Solid-phase synthesis of PhTX-3.2.4 and PhTX-2.3.3 derivatives

Article abstract of DOI:10.1016/S0040-4039(02)00928-0

A new solid-phase method for the synthesis of derivatives of the philanthotoxins is described. Diamines are attached as carbamates to hydroxymethyl polystyrene resin. Selective mono-alkylations by acid-labile, substituted benzhydryl chlorides, followed by reductive alkylations with Fmoc-protected amino aldehydes are employed to assemble the polyamine backbone. Different acid-stability of the benzhydrylic protective groups allows the selective removal from secondary amines for subsequent branching.

Full text of DOI:10.1016/S0040-4039(02)00928-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 4793–4796
Solid-phase synthesis of PhTX-3.2.4 and PhTX-2.3.3 derivatives
Daniel Jo¨nsson*
Department of Neurochemistry & Neurotoxicology, Stockholm University, S-10691 Stockholm, Sweden
Received 11 February 2002; revised 30 April 2002; accepted 13 May 2002
Abstract—A new solid-phase method for the synthesis of derivatives of the philanthotoxins is described. Diamines are attached
as carbamates to hydroxymethyl polystyrene resin. Selective mono-alkylations by acid-labile, substituted benzhydryl chlorides,
followed by reductive alkylations with Fmoc-protected amino aldehydes are employed to assemble the polyamine backbone.
Different acid-stability of the benzhydrylic protective groups allows the selective removal from secondary amines for subsequent
branching. © 2002 Elsevier Science Ltd. All rights reserved.
Polyamines terminally conjugated with different amino
acids such as tryptophan, tyrosine or arginine have
been found to be constituents in several arthropod
venom mixtures.1,2 The most extensively investigated
group of naturally occurring polyamine toxins is the
philanthotoxins (PhTXs), where the compound PhTX-
4.3.3 (Fig. 1) has been isolated from the venom of the
Egyptian digger wasp Philanthus triangulum.3,4 The
polyamine toxins have been found to interact with
cation selective ion channels and to have non-competi-
tive antagonistic properties towards ionotropic gluta-
mate receptors5 and towards the open pore of muscular
and neuronal nicotinergic acetylcholine receptors.6 The
PhTXs are low molecular weight polyamines, with one
terminus as a primary amine and the other terminus
linked to a relatively non-polar residue via an amide
bond. SAR studies of PhTXs as ligands to receptors
have shown that significant changes in affinity and
selectivity for different receptors can be observed by
altering the terminal amino group, the polyamine back-
bone, the aromatic residue or the hydrophobic terminus
of the ligand.7
analogues and other acylpolyamine toxins have been
published recently.9–11 The presented solid-phase
approaches use a polyamine part that is either a pre-
constructed polyamine9b–c selectively protected with
orthogonal protecting groups before introduction to the
solid support, or constructed sequentially using reduc-
tive alkylation,9d modified Mitsunobu reactions10 or
polypeptide reduction.11 In this communication, the
versatility of our previously published method for
polyamine synthesis,12 as an alternative route to
acylpolyamine toxins, is presented by the synthesis of
derivatives of the previously unpublished PhTX-3.2.4
and PhTX-2.3.3. The assembly of the polyamine back-
bones is performed by reductive alkylations of amines,
protected with acid-labile benzhydryl-type amino-pro-
tecting groups, with N-protected amino aldehydes
according to our previously reported protocol.12 This
method allows primary amines, attached to solid-phase,
to be selectively mono-alkylated with 4-methoxy dityl
(Mmd)13 and 4,4%-dimethoxy dityl (Dod)14,15 groups.
The Mmd and the Dod groups are used as semi-perma-
nent or temporary protecting groups, respectively, and
cleavage of the temporary Dod group introduces the
possibility of further derivatizing the secondary amines.
A variety of structural analogues of PhTX-4.3.3 have
been synthesized, and several different routes for solu-
tion synthesis have been presented.3,4,8 However, the
synthesis of acylpolyamine compounds has shifted
towards a solid-phase approach, largely as a result of
the difficulties of working with polyamines in solution.
A few solid-phase protocols for the synthesis of PhTX
* Tel.: +46-8-164266; fax: +46-8-161371; e-mail: daniel@
neurochem.su.se
Figure 1. Structure of PhTX-4.3.3.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00928-0

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D. Jo¨nsson / Tetrahedron Letters 43 (2002) 4793–4796
4-methoxydityl chloride and subsequent washes with 10%
trifluoroacetic acid (TFA) gave the mono-Mmd protected
3.12 The secondary amino group was then reductively
alkylated for 3×1 h with N-Fmoc-3-aminopropionalde-
hyde to give 4.17 After Fmoc-deprotection, the resin-
bound primary amine was alkylated with excess
4,4%-dimethoxydityl chloride, treated with 5% TFA form-
ing 5 and then reductively alkylated with N-Fmoc-2-
aminoacetaldehyde to give 6. Cleavage of the tertiary
Dod-group makes it possible to introduce branching on
the nitrogen and reductive alkylation with butyraldehyde
or 3-phenylpropionaldehyde gave 7a and 7b, respectively.
Fmoc-deprotection and acylation with activated Fmoc-
Tyr (OtBu)-OH and butyric acid employing standard
Fmoc-peptide protocol resulted in 8a and 8b. Cleavage
from the resin was accomplished with neat TFA for 2.5
h at 50°C. After filtration from the resin and evaporation
of the TFA solution, the crude products were dissolved
in0.5MaqueousHClandextractedwithEtOAc. Analysis
of the aqueous phase by RP-HPLC18 (Fig. 3) and
MALDI-TOF19 showed the expected products 9a (at 10.7
min) and 9b (at 12.0 min) in >80% purity and 75–80%
overall yield.20 In addition, small fractions of resins 4, 6
and 7 were withdrawn, cleaved with neat TFA and
analyzed by RP-HPLC in order to monitor the progress
of the reaction and for characterization of the interme-
diate products by MALDI-TOF.
Figure 2. 9a: R1=butyl, (M+H)+=464.3601; 9b: R1=3-phenyl-
propyl; 16: R2=isobutyl, (M+H)+=478.3757; 17: R2=H. (Cal-
culated monoisotopic molecular weights.)
The PhTX analogues 9a and 9b (Fig. 2) with the 2.3.3
backbone were synthesized as follows (Scheme 1). Con-
ventional polystyrene-based hydroxymethyl resin was
converted to the 4-nitrophenyl-carbonate resin 1.16 The
N-terminal 1,3-diaminopropane group was attached to
the solid support by incubating resin 1 with 1,3-
diaminopropane forming 2. Alkylation with an excess of
Scheme 1. (i) 10 equiv. of 4-nitrophenyl chloroformate, 10 equiv. of N-methylmorpholine (NMM), dichloromethane (DCM), 2 h,
rt; (ii) 10 equiv. of 1,3-diaminopropane, N,N-dimethylformamide (DMF), 1.5 h, rt; (iii) 4 equiv. of Mmd-Cl, 10 equiv. of
N,N-diisopropylethylamine (DIEA), DCM, 1.5 h; (iv) 10×10 s with 10% TFA in DCM; (v) 3 equiv. of N-Fmoc-3-aminopropi-
onaldehyde, 3 equiv. of NaCNBH3, 1-methyl-2-pyrrolidinone (NMP) with 3% AcOH, 40°C, 3×1 h; (vi) 20% piperidine in DMF,
30 min; (vii) 4 equiv. of Dod-Cl, 10 equiv. of DIEA, DCM, 1.5 h; (viii) 6×10 s with 5% TFA in DCM; (ix) 3 equiv. of
N-Fmoc-2-aminoacetaldehyde, 3 equiv. of NaCNBH3, NMP with 3% AcOH, 40°C, 3×1 h; (x) 5×10 s+1×15 min with 5% TFA
in DCM, (xi) a: 3 equiv. of butyraldehyde or b: 3-phenylpropionaldehyde, 3 equiv. of NaCNBH3, NMP with 3% AcOH, 40°C,
3×1 h; (xii) 1.5 equiv. of Fmoc-Tyr(OtBu)-OH, 1.5 equiv. of TBTU, 1.5 equiv. of HOBt, 3 equiv. of DIEA, DCM/DMF (1:1),
20 min, rt; (xiii) 1.5 equiv. of butyric acid, 1.5 equiv. of TBTU, 1.5 equiv. of HOBt, 3 equiv. of DIEA, DCM/DMF (1:1), 20 min,
rt; (xiv) TFA, 50°C, 2.5 h.

D. Jo¨nsson / Tetrahedron Letters 43 (2002) 4793–4796
4795
The PhTX analogues 16 and 17 (Fig. 2) were synthe-
sized using the same Dod/Mmd method as for 9a and
9b. However, the intermediate alkyl chains are altered
and both the branched and the unbranched analogue
possessing the same polyamine backbone are synthe-
sized according to Scheme 2. Resin bound 1,4-
diaminobutane, 10 was alkylated with either Dod-Cl or
Mmd-Cl and treated with 5% TFA to give 11a and 11b,
respectively. Subsequent reductive alkylation with N-
Fmoc-2-aminoacetaldehyde gave 12a and 12b. Washing
of resin 12a with 5% TFA and a subsequent reductive
alkylation with isobutyraldehyde gave 13a. Cleavage of
the Fmoc group and repetition of the protection with
Mmd-Cl and the reductive alkylation of the terminal
amine with N-Fmoc-3-aminopropionaldehyde gave 14a
and 14b. Acylation as above with activated Fmoc-Tyr
(OtBu)-OH and butyric acid formed 15a and 15b,
which were cleaved from the resin in neat TFA for 2.5
h at 50°C. The products 16 and 17 (Fig. 2) were treated
and analyzed as above. The main peaks at 9.5 or 8.8
min in the RP-HPLC analyses were of satisfying purity
(>75%) (Fig. 3) and displayed the correct molecular
weights, with an overall yield of 50%.
In conclusion, analogues of the polyamine toxins of the
philanthotoxin group can be conveniently and rapidly
synthesized on solid support. Sequential elongation and
Figure 3. HPLC chromatograms of 9a and 16, displaying
absorbance at 279 nm and the molecular weights of main
peaks.
Scheme 2. (i) 10 equiv. of 4-nitrophenyl chloroformate, 10 equiv. of NMM, DCM, 2 h, rt; (ii) 10 equiv. of 1,4-diaminobutane,
DMF, 1.5 h, rt; (iii) a: 4 equiv. of Dod-Cl or b: 4 equiv. of Mmd-Cl, 10 equiv. of DIEA, DCM, 1.5 h; (iv) a: 6×10 s with 5%
TFA in DCM, b: 10×10 s with 10% TFA in DCM; (v) 3 equiv. of N-Fmoc-2-aminoacetaldehyde, 3 equiv. of NaCNBH3, NMP
with 3% AcOH, 40°C, 3×1 h; (vi) only 12a: 5×10 s+1×15 min with 5% TFA in DCM; (vii) only 12a: 3 equiv. of isobutyraldehyde,
3 equiv. of NaCNBH3, NMP with 3% AcOH, 40°C, 3×1 h; (viii) 20% piperidine in DMF, 30 min; (ix) 4 equiv. of Mmd-Cl, 10
equiv. of DIEA, DCM, 1.5 h; (x) 10×10 s with 10% TFA in DCM; (xi) 3 equiv. of N-Fmoc-3-aminopropionaldehyde, 3 equiv.
of NaCNBH3, NMP with 3% AcOH, 40°C, 3×1 h; (xii) 1.5 equiv. of Fmoc-Tyr(OtBu)-OH, 1.5 equiv. of TBTU, 1.5 equiv. of
HOBt, 3 equiv. of DIEA, DCM/DMF (1:1), 20 min, rt; (xiii) 1.5 equiv. of butyric acid, 1.5 equiv. of TBTU, 1.5 equiv. of HOBt,
3 equiv. of DIEA, DCM/DMF (1:1), 20 min, rt; (xiv) TFA, 50°C, 2.5 h.

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D. Jo¨nsson / Tetrahedron Letters 43 (2002) 4793–4796
selective branching of the polyamine backbones, using
reductive alkylations with repetitive protocols, can be
used in order to obtain a large number of derivatives of
acylpolyamine toxins.
Stroemgaard, K.; Andersen, K.; Ruhland, T.; Krogs-
gaard-Larsen, P.; Jaroszewski, J. W. Synthesis 2001, 877–
884.
11. Wang, F.; Manku, S.; Hall, D. G. Org. Lett. 2000, 2,
1581–1583.
12. Jo¨nsson, D.; Unde´n, A. Tetrahedron Lett. 2002, 43, 3125–
3128.
Acknowledgements
13. 4-Methoxy dityl chloride (Mmd-Cl): 4-methoxy ben-
zophenone (10 g, 47.1 mmol) was dissolved in EtOH (250
ml). NaBH4 (0.9 g, 23.6 mmol, 0.5 equiv.) was added
slowly and the reduction was left to stir overnight. TLC
(petroleum ether/EtOAc, 9/1) indicated completeness of
the reduction and the mixture was poured onto water
(250 ml), stirred for 1 h and 4-methoxy benzhydrol could
be filtered off and dried. 4-Methoxy benzhydrol (5 g, 23.3
mmol) was dissolved in dried DCM (50 ml), oxalyl
chloride (2.25 ml, 25.7 mmol, 1.1 equiv.) was added
dropwise and the reaction was left stirring for 2 h at
room temperature. The solvent was evaporated and 4-
methoxydityl chloride was crystallized and recrystallized
from warm petroleum ether. Yield: 5.4 g, 99%.
14. 4,4%-Dimethoxy dityl chloride (Dod-Cl): 4,4%-dimethoxy
benzhydrol (6.0 g, 24.6 mmol) was dissolved in dried
DCM (60 ml), oxalyl chloride (2.36 ml, 27 mmol, 1.1
equiv.) was added dropwise and the reaction was left with
stirring for 1.5 h at room temperature. The solvent was
evaporated and the 4,4%-dimethoxydityl chloride was crys-
tallized and recrystallized from warm petroleum ether.
Yield: 5.8 g, 90%.
I wish to thank Assoc. Professor Anders Unde´n for
valuable scientific discussion and for his financial sup-
port with grants from the Magnus Bergwall Founda-
tion to A.U.
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