Solid-phase synthesis and biological evaluation of a combinatorial library of philanthotoxin analogues

Article abstract of DOI:10.1021/jm000220n

The modular structure of philanthotoxins was exploited for construction of the first combinatorial library of these compounds using solid-phase parallel synthesis. (S)-Tyrosine and (S) 3-hydroxyphenylalanine were used as amino acid components, spermine, 1,12-dodecanediamine, and 4,9-dioxa-1,12-dodecanediamine as amine components, and butanoyl, phenylacetyl, and cyclohexylacetyl as N-acyl groups. Following automated preparative HPLC, the resulting 18 compounds were isolated as the S-forms in 40-70% yields. The purity of the products was determined by HPLC with evaporative light scattering detection and by ¹H and ¹³C NMR. The thus obtained philanthotoxins were tested electrophysiologically for their antagonist properties on human muscle-type nicotinic acetylcholine receptors (nAChR) expressed in TE671 cells and on rat brain non-NMDA glutamate receptors (non-NMDAR) expressed in Xenopus oocytes. 4-Hydroxy analogues lacking the secondary amino groups (PhTX-12 and 4,9-dioxa-PhTX-12 and their analogues) were inactive on non-NMDAR, whereas the potency of the spermine derivatives (PhTX-343 and its analogues) increased with steric bulk of the N-acyl group. The analogue of PhTX-343 in which the N-butanoyl group was replaced by phenylacetyl group had IC₅₀ of 15 ± 4 nM on non-NMDAR. Increasing the steric bulk of the N-acyl group was not advantageous for activity at nAChR, and a sharp decrease in potency with increased steric bulk was observed with the derivatives of PhTX-12. 3-Hydroxy analogues generally exhibited lower activity and different response to alterations of the N-acyl groups as compared to the 4-hydroxy analogues. Since the acyl group alterations in PhTX-343 and 4,9-dioxa-PhTX-12 have a similar effect on potency, which is distinctly different from that observed for PhTX-12, the two former compounds may bind to nAChR in a similar fashion but differently from that of PhTX-12. The combinatorial library approach described in this work represents a prototype methodology for future exploration of structure-activity relationships of philanthotoxins.

Full text of DOI:10.1021/jm000220n

4526
J . Med. Chem. 2000, 43, 4526-4533
Solid -P h a se Syn th esis a n d Biologica l Eva lu a tion of a Com bin a tor ia l Libr a r y of
P h ila n th otoxin An a logu es
Kristian Strømgaard,† Tim J . Brier,‡ Kim Andersen,§ Ian R. Mellor,‡ Alex Saghyan,‡ Denis Tikhonov,‡
Peter N. R. Usherwood,‡ Povl Krogsgaard-Larsen,† and J erzy W. J aroszewski*,†
Department of Medicinal Chemistry and NeuroScience PharmaBiotec Research Center, Royal Danish School of Pharmacy,
Universitetsparken 2, DK-2100 Copenhagen, Denmark, Division of Molecular Toxicology, School of Biological Sciences,
University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom, and Department of Combinatorial
Chemistry, Medicinal Chemistry Research, H. Lundbeck A/ S, Ottiliavej 9, DK-2500 Valby, Denmark
Received May 24, 2000
The modular structure of philanthotoxins was exploited for construction of the first combina-
torial library of these compounds using solid-phase parallel synthesis. (S)-Tyrosine and (S)-
3-hydroxyphenylalanine were used as amino acid components, spermine, 1,12-dodecanediamine,
and 4,9-dioxa-1,12-dodecanediamine as amine components, and butanoyl, phenylacetyl, and
cyclohexylacetyl as N-acyl groups. Following automated preparative HPLC, the resulting 18
compounds were isolated as the S-forms in 40-70% yields. The purity of the products was
1
determined by HPLC with evaporative light scattering detection and by H and 13C NMR. The
thus obtained philanthotoxins were tested electrophysiologically for their antagonist properties
on human muscle-type nicotinic acetylcholine receptors (nAChR) expressed in TE671 cells and
on rat brain non-NMDA glutamate receptors (non-NMDAR) expressed in Xenopus oocytes.
4-Hydroxy analogues lacking the secondary amino groups (PhTX-12 and 4,9-dioxa-PhTX-12
and their analogues) were inactive on non-NMDAR, whereas the potency of the spermine
derivatives (PhTX-343 and its analogues) increased with steric bulk of the N-acyl group. The
analogue of PhTX-343 in which the N-butanoyl group was replaced by phenylacetyl group had
IC50 of 15 ( 4 nM on non-NMDAR. Increasing the steric bulk of the N-acyl group was not
advantageous for activity at nAChR, and a sharp decrease in potency with increased steric
bulk was observed with the derivatives of PhTX-12. 3-Hydroxy analogues generally exhibited
lower activity and different response to alterations of the N-acyl groups as compared to the
4-hydroxy analogues. Since the acyl group alterations in PhTX-343 and 4,9-dioxa-PhTX-12
have a similar effect on potency, which is distinctly different from that observed for PhTX-12,
the two former compounds may bind to nAChR in a similar fashion but differently from that
of PhTX-12. The combinatorial library approach described in this work represents a prototype
methodology for future exploration of structure-activity relationships of philanthotoxins.
Ch a r t 1
In tr od u ction
Philanthotoxins, a group of noncompetitive antago-
nists of ionotropic receptors, are composed of long-chain
polyamines connected to a relatively nonpolar head-
group via an amide bond.1-6 The prototype structure is
philanthotoxin-433 [PhTX-433 (1), Chart 1], a venom
constituent of the Egyptian digger wasp Philanthus
triangulum.1 The interest in medicinal chemistry and
pharmacology of philanthotoxins has recently been
highlighted by the observation that specificity of their
antagonist action on various classes of ionotropic recep-
tors can be achieved by modification of the polyamine
portion of the molecule.7,8 Thus, the natural toxin PhTX-
433 (1) and its synthetic analogue PhTX-343 (2) an-
tagonize various types of nicotinic acetylcholine recep-
tors (nAChRs)3,6,9-11 and ionotropic glutamate receptors
(iGluRs)2-5,12-16 with rather similar potency. On the
other hand, the analogues 3 (PhTX-12) and 4 (4,9-dioxa-
PhTX-12), in which the secondary amino groups are
* To whom correspondence should be addressed. Phone: 45 3530
6372. Fax: 45 3530 6040. E-mail: jj@dfh.dk.
† Royal Danish School of Pharmacy.
‡ University of Nottingham.
replaced by methylene groups or oxygen atoms, exhibit
enhanced antagonist activity at mammalian muscle-
type nAChR and Torpedo nAChR while being inactive
§ H. Lundbeck A/S.
10.1021/jm000220n CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/26/2000

Combinatorial Library of Philanthotoxin Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4527
on several types of iGluR.7,8,17 The latter selectivity is
not obtained when the aromatic headgroup portion of
the philanthotoxin molecule is modified.3,4,10
Ta ble 1. Antagonist Effect of Philanthotoxin Analogues on
nAChR Expressed in TE671 Cellsa
In previous structure-activity investigations, a con-
siderable number of synthetic analogues of 2 containing
the symmetrical spermine moiety or a closely relat-
ed polyamine have been tested on iGluR and
nAChR.2-6,9,10,15 The results of these investigations
emphasize the importance of the hydrophobic character
of the headgroup. By contrast, no information about the
influence of the structure of the headgroup on the
potency of philanthotoxin analogues that lack inner
basic sites (such as 3 and 4) is available. This issue is
addressed in the studies described herein. A comparison
of structure-activity relationships (SARs) for 2-4 might
indicate whether the three compounds bind to nAChR
in a similar fashion.
IC50 (µM)
compd
X
R1
R2
peak value
end value
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
CH2 3-OH CH2CH3 104 ( 14 (11)
2.43 ( 0.33 (11)
8.05 ( 5.26 (11)
nt
CH2 3-OH C6H5
CH2 3-OH C6H11
105 ( 12 (10)
nt
CH2 4-OH CH2CH3 15.1 ( 1.4 (12) 0.79 ( 0.16 (12)
CH2 4-OH C6H5
CH2 4-OH C6H11
388 ( 130 (12) 17.2 ( 2.3(12)
>1000 (10)
151 ( 13 (10)
42.6 ( 2.3 (10)
25.3 ( 1.2 (9)
O
O
O
O
O
O
3-OH CH2CH3 110 ( 12 (10)
3-OH C6H5
3-OH C6H11
50.9 ( 3.2 (9)
30.7 ( 1.6 (12) 4.47 ( 0.36 (12)
4-OH CH2CH3 72.7 ( 4.4 (20) 10.9 ( 0.8 (20)
Resu lts
4-OH C6H5
4-OH C6H11
35.0 ( 4.4 (12) 12.6 ( 1.8 (12)
25.9 ( 1.2 (12) 5.69 ( 0.61 (12)
Syn th esis. Because of the presence of three distinct
parts of the molecule (an amino acid moiety, an acyl
group, and a long-chain polyamine moiety) joined by
amide bonds, philanthotoxins are particularly suitable
for synthesis using solid-phase methodology similar to
that used in peptide synthesis. Moreover, variation of
the structures of the three building blocks can provide
a combinatorial library of philanthotoxins for explora-
tion of SARs. In the work described herein, two different
amino acids [(S)-tyrosine and (S)-3-hydroxyphenylala-
nine], three carboxylic acids (butanoic, phenylacetic, and
cyclohexylacetic acid), and three amines (spermine, 1,12-
dodecanediamine, and 4,9-dioxa-1,12-dodecanediamine)
were used for parallel synthesis of a library of 18
analogues. Solid-phase methods employed in this work
are essentially the same as those recently published by
Strømgaard et al. for synthesis of the enantiomers of 2
and 3.8
The synthetic procedure is shown in Scheme 1. The
diamines 5-7 were attached to trityl chloride resin to
afford the resins 8-10, respectively. The required
diamines 5 and 6 were commercial products, whereas
7 was synthesized from spermine in three steps as
previously described.8 A 10-fold molar excess of the
diamines was used in the resin derivatization step to
minimize cross-linking reaction.8
The coupling of either (S)-N-(9-fluorenylmethoxycar-
bonyl)-3-(tert-butoxy)phenylalanine (11) or (S)-N-(9-
fluorenylmethoxycarbonyl)-O-(tert-butyl)tyrosine (12) to
the free amino groups of the resins 8-10 was ac-
complished with excess of HATU (13) and collidine,18-21
to give the respective resins 14-19. Deprotection of the
primary amino group was carried out with piperidine,
which gave the resins 20-25. In the subsequent step,
the N-acyl groups were attached using butanoic acid
(26), phenylacetic acid (27), or cyclohexylacetic acid (28),
again using HATU (13) and collidine. This gave the
resins 29-46.
Finally, cleavage from the resins with simultaneous
deprotection of the phenol groups, and in the case of
41-46 also deprotection of the secondary amino groups,
afforded philanthotoxin analogues 47-64. This step was
accomplished with a mixture of trifluoroacetic acid
(TFA) and the scavengers triisopropylsilane and H2O.
The crude products were subjected to automated pre-
NH 3-OH CH2CH3 >1000
NH 3-OH C6H5
NH 3-OH C6H11
117 ( 23 (9)
98.2 ( 2.0 (10) 10.1 ( 0.6 (10)
59.8 ( 4.5 (10) 5.33 ( 0.62 (10)
NH 4-OH CH2CH3 56.7 ( 21.3 (12) 11.8 ( 5.9 (12)
NH 4-OH C6H5
NH 4-OH C6H11
249 ( 58 (9)
22.3 ( 1.4 (10)
86.9 ( 4.5 (11) 13.2 ( 2.0 (11)
a
From decrease of the current elicited by 10 µM acetylcholine
(holding potential VH ) -100 mV) by simultaneous co-application
of the antagonist, at the peak value of the current and at the end
of a 1-s application period (number of experiments given in
parentheses); nt, not tested.
parative HPLC-MS.22 The yields of purified 47-52
were about 40%, based on 8, and those of 53-58 and
59-64 about 60-70%, based on 9 and 10, respectively.
Purity of the final products was in the range of 97-
100%, as determined by HPLC with evaporative light
scattering (ELS)23 detection. This method is capable of
detection and quantification of amine impurities in the
final products. The amines originate from the resin
cross-linking reactions and cannot be detected by con-
ventional UV monitoring of the HPLC eluate.
1
All final products were characterized by H and 13C
NMR spectroscopy as well as mass spectrometry. Since
enantiomerically pure amino acids 11 and 12 were used
as the starting materials, and the synthesis as described
in Scheme 1 was previously demonstrated to take place
without racemization,8 the products 47-64 are assumed
to be pure S-enantiomers.
In Vitr o Electr op h ysiology. Whole-cell patch-clamp
recordings from TE671 cells, which express embryonic
muscle-type nAChR,24,25 were undertaken as previously
described.7,26 The effects of the test compounds (47-
64) on whole-cell currents elicited by 10 µM acetylcho-
line, applied as 1-s pulses, were investigated. All data
were obtained at a holding potential (VH) of -100 mV.
Because responses of TE671 cells to acetylcholine
rapidly desensitize, the effects of the compounds on the
maximum amplitude of the acetylcholine-induced cur-
rent (peak value) and on the current remaining at the
end of the 1-s application of agonist (end value) were
determined. The resulting IC50 values are summarized
in Table 1.
Inhibition of non-NMDA receptors (non-NMDAR) by
philanthotoxin analogues was determined by measuring
the reduction of the response to 100 µM kainic acid of

4528 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Strømgaard et al.
Sch em e 1
Xenopus laevis oocytes injected with rat brain RNA and
voltage-clamped at -80 mV, as previously described.12
The IC50 values are given in Table 2.
An objective of this work was to investigate whether
the structural changes to the headgroup of compounds
lacking inner basic sites, such as PhTX-12 (3) and 4,9-
dioxa-PhTX-12 (4), lead to changes in biological activity
similar to those obtained following modifications of the
headgroup of PhTX-343 (2). Previous studies with 2 and
its analogues have shown that replacement of the
N-butanoyl moiety with a longer acyl group invariably
leads to increased antagonist potency at nAChR and
iGluR. This has been shown by [3H]HTX binding to
Torpedo electric organ membranes,3 by [3H]MK-801
binding to rat brain membranes,3 by antagonism of
nAChR of locust neuronal somata10 and cockroach
cholinergic synapse,10 and by antagonism of iGluR
sensitive to quisqualate (qGluR) of locust muscle.2
Similarly, replacement of the N-butanoyl moiety of 2
with an N-benzoyl2,3,10 or N-cyclohexylcarbonyl2,3 group
Discu ssion
Solid-phase synthesis of philanthotoxins8,27 offers
distinct advantages as compared to traditional solution-
phase chemistry2,6,7,15,28-33 in terms of speed, yield, and
purity of the products. Enantiomerically pure com-
pounds were previously shown to be formed on solid
phase from (S)-tyrosine by use of HATU (13) and
collidine as coupling reagents.8 Moreover, the modular
composition of philanthotoxins enables the easy con-
struction of libraries of analogues by parallel synthesis.
These advantages were exploited in the present work
to obtain a small philanthotoxin library containing
compounds 47-64 (Scheme 1).

Combinatorial Library of Philanthotoxin Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4529
Ta ble 2. Antagonist Effect of Philanthotoxin Analogues on
Non-NMDAR Expressed in Xenopus Oocytes from Rat Brain
mRNAa
non-NMDAR (Table 2). Also in accordance with previous
studies,2,3,6,10,11 potency within the polyamine series 62-
64 increased with the steric bulk of the N-acyl groups
(Table 2). The analogue 63 is 50 times more potent than
the lead structure 62 (S-enantiomer of 2). In the latter
series (62-64), the steric bulk did not cause significant
changes of potency when tested at nAChR (Table 1). A
similar situation was observed for the 4,9-dioxa-PhTX-
12 analogues 56-58. In contrast, a pronounced decrease
of potency with increased steric bulk was observed for
the derivatives of PhTX-12 (50-52). It can thus be
concluded that in contrast to the action of 2 or 62 on
iGluR, introduction of a bulky apolar substituent into
the acyl group of 50 and 56 does not increase antagonist
potency at nAChR (Table 1). Since the acyl group
alterations in 56 and 62 have a similar effect on potency,
which is distinctly different from that for 50 (Table 1),
the two compounds containing heteroatoms within the
polyamine chain may bind to nAChR in a similar
fashion but differently from that of 50.
In the case of antagonist action of 3-hydroxy ana-
logues of 2 (compounds 59-61) on nAChR, an increase
of the potency with the steric bulk of the acyl group was
observed (Table 1). Interestingly, the 3-hydroxy ana-
logue 59 is about 10-fold less potent than the 4-hydroxy
analogue 62, but this difference is largely removed by
the presence of a benzene (compounds 60 and 63) or
cyclohexane (compounds 61 and 64) ring. A similar
trend is observed with the dioxa analogues 53-55.
There is a general trend that the 4-hydroxy analogues
are more potent than the 3-hydroxy analogues, although
some exceptions exist (Table 1).
In conclusion, N-acyl group modifications of the lead
structure 3 (compounds 47-52) did not improve the
potency at nAChR, contrary to what has been observed
with analogues of 2 at iGluR. The SARs described in
this work contribute to our understanding of the an-
tagonism of nAChR by philanthotoxin analogues lacking
the inner basic sites. Further electrophysiological stud-
ies are necessary to delineate potential differences in
the interaction of these analogues with nAChR.
Our previous work has demonstrated that solid-phase
synthesis is an efficient source of enantiomerically pure
philanthotoxin analogues.8 We believe that the combi-
natorial library approach described in the present work
will be of crucial importance in future studies of SARs
in the field of neuroactive polyamine toxins and in
further optimization of the structure of PhTX-12 (3).
compd
X
R1
R2
IC50 (µM)
50
56
62
63
64
CH2
O
NH
NH
NH
4-OH
4-OH
4-OH
4-OH
4-OH
CH2CH3
CH2CH3
CH2CH3
C6H5
>100
>100
0.75 ( 0.08 (5)
0.015 ( 0.004 (10)
0.18 ( 0.12 (4)
C6H11
a
From decrease of the current elicited by 100 µM kainic acid
(holding potential VH ) -80 mV) by simultaneous co-application
of the antagonist (number of experiments given in parentheses).
increased potency in all these assays. In contrast, the
presence of the N-phenylacetyl group increased potency
on nAChR and NMDAR, as reflected by the [3H]HTX
and [3H]MK-801 binding assay,3 respectively, but slightly
decreased potency at locust muscle qGluR.2 Generally,
an increase of the hydrophobic character of the N-acyl
group raises the potency of the analogues of 2, and steric
bulk in the acyl group (such as a phenyl or cyclohexyl
group) is tolerated very well.
Previous modifications of the tyrosine phenol function
of 2 revealed a somewhat complex pharmacology. Acety-
lation or benzylation of the phenol group led to un-
changed or slightly decreased potency on qGluR.11 On
the other hand, acetylation did not change and benzy-
lation strongly increased the potency on insect nAChR.10
Removal of the phenol group resulted in a 3-fold
increase of the potency on qGluR2 and a 16-fold increase
on locust nAChR.10 The latter structural change did not
affect binding to Torpedo electric organ membranes
([3H]HTX binding assay) but abolished binding of the
compound to rat brain membranes ([3H]MK-801 binding
assay).3 There have been no studies of the effect on
potency of the position of the hydroxy group on the
benzene ring of 2.
On the basis of these previous investigations, a library
was constructed using (S)-tyrosine and (S)-3-hydroxy-
phenylalanine as the amino acid residues, spermine, 1,-
12-dodecanediamine (5), and 4,9-dioxa-1,12-dodecanedi-
amine (6) as the amine moieties, and butanoyl, pheny-
lacetyl, and cyclohexylacetyl as the N-acyl groups. Use
of the two amino acids addresses the question of the
importance of the position of the hydroxy group in the
amino acid moiety. The three carboxylic acids were used
since considerable amount of information about the
effect of a benzene and cyclohexane ring in the sper-
mine-derived philanthotoxins exists, as described above.
The results of the electrophysiological assays with
47-64 are summarized in Tables 1 and 2. The nAChR
assays show that for all compounds tested, the IC50
values for end currents were lower than those for peak
currents. The ratio of the IC50 for peak current and end
current varied between the compounds tested, reflecting
the different onset rates for use-dependent antagonism
that occurs during co-application of all of the analogues
with acetylcholine. In accordance with previous studies
with iGluR,7,8,19 the monoamines 50 and 56 (S-enanti-
omers of 3 and 4) were inactive when tested on rat brain
Exp er im en ta l Section
Ch em istr y. Gen er al P r ocedu r es. Unless otherwise stated,
starting materials were obtained from commercial suppliers
and were used without further purification. Polystyrene-based
trityl chloride resin (1% divinylbenzene, 200-400 mesh) and
(S)-N-Fmoc-O-(tert-butyl)tyrosine were obtained from Nova-
biochem (La¨ufelingen, Switzerland). (S)-N-Fmoc-3-(tert-but-
oxy)phenylalanine was obtained from RSP Amino Acid Ana-
logues, Inc. (Worchester, MA). N,4N9-Bis(tert-butoxycarbonyl)-
spermine (7) was synthesized as previously described.8 Tet-
rahydrofuran (THF) was distilled under N2 from sodium/
benzophenone immediately prior to use. 1H and 13C NMR
spectra were recorded at 500.13 and 125.67 MHz, respectively,
on a Bruker Avance DRX 500 spectrometer, using CD3OD as
solvent and tetramethylsilane (TMS) as internal reference.
1
Coupling constants (nJ values) are in Hz. Multiplicities of H

4530 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Strømgaard et al.
NMR signals are given as follows: s, singlet; d, doublet; t,
triplet; q, quartet; p, pentet; s, sextet; m, multiplet.
(S)-N-(12-Am in od od ecyl)-3-h yd r oxy-r-[(1-oxob u t yl)-
a m in o]ben zen ep r op a n a m id e Tr iflu or oa ceta te (47).
Yield: 39%. 1H NMR: δ 0.84 (t), 1.54 (s), 2.15 (m) (respectively
4-CH3, 3-CH2 and 2-CH2 of the 1-oxobutyl moiety, both 3J )
Analytical and preparative high-performance liquid chro-
matography-mass spectrometry (HPLC-MS) were performed
on a Perkin-Elmer API 150EX instrument equipped with a
turbo ionspray (electrospray ionization) source. The HPLC
system consisted of two Shimadzu LC8A pumps. UV trace was
obtained with a Shimadzu SPD10A detector operating at 274
nm. Evaporating light scattering (ELS) trace was obtained
with an Eurosep DDL 31 light scattering detector and was
used for estimation of purity of final products. Analytical
HPLC-MS was performed on a 50 × 4.6-mm YMC RP18
column, using 2 mL/min of water/acetonitrile/TFA 90:10:0.05
raising to 10:90:0.05 during 7 min, with 10-µL injections.
Preparative HPLC-MS (split-flow MS detection) was run with
190-µL injections (50-mg samples in 1.0 mL of MeOH) to a 50
× 20-mm YMC RP18 column eluted with the same solvent
gradient at 22.7 mL/min.
Atta ch m en t of Am in es 5-7 to Tr ityl Ch lor id e Resin .
1,12-Dodecanediamine (5; 1.00 g, 5.00 mmol) was attached to
the resin (0.50 mmol) as previously described.8 The loading of
the resulting resin 8 was 1.53 mmol/g under the assumption
that the reaction had gone to completion. 4,9-Dioxa-1,12-
dodecanediamine (6; 1.02 g, 5.00 mmol) was dissolved in THF
(10 mL), and trityl chloride resin (0.50 mmol) was added to
the solution in five portions. The suspension was stirred at
room temperature for 2 h. The solvent was removed by
filtration and the resin was treated with a 10% solution of
diisopropylethylamine (DIEA) in MeOH (10 mL) for 5 min to
cap any remaining chlorine substituents. The resin was
washed with DMF (3 × 5 mL), CH2Cl2 (3 × 5 mL), MeOH (3
× 5 mL) and CH2Cl2 (3 × 5 mL), and dried in vacuo. The
loading of the resulting resin 9 was 1.52 mmol/g under the
assumption that the reaction had gone to completion. Reaction
of N4,N9-bis(tert-butoxycarbonyl)spermine (7; 2.01 g, 5.00
mmol) with trityl chloride resin (0.50 mmol) was carried out
as previously described.8 The loading of the resulting resin 10
was 1.17 mmol/g under the assumption that the reaction had
gone to completion.
2
3
3
7.5), 2.80 and 2.98 (each dd, J AB ) 13.7, J AX ) 8.5, J BX
)
7.1, â-CH2) 4.16 (dd, R-CH), 6.61-6.64 (m, 1 H), 6.65-6.66 (m,
1 H), 6.68-6.71 (m, 1 H) and 7.07 (m, 1 H) (aromatic H), 2.90
(t, 3J ) 7.4, 12-CH2), 3.03-3.17 (m, 1-CH2), 1.60-1.67 (m, 2H)
and 1.19-1.42 (m, 18 H) (the remaining CH2 of the diamine
moiety). 13C NMR: δ 13.9, 20.3, 27.5, 27.9, 28.7, 30.3, 30.4,
30.5, 30.7 (3 C), 38.8, 39.2, 40.4, 40.8, 56.2, 114.7, 117.2, 121.5,
130.4, 139.9, 158.6, 173.5, 175.9. HPLC-ELS: 97.7%. MS
(ES): 434.4 (M + 1).
(S)-N-(12-Am in od od ecyl)-3-h yd r oxy-r-[(1-oxo-2-p h en -
ylet h yl)a m in o]b en zen ep r op a n a m id e Tr iflu or oa cet a t e
1
2
(48). Yield: 40%. H NMR: δ 2.82 and 2.98 (each dd, J AB
)
13.7, 3J AX ) 8.4, 3J BX ) 6.9, â-CH2), 3.47 (d) and 3.52 (d) (2J )
14.5, benzylic H), 4.52 (dd, R-CH), 6.63-6.65 (m, 3 H), 7.03-
7.06 (m, 1 H), 7.13-7.15 (m, 2 H) and 7.18-7.31 (m, 3 H)
(aromatic H), 2.90 (t, 3J ) 7.4, 12-CH2), 3.05-3.14 (m, 1-CH2),
1.61-1.67 (m, 2H) and 1.19-1.40 (m, 18 H) (the remaining
CH2 of the diamine moiety). 13C NMR: δ 27.5, 27.9, 28.6, 30.3,
30.4, 30.5, 30.6, 30.7, 39.2, 40.4, 40.8, 43.6, 44.0, 56.3, 114.8,
117.2, 121.5, 127.9, 129.6 (2 C), 130.1, 130.2, 130.5, 136.7,
139.7, 158.6, 173.3, 173.8. HPLC-ELS: 98.1%. MS (ES): 482.4
(M + 1).
(S)-N-(12-Am in od od ecyl)-3-h yd r oxy-r-[(1-oxo-2-cyclo-
h exyleth yl)a m in o]ben zen ep r op a n a m id e Tr iflu or oa ce-
ta te (49). Yield: 39%. 1H NMR: δ 2.77 and 2.99 (each dd,
3
3
2J AB ) 13.7, J AX ) 8.4, J BX ) 6.9, â-CH2), 4.54 (dd, R-CH),
6.61-6.61 (m, 1 H), 6.67 (m, 1 H), 6.69-6.71 (m, 1 H) and
7.05-7.09 (m, 1 H) (aromatic H), 2.03 (d, 2-CH2 of the
cyclohexylacetyl moiety), 2.90 (t, 3J ) 7.5, 12-CH2), 3.09-3.14
(m, 1-CH2), 1.61-1.67 (m, 8 H), 1.19-1.40 (m, 22 H) and 0.77-
0.91 (m, 1 H) (the remaining CH and CH2 of the diamine and
the cyclohexylacetyl moiety).13C NMR: δ 27.3, 27.4, 27.5, 27.9,
28.7, 30.3, 30.4, 30.5, 30.7, 30.7, 34.0, 34.1, 34.2, 36.8, 39.1,
40.4, 40.8, 45.0, 56.2, 114.7, 117.2, 121.4, 130.4, 140.0, 158.6,
173.6, 175.3. HPLC-ELS: 98.6%. MS (ES): 488.7 (M + 1).
Gen er a l Syn th etic P r oced u r e. A solution of either (S)-
N-Fmoc-3-(tert-butoxy)phenylalanine (11) or (S)-N-Fmoc-O-
(tert-butyl)tyrosine (12) (1.54 mmol) and HATU (13; 1.54
mmol) in DMF (3 mL), and of collidine (2.32 mmol) in DMF (2
mL), was added to each of the resins 8-10 (0.39 mmol) placed
in 10-mL syringes equipped with a polypropylene filter and a
stopper. The syringes were agitated for 2 h at room temper-
ature, the solvent removed, and the resins washed with DMF
(3 × 5 mL), CH2Cl2 (3 × 5 mL), MeOH (3 × 5 mL) and CH2Cl2
(3 × 5 mL), to give the resins 14-19.
(S)-N-(12-Am in od od ecyl)-4-h yd r oxy-r-[(1-oxob u t yl)-
a m in o]b en zen ep r op a n a m id e Tr iflu or oa cet a t e (50).7,9
Yield: 41%. 1H NMR: δ 0.84 (t), 1.54 (s), 2.15 (m) (respectively
4-CH3, 3-CH2 and 2-CH2 of the 1-oxobutyl moiety, both 3J )
2
3
3
7.5), 2.77 and 2.95 (each dd, J AB ) 13.7, J AX ) 8.5, J BX
)
7.1, â-CH2) 4.48 (dd, R-CH), 6.68 and 7.03 (each 2H, AA′BB′
system, aromatic H), 2.90 (t, 3J ) 7.4, 12-CH2), 3.02-3.16 (m,
1-CH2), 1.64 (m, 2H) and 1.19-1.42 (m, 18 H) (the remaining
CH2 of the diamine moiety). 13C NMR: δ 13.9, 20.3, 27.5, 27.9,
28.6, 30.2, 30.3, 30.4, 30.5, 30.66, 30.67, 30.71, 38.5, 38.8, 40.4,
40.8, 56.5, 116.2 (2 C), 129.1, 131.3 (2 C), 157.3, 173.7, 175.9.
HPLC-ELS: 98.1%. MS (ES): 434.6 (M + 1).
A solution of 20% piperidine in DMF (v/v, 5 mL) was added
to each of the resins 14-19, and the mixtures agitated for 3
min at room temperature. The resins were subsequently
washed with DMF (3 × 5 mL), treated with 20% piperidine in
DMF (5 mL) for further 20 min, and washed with DMF (3 ×
5 mL), CH2Cl2 (3 × 5 mL), MeOH (3 × 5 mL) and CH2Cl2 (3 ×
5 mL), to give the resins 20-25.
A solution of butanoic acid (26), phenylacetic acid (27) or
cyclohexylacetic acid (28) (1.54 mmol) and HATU (1; 1.54
mmol) in DMF (3 mL) was added to each of the resins 20-25,
followed by addition of collidine (2.32 mmol) in DMF (2 mL).
The mixtures were agitated for 2 h at room temperature, and
the resins washed with DMF (3 × 5 mL), CH2Cl2 (3 × 5 mL),
MeOH (3 × 5 mL) and CH2Cl2 (3 × 5 mL), to give resins 29-
34, 35-40 and 41-46, respectively.
(S)-N-(12-Am in od od ecyl)-4-h yd r oxy-r-[(1-oxo-2-p h en -
ylet h yl)a m in o]b en zen ep r op a n a m id e Tr iflu or oa cet a t e
1
2
(51). Yield: 43%. H NMR: δ 2.78 and 2.96 (each dd, J AB
)
13.7, 3J AX ) 8.5, 3J BX ) 6.6, â-CH2), 3.46 (d) and 3.52 (d) (2J )
14.1, benzylic H), 4.48 (dd, R-CH), 6.65 (m, 2 H), 6.97 (m, 2
H), 7.12 (m, 2 H) and 7.19-7.29 (m, 3 H) (aromatic H), 2.90
(t, 3J ) 7.4, 12-CH2), 3.03-3.17 (m, 1-CH2), 1.60-1.66 (m, 2H)
and 1.18-1.41 (m, 18 H) (the remaining CH2 of the diamine
moiety). 13C NMR: δ 27.5, 27.9, 28.6, 30.3, 30.3, 30.4, 30.5,
30.6, 30.7, 38.4, 40.4, 40.8, 43.7, 44.0, 47.9, 56.6, 116.3, 127.9,
128.9, 129.6 (2 C), 130.1, 130.1, 131.3, 136.7, 157.4, 173.5,
173.8. HPLC-ELS: 98.7%. MS (ES): 482.2 (M + 1).
TFA/iPr3SiH/H2O (95:2.5:2.5) (5 mL) was added to each of
the above resins, and the mixtures were agitated for 2 h at
room temperature. The solvent was removed, and the resins
washed with MeOH (2 × 5 mL) and CH2Cl2 (2 × 5 mL). The
combined solvents were concentrated in vacuo and triturated
with Et2O to give the crude products 47-64, which were
purified using automated preparative HPLC using split-flow
MS detection for identification of peaks, giving the products
as trifluoroacetates in yields from 39-43%, 63-69% and 53-
59% for compounds 47-52, 53-58 and 59-64, respectively.
(S)-N-(12-Am in od od ecyl)-4-h yd r oxy-r-[(1-oxo-2-cyclo-
h exyleth yl)a m in o]ben zen ep r op a n a m id e Tr iflu or oa ce-
ta te (52). Yield: 43%. 1H NMR: δ 2.75 and 2.97 (each dd,
2J AB ) 13.7, J AX ) 8.4, J BX ) 6.9, â-CH2), 4.50 (dd, R-CH),
6.69 (m, 2 H) and 7.04 (m, 2 H) (aromatic H), 2.02 (d, 2-CH2 of
the cyclohexylacetyl moiety), 2.90 (t, 3J ) 7.5, 12-CH2), 3.10-
3.14 (m, 1-CH2), 1.61-1.67 (m, 8 H), 1.15-1.42 (m, 22 H) and
0.77-0.91 (m, 1 H) (the remaining CH and CH2 of the diamine
and the cyclohexylacetyl moiety). 13C NMR: δ 27.3, 27.4, 27.5,
27.9, 28.7, 30.3, 30.4, 30.5, 30.7, 30.7, 34.0, 34.1, 34.2, 36.8,
3
3

Combinatorial Library of Philanthotoxin Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4531
39.1, 40.4, 40.8, 45.0, 56.2, 114.7, 117.2, 121.4, 130.4, 140.0,
158.6, 173.6, 175.3. HPLC-ELS: 96.8%. MS (ES): 488.5 (M +
1).
H), 1.09-1.25 (m, 3 H), 1.46 (d, 1 H), 1.56-1.69 (m, 6 H from
polyamine moiety and 5 H from the cyclohexylacetyl moiety),
1.89 (p, 2 H) (2-, 6-, 7- and 11-CH2), 3.05 (t, 2 H, 3J ) 7.1),
3.15-3.26 (m, 2 H), 3.33-3.38 (m, 2 H), 3.41 (t, 2 H, 3J ) 6.1),
3.48 (t, 2 H, J ) 6.1) and 3.56 (t, 2 H, J ) 5.7) (1-, 3-, 5-, 8-,
10- and 12-CH2). 13C NMR: δ 27.2, 27.3, 27.3, 27.5, 27.5, 28.6,
30.4, 34.0, 34.1, 36.9, 37.8, 38.4, 39.5, 45.0, 56.5, 69.4, 69.4,
71.8, 72.1, 116.3 (2 C), 129.1, 131.3 (2 C), 157.4, 173.8, 175.3.
HPLC-ELS: 99.9%. MS (ES): 492.5 (M + 1).
(S)-N-[3-[[4-[(3-Am in opr opyl)a m in o]bu tyl]a m in o]p r op -
yl]-3-h ydr oxy-r-[(1-oxobu tyl)am in o]ben zen epr opan am ide
Tr is(tr iflu or oa ceta te) (59). Yield: 57%. 1H NMR: δ 0.87 (t),
1.56 (s), 2.18 (t) (respectively 4-CH3, 3-CH3 and 2-CH3 of the
1-oxobutyl moiety, both 3J ) 7.5), 4.42 (t, R-CH), 6.66 (m, 2
H), 6.71 (m, 1 H) and 7.10 (m, 1 H) (aromatic H), 1.78-1.81
(m, 6 H) and 2.08 (m, 2 H) (2-, 6-, 7- and 11-CH2), 2.84-2.94
(m, 3 H), 2.97-3.02 (m, 3 H), 3.03-3.09 (m, 4 H), 3.13 (t, 2 H,
3J ) 8.0) and 3.18-3.30 (m, 2 H) (â-CH2 and remaining CH2
of the polyamine moiety). 13C NMR: δ 14.0, 20.3, 24.4, 24.5,
25.6, 27.5, 36.8, 38.0, 38.6, 38.7, 46.0, 46.2, 48.2, 48.4, 49.7,
56.8, 114.9, 117.2, 121.4, 130.6, 139.8, 158.7, 175.2, 176.3.
HPLC-ELS: 100%. MS (ES): 436.6 (M + 1).
(S)-N-(4,9-Dioxa-12-am in ododecyl)-3-h ydr oxy-r-[(1-oxo-
bu tyl)am in o]ben zen epr opan am ide Tr iflu or oacetate (53).
Yield: 66%. 1H NMR: δ 0.85 (t), 1.55 (s), 2.16 (t) (respectively
4-CH3, 3-CH3 and 2-CH3 of the 1-oxobutyl moiety, both 3J )
3
3
2
3
3
7.5), 2.81 and 2.99 (each dd, J AB ) 13.7, J AX ) 8.0, J BX
)
7.1, â-CH2), 4.50 (t, R-CH), 6.62-6.70 (m, 3 H) and 7.07 (m, 1
H) (aromatic H), 1.58-1.65 (m, 6 H) and 1.91 (p, 2 H) (2-, 6-,
7- and 11-CH2), 3.05 (t, 2 H, 3J ) 7.1), 3.13-3.27 (m, 2 H),
3
3
3.32-3.37 (m, 2 H), 3.41 (t, 2 H, J ) 5.7), 3.47 (t, 2 H, J )
3
6.1) and 3.56 (t, 2 H, J ) 5.7) (1-, 3-, 5-, 8-, 10- and 12-CH2).
13C NMR: δ 14.0, 20.3, 27.5, 27.6, 28.6, 30.4, 37.9, 38.8, 39.2,
39.5, 56.3, 69.4, 69.4, 71.8, 72.1, 114.8, 117.3, 121.5, 130.5,
139.9, 158.6, 173.6, 176.0. HPLC-ELS: 98.3%. MS (ES): 438.4
(M + 1).
(S)-N-(4,9-Dioxa-12-am in ododecyl)-3-h ydr oxy-r-[(1-oxo-
2-p h en ylet h yl)a m in o]b en zen ep r op a n a m id e Tr iflu or o-
a ceta te (54). Yield: 65%. 1H NMR: δ 2.81 and 2.99 (each dd,
2J AB ) 13.7, 3J AX ) 8.0, 3J BX ) 7.1, â-CH2), 4.50 (t, R-CH), 6.62-
6.70 (m, 3 H) and 7.07 (m, 1 H) (aromatic H), 1.58-1.65 (m, 6
3
H) and 1.91 (p, 2 H) (2-, 6-, 7- and 11-CH2), 3.05 (t, 2 H, J )
(S)-N-[3-[[4-[(3-Am in op r opyl)a m in o]bu tyl]a m in o]p r op -
yl]-3-hydroxy-r-[(1-oxo-2-phenylethyl)amino]benzenepropan-
3
7.1), 3.13-3.27 (m, 2 H), 3.32-3.37 (m, 2 H), 3.41 (t, 2 H, J
) 5.7), 3.47 (t, 2 H, 3J ) 6.1) and 3.56 (t, 2 H, 3J ) 5.7) (1-, 3-,
5-, 8-, 10- and 12-CH2). 13C NMR: δ 27.5, 27.5, 28.6, 30.3, 37.8,
39.1, 39.5, 43.6, 56.4, 69.3, 69.3, 71.8, 72.1, 114.8, 117.2, 121.5,
127.9, 129.6 (2 C), 130.2 (2 C), 130.5, 136.7, 139.7, 158.7, 173.4,
173.8. HPLC-ELS: 99.8%. MS (ES): 486.4 (M + 1).
a m id e Tr is(tr iflu or oa ceta te) (60). Yield: 53%. H NMR δ
1
3.01 (dd, 2J AB ) 13.7, 3J BX ) 7.1, HB proton of â-CH2), 3.51 (d)
and 3.55 (d) (2J ) 14.1, benzylic H), 4.42 (t, R-CH), 6.65-6.68
(m, 3 H), 7.06-7.10 (m, 1 H), 7.16-7.19 (m, 2 H), 7.20-7.24
(m, 1 H) and 7.25-7.29 (m, 2 H) (aromatic H), 1.70-1.81 (m,
6 H) and 2.07 (p, 2 H) (2-, 6-, 7- and 11-CH2), 2.80-2.92 (m, 5
(S)-N-(4,9-Dioxa-12-am in ododecyl)-3-h ydr oxy-r-[(1-oxo-
3
H), 3.05 (apparent t, 4 H), 3.11 (t, 2 H, J ) 7.5) and 3.16 (m,
2-cycloh exyleth yl)a m in o]ben zen ep r op a n a m id e Tr iflu o-
1
2 H) (HA proton of â-CH2 and remaining CH2 of the polyamine
moiety). 13C NMR: δ 24.4, 24.4, 25.6, 27.5, 36.8, 38.0, 38.5,
43.5, 46.0, 46.1, 48.2, 48.4, 57.1, 115.0, 117.2, 121.4, 128.0,
129.7 (2 C), 130.3 (2 C), 130.7, 136.7, 139.6, 158.7, 174.2, 175.0.
HPLC-ELS: 99.0%. MS (ES): 484.4 (M + 1).
r oa ceta te (55). Yield: 63%. H NMR: δ 2.78 and 3.01 (each
dd, 2J AB ) 13.7, 3J AX ) 8.5, 3J BX ) 7.1, â-CH2), 4.52 (dd, R-CH),
6.62-6.71 (m, 3 H) and 7.07 (m, 1 H) (aromatic H), 0.79-0.92
(m, 2 H), 1.08-1.25 (m, 3 H), 1.47 (d, 1 H), 1.56-1.69 (m, 6 H
from polyamine moiety and 5 H from the cyclohexylacetyl
moiety), 1.91 (p, 2 H) (2-, 6-, 7- and 11-CH2), 3.05 (t, 2 H, 3J )
(S)-N-[3-[[4-[(3-Am in opr opyl)a m in o]bu tyl]a m in o]p r op -
yl]-3-hydroxy-r-[(1-oxo-2-phenylethyl)amino]benzenepropan-
a m id e Tr is(tr iflu or oa ceta te) (61). Yield: 54%. 1H NMR: δ
3
7.1), 3.15-3.27 (m, 2 H), 3.34-3.39 (m, 2 H), 3.42 (t, 2 H, J
) 5.7), 3.48 (t, 2 H, 3J ) 6.1) and 3.56 (t, 2 H, 3J ) 5.7) (1-, 3-,
5-, 8-, 10- and 12-CH2). 13C NMR: δ 27.3, 27.3, 27.4, 27.5, 27.5,
28.6, 30.4, 34.0, 34.2, 36.8, 37.9, 39.1, 39.5, 45.0, 56.3, 69.4,
69.4, 71.8, 72.1, 114.7, 117.2, 121.5, 130.5, 140.0, 158.6, 1743.7,
175.3. HPLC-ELS: 99.8%. MS (ES): 492.3 (M + 1).
3
4.45 (t, J ) 7.8, R-CH), 6.64-6.72 (m, 3 H) and 7.10 (m, 1 H)
(aromatic H), 0.86-0.89 (m, 2 H), 1.16-1.20 (m, 3 H), 1.47
(m, 1 H) and 1.57-1.65 (m, 5 H) (CH and CH2 of the diamine
and the cyclohexylacetyl moiety),1.80 (m, 2-, 6- and 8-CH2),
2.08 (m, 11-CH2 and 2-CH2 of the cyclohexylacetyl moiety),
2.80-3.27 (m, 14H, â-CH2 and the remaining CH2 of the
polyamine moiety). 13C NMR: δ 24.4, 25.5, 27.2, 27.3 (2 C),
27.5, 33.9, 34.2, 36.7, 36.8, 37.9, 38.5, 44.9, 45.9, 46.1, 48.2,
56.7, 114.8, 117.2, 121.3, 130.6, 139.8, 158.6, 175.2, 175.7.
HPLC-ELS: 98.7%. MS (ES): 490.5 (M + 1).
(S)-N-(4,9-Dioxa-12-am in ododecyl)-4-h ydr oxy-r-[(1-oxo-
bu tyl)am in o]ben zen epr opan am ide Tr iflu or oacetate (56).7
Yield: 69%. 1H NMR: δ 0.85 (t), 1.55 (s), 2.15 (t) (respectively
4-CH3, 3-CH3 and 2-CH3 of the 1-oxobutyl moiety, both 3J )
2
3
3
7.5), 2.79 and 2.96 (each dd, J AB ) 13.7, J AX ) 8.0, J BX
)
7.1, â-CH2), 4.52 (t, R-CH2), 6.69 and 7.03 (each 2 H, ArH),
1.58-1.68 (m, 6 H) and 1.91 (p, 2 H) (2-, 6-, 7- and 11-CH2),
3.05 (t, 2 H, 3J ) 6.6), 3.13-3.26 (m, 2 H), 3.31-3.36 (m, 2 H),
(S)-N-[3-[[4-[(3-Am in opr opyl)a m in o]bu tyl]a m in o]p r op -
yl]-4-h ydr oxy-r-[(1-oxobu tyl)am in o]ben zen epr opan am ide
Tr is(tr iflu or oa ceta te) (62).8 Yield: 59%. 1H NMR: δ 0.87
(t), 1.55 (s), 2.17 (m) (respectively 4-CH3, 3-CH2 and 2-CH2 of
3
3
3.41 (t, 2 H, J ) 5.7), 3.48 (t, 2 H, J ) 6.1) and 3.56 (t, 2 H,
3J ) 5.7) (2-, 6-, 7- and 11-CH2). 13C NMR: δ 14.0, 20.3, 27.5,
27.5, 28.6, 30.4, 37.8, 38.5, 38.8, 39.5, 56.6, 69.4, 71.8, 72.1,
116.3 (2 C), 129.1, 131.3 (2 C), 157.4, 173.8, 175.9. HPLC-
ELS: 99.7%. MS (ES): 438.4 (M + 1).
3
the 1-oxobutyl moiety, both J ) 7.4), 4.37 (t, 3J ) 7.8, R-CH),
6.71 and 7.05 (each 2H, aromatic H), 1.78 (m, 2-, 6- and 8-CH2),
2.08 (p, 11-CH2), 2.79-3.28 (m, 14H, â-CH2 and the remaining
CH2 of the polyamine moiety). 13C NMR: δ 14.0, 20.3, 24.3,
24.4, 25.5, 27.4, 36.8, 37.9, 37.9, 38.7, 45.9, 46.2, 48.2, 48.3,
57.1, 116.3 (2 C), 128.9, 131.3 (2 C), 157.5, 175.2, 176.3. HPLC-
ELS: 99.5%. MS (ES): 435.5 (M + 1).
(S)-N-(4,9-Dioxa-12-am in ododecyl)-4-h ydr oxy-r-[(1-oxo-
2-p h en ylet h yl)a m in o]b en zen ep r op a n a m id e Tr iflu or o-
a ceta te (57). Yield: 67%. 1H NMR: δ 2.80 and 2.97 (each dd,
2J AB ) 13.7, 3J AX ) 8.5, 3J BX ) 7.1, â-CH2), 3.50 (two d, benzylic
H), 4.46 (t, R-CH), 6.66 (m, 2 H), 6.98 (m, 2 H), 7.14 (m, 2 H),
7.20-7.22 (m, 1 H) and 7.23-7.27 (m, 1 H) (aromatic H), 1.59-
1.66 (m, 6 H) and 1.89 (p, 2 H) (2-, 6-, 7- and 11-CH2), 3.03 (t,
2 H, 3J ) 6.6), 3.13-3.25 (m, 2 H), 3.30-3.35 (m, 2 H), 3.40 (t,
2 H, 3J ) 6.1), 3.47 (t, 2 H, 3J ) 6.1) and 3.56 (t, 2 H, 3J ) 6.1)
(1-, 3-, 5-, 8-, 10- and 12-CH2). 13C NMR: δ 27.5, 27.5, 28.6,
30.4, 37.9, 38.3, 39.5, 43.7, 56.7, 69.4, 71.8, 72.1, 116.3, 127.9,
128.9, 129.6, 130.2, 131.3, 136.7, 157.4, 173.5, 173.8. HPLC-
ELS: 99.8%. MS (ES): 486.4 (M + 1).
(S)-N-[3-[[4-[(3-Am in op r opyl)a m in o]bu tyl]a m in o]p r op -
yl]-4-hydroxy-r-[(1-oxo-2-phenylethyl)amino]benzenepropan-
a m id e Tr is(tr iflu or oa ceta te) (63).3 Yield: 59%. 1H NMR:
δ 4.36 (t, 3J ) 7.8, R-CH), 6.69 (m, 2 H), 7.01 (m, 2 H), 7.17
(m, 2 H) and 7.22-7.28 (m, 3 H) (aromatic H), 1.77 (m, 2-, 6-
and 8-CH2), 2.08 (p, 11-CH2), 2.79-3.28 (m, 14H, â-CH2 and
the remaining CH2 of the polyamine moiety), 3.49 (d) and 3.54
(d) (2J ) 14.5, benzylic H). 13C NMR: δ 24.3 (2 C), 25.4, 27.4,
36.8, 37.8, 37.8, 43.5, 45.9, 46.2, 48.1, 48.3, 57.3, 116.4 (2 C),
128.0, 128.7, 129.7 (2 C), 130.2 (2 C), 131.3 (2 C), 136.7, 157.5,
174.2, 175.0. HPLC-ELS: 99.4%. MS (ES): 484.4 (M + 1).
(S)-N-(4,9-Dioxa-12-am in ododecyl)-4-h ydr oxy-r-[(1-oxo-
2-cycloh exyleth yl)a m in o]ben zen ep r op a n a m id e Tr iflu o-
1
r oa ceta te (58). Yield: 69%. H NMR: δ 2.76 and 2.98 (each
(S)-N-[3-[[4-[(3-Am in op r opyl)a m in o]bu tyl]a m in o]p r op -
yl]-4-hydroxy-r-[(1-oxo-2-cyclohexylethyl)amino]benzenepropan-
a m id e Tr is(tr iflu or oa ceta te) (64). Yield: 57%. 1H NMR: δ
dd, 2J AB ) 13.7, 3J AX ) 9.0, 3J BX ) 6.6, â-CH2), 4.48 (dd, R-CH),
6.69 (m, 2 H) and 7.04 (m, 2 H) (aromatic H), 0.78-0.93 (m, 2

4532 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Strømgaard et al.
4.41 (t, 3J ) 7.8, R-CH), 6.71 (m, 2 H) and 7.06 (m, 2 H)
(aromatic H), 0.80-0.92 (m, 2 H), 1.12-1.23 (m, 3 H), 1.48
(m, 1 H) and 1.57-1.68 (m, 5 H) (CH and CH2 of the diamine
and the cyclohexylacetyl moiety),1.80 (m, 2-, 6- and 8-CH2),
2.08 (m, 11-CH2 and 2-CH2 of the cyclohexylacetyl moiety),
2.79-3.28 (m, 14H, â-CH2 and the remaining CH2 of the
polyamine moiety). 13C NMR: δ 24.3, 24.4, 25.4, 27.2, 27.3,
27.3, 27.4, 34.0, 34.2, 36.8, 37.8, 37.9, 44.9, 45.9, 46.3, 48.2,
48.3, 57.0, 116.3 (2 C), 129.0, 131.3 (2 C), 157.5, 175.2, 175.7.
HPLC-ELS: 99.3%. MS (ES): 490.4 (M + 1).
(7) Strømgaard, K.; Brierley, M. J .; Andersen, K.; Sløk, F. A.; Mellor,
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Electr op h ysiology. n ACh R Assa y. The nAChR assay was
performed essentially as previously described.7,26 TE671 cells
were grown in Petri dishes and transferred to a perfusion bath
mounted on the stage of an inverted microscope. Patch pipets
filled with 140 mM CsCl, 1 mM CaCl2, 11 mM EDTA and 5
mM HEPES (pH adjusted to 7.2 with 1 M CsOH) were used
for whole-cell recording. Cells were constantly perfused at ca.
5 mL/min with saline containing 135 mM NaCl, 5.4 mM KCl,
1 mM CaCl2, 1 mM MgCl2, 5 mM HEPES (pH adjusted to 7.4
with 3 M NaOH). Ligands (10 µM acetylcholine alone or 10
µM acetylcholine plus test compound) were applied as 1-s
pulses at intervals of at least 30 s to allow the nAChR to
recover from the desensitizing effect of agonist and the
blocking effect of the antagonist. Whole-cell currents were
monitored using an Axopatch 200 patch-clamp amplifier (Axon
Instruments) and the output was recorded on the hard disk
of a PC using pClamp 5.7.2 software (Axon Instruments).
Whole-cell recordings were performed at ambient laboratory
temperature (17-23 °C).
Non -NMDAR Assa y. X. laevis oocytes were injected with
rat brain RNA and incubated at 18 °C for at least 3 days.12
Single oocytes were transferred to a perfusion bath and
continuously perfused with saline containing 120 mM NaCl,
2 mM KCl, 1.8 mM CaCl2, 10 mM HEPES (pH adjusted to 7.5
with NaOH). The oocytes were voltage-clamped at -80 mV
using an Axoclamp 2A (Axon Instruments) and current output
was digitized with a Sony PCM and recorded to videotape with
a Sony VCR. Responses of non-NMDAR were elicited by
perfusion of 100 µM kainic acid for 120 s. PhTX-343 analogues
were co-applied from 40-80 s of this kainic acid application.
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Ack n ow led gm en t. This work was supported in part
by a grant from EU (BIOMED-2 PL962395). We thank
Mr. Peter Brøsen, H. Lundbeck A/S, for the assistance
with analytical and preparative HPLC-MS.
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