Effects on the Glutamate Gated Ion Channels of
the Locust Muscle Fiber Membrane. Comp. Biochem. Physiol.
1991, 98C, 479-489.
(10) Bruce, M.; Bukownik, R.; Eldefrawi, A. T.; Eldefrawi, M. E.;
Goodnow, R., Jr.; Kallimopoulos, T.; Konno, K.; Nakanishi, K.;
Niwa, M.; Usherwood, P. N. R. Structure-Activity Relationships
of Analogs of the Wasp Toxin Philanthotoxin: Non-competitive
Antagonists of Quisqualate Receptors. Toxicon 1990, 28, 1333-
1346.
(11) Anis, N.; Sherby, S.; Goodnow, R., Jr.; Niwa, M.; Konno, K.;
Kallimopoulos, T.; Bukownik, R.; Nakanishi, K.; Usherwood, P.
Structure-Activity Relationships of Philanthotoxin Analogs and
Polyamines on N-Methyl-D-aspartate and Nicotinic Acetylcholine
Receptors. J. Pharm. Exp. Ther. 1990, 254, 764-773.
(12) Choi, S. K.; Kalivretenos, A. G.; Usherwood, P. N.; Nakanishi,
K. Labeling Studies of Photolabile Philanthotoxins with Nicotinic
Acetylcholine Receptors: Mode of Interaction between Toxin and
Receptor. Chem. Biol. 1995, 2, 23-32.
(13) Tikhonov, D. B.; Mellor, I. R.; Usherwood, P. N. R.; Magazanik,
L. G. Modeling of the Pore Domain of the GLUR1 Channel:
Homology with K+ Channel and Binding of Channel Blockers.
Biophys. J. 2002, 82, 1884-1893.
(14) Washburn, M. S.; Dingledine, R. Block of R-Amino-3-hydroxy-
5-methyl-4-isoxazolepropionic acid (AMPA) Receptors by Poly-
amines and Polyamine Toxins. J. Pharm. Exp. Ther. 1996, 278,
669-678.
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and Block of Rat GluR6 Glutamate Receptor Channels by
Internal and External Polyamines. J. Physiol. 1997, 502, 575-
589.
(16) Shao, Z. Y.; Mellor, I. R.; Brierley, M. J.; Harris, J.; Usherwood,
P. N. R. Potentiation and Inhibition of Nicotinic Acetylcholine
Receptors by Spermine in the TE671 Human Muscle Cell Line.
J. Pharm. Exp. Ther. 1998, 286, 1269-1276.
(17) Karst, H.; Joeels, M.; Wadman, W. J.; Piek, T. Philanthotoxin
Inhibits Ca2+ Currents in Rat Hippocampal CA1 Neurons. Eur.
J. Pharmacol. 1994, 270, 357-360.
m, Hax-R), 3.32-3.02 (8H, m, H-1, H-4, H-8, and H-10), 2.90
(2H, t, J ) 7.0 Hz, H-7), 2.72 (2H, t, J ) 7.3 Hz, H-3), 2.61-
2.43 (2H, m, Heq-â and Heq-â′), 2.39 (2H, t, J ) 7.4 Hz, H-2′),
2.10 (2H, p, J ) 7.4 Hz, H-9), 2.07-1.92 (2H, m, Hax-â and
Hax-â′), 1.86-1.68 (6H, m, H-2, H-5 and H-6), 1.62 (2H, sx, J
) 7.4 Hz, H-3′), 0.97 (3H, t, J ) 7.4 Hz, H-4′). 13C NMR (75
MHz, CD3OD): δ 177.1 (C-1′), 173.5 (CdO, amino acid), 143.5
(C-d), 129.7 (C-f), 128.2 (C-b), 126.7 (C-a), 50.3 (C-γ), 48.2
(C-4), 48.1 (C-7), 46.2 (C-3), 45.8 (C-8), 44.6 and 40.4 (C-R and
C-R′), 37.8 (C-10), 37.1 (C-1), 36.0 (C-2′), 35.2 and 34.1 (C-â
and C-â′), 27.5 (C-2), 25.4 (C-9), 24.3 (2C, C-5 and C-6), 20.0
(C-3′), 14.3 (C-4′). HRMS: calcd for C26H46N5O2 [M + H]+
460.36460, found 460.36413.
Electrophysiology. AMPAR Assay. A two-electrode volt-
age clamp (TEVC) was used to record responses to 100 µM
kainite of X. laevis oocytes expressing AMPAR. X. laevis
oocytes were injected with rat brain RNA and incubated at
18 °C for at least 3 days.3 Single oocytes were transferred to
a perfusion bath and continuously washed with saline contain-
ing 120 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, and 5 mM
HEPES (pH adjusted 7.5 with NaOH). Microelectrodes were
pulled from borosilicate glass capillaries (GC150TF-10, Clark
Electromedical Instruments) using a Sutter P-97 program-
mable puller and had resistances of ∼0.5 MΩ when filled with
3.0 M KCl. The oocytes were voltage clamped at -80 mV using
an Axoclamp (Axon Instruments), and output currents were
digitized with Sony PCM and recorded to videotape with a
Sony VCR. Responses of AMPAR were elicited by perfusion of
100 µM kainic acid for 120 s. Philanthotoxins (2 and 4-20)
were coapplied from 40 to 80 s of this kainic acid application.
Acknowledgment. This work was supported by the
Danish Technical Research Council (Grant No. 26-00-
0312), Danish Medical Research Council (Grant No. 22-
00-0372), the Wellcome Trust (Grant No. 067496), the
Carlsberg Foundation, and the Novo Nordisk Founda-
tion. Technical assistance of Ms. Uraiwan Ngamrabiab
Adamsen is gratefully acknowledged.
(18) Bra¨uner-Osborne, H.; Egebjerg, J.; Nielsen, E. O.; Madsen, U.;
Krogsgaard-Larsen, P. Ligands for Glutamate Receptors: Design
and Therapeutic Prospects. J. Med. Chem. 2000, 43, 2609-2645.
(19) Fang, K.; Hashimoto, M.; Jockusch, S.; Turro, N. J.; Nakanishi,
K.
A Bifunctional Photoaffinity Probe for Ligand/Receptor
Interaction Studies. J. Am. Chem. Soc. 1998, 120, 8543-8544.
(20) Strømgaard, K.; Brierley, M. J.; Andersen, K.; Sløk, F. A.; Mellor,
I. R.; Usherwood, P. N. R.; Krogsgaard-Larsen, P.; Jaroszewski,
J. W. Analogues of Neuroactive Polyamine Wasp Toxins That
Lack Inner Basic Sites Exhibit Enhanced Antagonism Toward
a Muscle-Type Mammalian Nicotinic Acetylcholine Receptor. J.
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