Solid-phase synthesis of rigid acylpolyamines using temporary N-4,4′-dimethoxytrityl protection in the presence of trityl linkers

Article abstract of DOI:10.1021/jo049278q

An N-protection protocol employing the 4,4′-dimethoxytrityl (Dmt) group in combination with borane reduction of resin-bound polyamides was shown to be an efficient methodology that enables synthesis of novel analogues of natural acylpolyamine toxins. Thus, three philanthotoxins containing polyamine chains with piperidyl and cyclohexyl structural elements, which introduce conformational rigidity, increased lipophilicity, and altered proteolytic properties, were obtained in 39-44% overall yield.

Full text of DOI:10.1021/jo049278q

Solid -P h a se Syn th esis of Rigid
Acylp olya m in es Usin g Tem p or a r y
N-4,4′-Dim eth oxytr ityl P r otection in th e
P r esen ce of Tr ityl Lin k er s
Christian A. Olsen,† Matthias Witt,‡
J erzy W. J aroszewski,† and Henrik Franzyk*,†
Department of Medicinal Chemistry, The Danish University
of Pharmaceutical Sciences, Universitetsparken 2,
DK-2100 Copenhagen, Denmark, and Bruker Daltonik
GmbH, Fahrenheitstrasse 4, D-28359 Bremen, Germany
hf@dfuni.dk
Received April 29, 2004
Abstr a ct: An N-protection protocol employing the 4,4′-
dimethoxytrityl (Dmt) group in combination with borane
reduction of resin-bound polyamides was shown to be an
efficient methodology that enables synthesis of novel ana-
logues of natural acylpolyamine toxins. Thus, three philan-
thotoxins containing polyamine chains with piperidyl and
cyclohexyl structural elements, which introduce conforma-
tional rigidity, increased lipophilicity, and altered proteolytic
properties, were obtained in 39-44% overall yield.
F IGURE 1. Structures of philanthotoxins1-4.
phase synthesis6,7 (SPS) of acylpolyamine toxins has
attracted considerable interest, and several structure-
activity relationship (SAR) studies have been performed
with philanthotoxins on various receptor types.6,7
Simple natural polyamines, such as spermidine and
spermine, are ubiquitous in eukaryotic cells, and they
play important roles in cellular physiology.1 Polyamine
derivatives have been the subject of extensive investiga-
tions due to their ability to interact with ion channels in
the central and in the peripheral nervous system.2-4
Among the naturally occurring polyamine derivatives,
acylpolyamine toxins comprise a class of compounds that
antagonize various types of ionotropic receptors, e.g.,
nicotinic acetylcholine receptors (nAChR) and ionotropic
glutamate receptors (iGluR).5-7 An example of a natural
neuroactive acylpolyamine is philanthotoxin-433 (PhTX-
433, 1, Figure 1) from the venom of the female Egyptian
digger wasp Philanthus triangulum.8,9 Due to the thera-
peutic potential of such polyamine-containing com-
pounds,10-12 solution-phase synthesis13,14 as well as solid-
In particular, the development of sequential SPS
methodologies has facilitated the synthesis of polyamine
moieties, whereas the attachment of amino acid or acyl
residues is readily achieved by conventional solid-phase
peptide synthesis (SPPS) procedures. The synthetic
strategies previously used for SPS of polyamines may be
classified into three major groups: (i) N-alkylation
reactions,15-18 (ii) Mitsunobu reactions,19-21 and (iii)
reductive methods based on intermediary imines22,23 or
amides.24
In the present paper, we report on the development of
a novel N-protection strategy, which in combination with
* To whom correspondence should be addressed. Phone: +45-
35306255. Fax: +45-35306040.
(11) Blagbrough, I. S.; Carrington, S.; Geall, A. J . Pharm. Sci. 1997,
3, 223-233.
† The Danish University of Pharmaceutical Sciences.
‡ Bruker Daltonik GmbH.
(12) Casero, R. A.; Woster, P. M. J . Med. Chem. 2001, 44, 1-26.
(13) Karigiannis, G.; Papaioannou, D. Eur. J . Org. Chem. 2000,
1841-1863.
(1) Cohen, S. S. A Guide to Polyamines; Oxford University Press:
New York, 1998.
(14) Kuksa, V.; Buchan, R.; Lin, P. K. T. Synthesis 2000, 1189-
1207.
(15) Manov, N.; Bienz, S. Tetrahedron 2001, 57, 7893-7898.
(16) Manov, N.; Tzouros, M.; Chesnov, S.; Bigler, L.; Bienz, S. Helv.
Chim. Acta 2002, 85, 2827-2846.
(17) Kan, T.; Kobayashi, H.; Fukuyama, T. Synlett 2002, 8, 1338-
1340.
(18) Olsen, C. A.; Witt, M.; J aroszewski, J . W.; Franzyk, H. Org.
Lett. 2004, 6, 1935-1938.
(19) Hone, N. D.; Payne, L. J . Tetrahedron Lett. 2000, 41, 6149-
6152.
(20) Strømgaard, K.; Andersen, K.; Ruhland, T.; Krogsgaard-Larsen,
P.; J aroszewski, J . W. Synthesis 2001, 877-884.
(21) Olsen, C. A.; Witt, M.; J aroszewski, J . W.; Franzyk, H. Synlett
2004, 473-476.
(2) Scott, R. H.; Sutton, K. G.; Dolphin, A. C. Trends Neurosci. 1993,
16, 153-160.
(3) Williams, K. Biochem. J . 1997, 325, 289-297.
(4) Igarashi, K.; Kashiwagi, K. Biochem. Biophys. Res. Commun.
2000, 271, 559-564.
(5) Mueller, A. L.; Roeloffs, R.; J ackson, H. In The Alkaloids; Cordell,
G. A., Ed.; Academic Press: New York, 1995; Vol. 46, p 63-94.
(6) Strømgaard, K.; Andersen, K.; Krogsgaard-Larsen, P.; J arosze-
wski, J . W. Mini Rev. Med. Chem. 2001, 1, 317-338.
(7) Strømgaard, K.; Mellor, I. R. Med. Res. Rev. 2004, 24, 589-620.
(8) Eldefrawi, A. T.; Eldefrawi, M. E.; Konno, K.; Mansour, N. A.;
Nakanishi, K.; Eugene, O.; Usherwood, P. N. R. Proc. Natl. Acad. Sci.
U.S.A. 1988, 85, 4910-4913.
(9) Piek, T.; Fokkens, R. H.; Karst, H.; Kruk, C.; Lind, A.; van Marle,
J .; Tong, Y. C. In Neurotox 8: Molecular Basis of Drug and Pesticide
Action; Lunt, G. G., Ed.; Elsevier: Amsterdam, 1988; pp 61-76.
(10) Green, A. C.; Nakanishi, K.; Usherwood, P. N. R. Brain Res.
1996, 717, 135-146.
(22) Chhabra, S. R.; Khan, A. N.; Bycroft, B. W. Tetrahedron Lett.
2000, 41, 1095-1098.
(23) J o¨nsson, D. Tetrahedron Lett. 2002, 43, 4793-4796.
(24) Wang, F.; Manku, S.; Hall, D. G. Org. Lett. 2000, 2, 1581-1583.
10.1021/jo049278q CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/12/2004
J . Org. Chem. 2004, 69, 6149-6152
6149

TABLE 1. Sta bility of Tr t a n d Dm t Gr ou p s tow a r d
Selected Dilu te Acid s in Dich lor om eth a n ea
borane reduction of polyamides supported on trityl resins
enabled efficient SPS of three novel philanthotoxins 2-4
(Figure 1). This method allows SPS of compounds with
a high degree of structural diversity of the polyamine
chain, as the incorporation of piperidyl and cyclohexyl
building blocks as well as acyclic aliphatic fragments was
feasible.
Recently, two reports on solution-phase synthesis of
monoacylated polyamines containing ring structures in
the polyamine chain have been published, and incorpora-
tion of piperidyl and cyclohexyl moieties resulted indeed
in interesting biological activities.25,26 Another SPS pro-
tocol has been applied for the synthesis of philanthotoxins
with similar structures; however, this approach is re-
stricted to the synthesis of compounds with tertiary inner
amino functionalities.27
A novel protecting group strategy was necessary in
order to transform a resin-bound polyamide into the
corresponding resin-bound polyamine having only the
terminal secondary amine unprotected. Wang et al.24
have employed a post-reductive bis-N-[1-(4,4-dimethyl-
2,6-dioxocyclohexylidene)ethyl] (Dde)28 protection of the
terminal primary amino group, followed by Boc protection
of the inner secondary amino groups, and final selective
liberation of the terminal primary amino group.24 For the
present purpose of SPS of polyamine derivatives having
secondary internal and/or terminal amino groups in the
resin-bound polyamine intermediate, this strategy is not
applicable.
Trt
Dmt
acid (0.1 M) 10 min 30 min 20 h 10 min 30 min 20 h
TFA
DFA
ClAcOH
AcOH
PPTS
-
(
+
+
+
-
-
+
+
+
-
-
(
+
+
-
-
(
+
+
-
-
-
(
+
-
-
-
(
(
a
Conditions: N1-trityl-1,4-butanediamine and N1-(4,4′-dimeth-
oxytrityl)-N4-trityl-1,4-butanediamine were each agitated with 0.1
M acid in CH2Cl2, and TLC was performed after 10 min, 30 min,
and 20 h. DFA ) difluoroacetic acid, ClAcOH ) chloroacetic acid,
AcOH ) acetic acid, and PPTS ) pyridinium p-toluenesulfonate;
+ denotes full stability (no cleavage), ( denotes partial stability,
and - denotes full cleavage.
SCHEME 1. Bu ild in g Block Syn th esisa
A previous analytical study has indicated that the
4-monomethoxytrityl (Mmt) and Trt groups might be
useful as a protecting scheme during SPPS using acetic
acid/trifluoroethanol/dichloromethane 1:2:7 for selective
deprotection.29 Considering this observation, it was en-
visioned that introduction of a Dmt group prior to borane
reduction, followed by post-reductive Boc protection of
the resulting secondary amines, and a final selective Dmt
removal, would constitute a simple solution to the
synthetic aim addressed. To implement this strategy, a
mild and selective method for deprotection of the N-Dmt
group on a polyamine attached to a solid support via trityl
or 2-chlorotrityl linkers was developed. Qualitative TLC
investigations in solution indicated that the desired
selectivity could be obtained by using 0.1 M chloroacetic
acid in dichloromethane (Table 1). Further studies showed
that Boc groups were stable under these conditions. To
address the possibility of premature cleavage from the
resin during the chloroacetic acid treatment, a resin-
bound diamine was acylated and treated with 0.1 M
chloroacetic acid in dichloromethane. NMR analysis of
the drained solvent pooled with MeOH washings showed
no trace of amine.30 These findings were utilized in the
synthetic sequences leading to compounds 2-4.
a
Reagents and conditions: (a) Fmoc-Cl, dioxane, aqueous
Na2CO3, 16 h (91%); (b) TMS-Cl, DMF, N2, 60 °C, 1 h; (c) Et3N,
2-chlorotrityl chloride resin, N2, 16 h; (d) ethyl trifluoroacetate,
-60 f 0 °C, MeOH, N2, then 2-(trimethylsilyl)ethyl p-nitrophenyl
carbonate, CH2Cl2, then 2 M aqueous NaOH (48% overall); (e)
2-(trimethylsilyl)ethyl p-nitrophenyl carbonate, MeOH/CH2Cl2
(1:1), 16 h (93%).
The intermediate resin-bound polyamides for synthesis
of structures 2-4 were obtained from building blocks and
resins 5-8 (Scheme 1), along with a commercially avail-
able trityl resin preloaded with dicyclohexylmethane-4,4′-
diamine (9). N-(9-Fluorenylmethoxycarbonyl) (Fmoc) pro-
tected isonipecotic acid (5) was obtained as previously
described.31 A 2-chlorotrityl chloride resin was loaded
with O-trimethylsilyl (TMS)-protected isonipecotic acid
to give resin 6 upon desilylation, as described by
Barlos.32 For the mono-N-protection of the two diamines,
the 2-(trimethylsilyl)ethoxycarbonyl (Teoc)33 group was
chosen due to its stability under acylation conditions, and
its conveniently mild removal with tetrabutylammonium
fluoride (TBAF).34 Preparation of compound 7 required
a selective, temporary protection of the primary amine,
for which trifluoroacetylation was employed.27 Subse-
quent treatment with 2-(trimethylsilyl)ethyl p-nitro-
phenyl carbonate and removal of the trifluoroacetyl group
with 3 M aqueous NaOH afforded building block 7 (48%,
(25) Yoneda, Y.; Mimura, T.; Kawagoe, K.; Yasukouchi, T.; Tatematu,
T.; Ito, M.; Saito, M.; Sugimura, M.; Kito, F.; Kawajiri, S. Bioorg. Med.
Chem. 2002, 10, 1347-1359.
(26) Fixon-Owoo, S.; Levasseur, F.; Williams, K.; Sabado, T. N.;
Lowe, M.; Klose, M.; Mercier, A. J .; Fields, P.; Atkinson, J . Phytochem-
istry 2003, 63, 315-334.
(30) Consult the Supporting Information for details.
(31) Combs, A. P.; Kapoor, T. M.; Feng, S.; Chen, J . K.; Daude´-Snow,
L. F.; Schreiber, S. L. J . Am. Chem. Soc. 1996, 118, 287-288.
(32) Barlos, P. Liebigs Ann. Chem. 1988, 1079-1081.
(33) Carpino, L. A.; Tsao, J .-H. J . Chem. Soc., Chem. Commun. 1978,
358-359.
(27) Olsen, C. A.; Witt, M.; J aroszewski, J . W.; Franzyk, H. Org.
Lett. 2003, 5, 4183-4185.
(28) Nash, I. A.; Bycroft, B. W.; Chan, W. C. J . Chem. Soc., Chem.
Commun 1993, 37, 2625-2628.
(29) Matysiak, S.; Bøldicke, T.; Tegge, W.; Frank, R. Tetrahedron
Lett. 1998, 39, 1733-1734.
(34) Koh, J . S.; Ellman, J . A. J . Org. Chem. 1996, 61, 4494-4495.
6150 J . Org. Chem., Vol. 69, No. 18, 2004

SCHEME 2. SP S of P h ila n th otoxin 2a
TABLE 2. Test of Am id e Cou p lin g on Resin 6
Em p loyin g Selected Rea gen ts
entry
reagentsa (equiv)
solvent
time (h) yieldb (%)
1
2
3
4
DIC/HOBt (5:5)
DMF
DMF
DMF
DMF
18
18
18
18
90
32
66
91
HATU/DIPEA (5:10)
TBTU/DIPEA (5:10)
PyBOP/DIPEA (5:10)
a
The crude material was cleaved from the resin using TFA/
b
CDCl3 (1:1) for 1 h. Filtrates and washings with CDCl3 were
combined for each entry and analyzed quantitatively by 1H NMR
using toluene as internal standard. Yields were estimated by
comparison with a sample of resin 6, cleaved under identical
conditions.
Various coupling reagents were examined in order to
identify the optimal conditions for chain elongation by
amide bond formation on resin 6 using primary amines
(Table 2). The yield of 13 using the DIC/HOBt reagent
pair was compared to the yields obtained when employing
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroni-
um hexafluorophosphate (HATU),38 O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate (TB-
TU),39 or benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP)40 in combination with di-
isopropylethylamine (DIPEA). Surprisingly, HATU/DI-
PEA afforded the lowest yield, and TBTU/DIPEA did not
give an acceptable yield of 13 either. These results are
contradictory to previous findings, where uronium salts
such as HATU were reported to give better results than
phosphonium salts and carbodiimide activation.38,39 As
DIC/HOBt and PyBOP/DIPEA appeared to perform
equally well in this particular coupling, these reagents
were employed in the total syntheses of 3 and 4 (Scheme
3), respectively, to compare the overall yields.
Starting from resin 6, the amide bond formation (DIC
and HOBt) using diamine building block 7 afforded resin
14 (sequence A). Removal of the Teoc group with TBAF,
followed by acylation with 5, and subsequent Fmoc group
removal were performed as in the synthesis of 2. Intro-
duction of the terminal Dmt group was achieved with
Dmt-Cl to give resin 15. Then borane reduction was
performed at 60 °C.
After dissociation of the borane-polyamine complex,
the thus-formed inner secondary amine was Boc pro-
tected, and then the terminal Dmt group was selectively
removed with 0.1 M chloroacetic acid in dichloromethane
to give resin 16. Attachment of the amino acid (Tyr)
residue and the butanoyl group by Pfp ester couplings
afforded resin 17. Finally, simultaneous deprotection and
cleavage from the linker with TFA/CH2Cl2 (1:1) afforded
crude 3, which was purified by reversed-phase VLC to
give its tris(TFA) salt in 43% overall yield.
a
Reagents and conditions: (a) 5, DIC, HOBt, DMF, N2, 3 h; (b)
20% piperidine in DMF, 2 × 10 min; (c) Dmt-Cl, DIPEA, CH2Cl2,
N2, 1.5 h; (d) BH3‚THF, 60 °C, 16 h; (e) piperidine, 60 °C, 3 × 2 h;
(f) Boc2O, DIPEA, CH2Cl2, N2, 16 h; (g) chloroacetic acid (0.1 M in
CH2Cl2), 3 × 30 min; (h) (S)-Fmoc-Tyr(tBu)-OPfp, HODhbt,
DIPEA, DMF, N2, 5 h; (i) conditions as in (b); (j) C3H7COOPfp,
HODhbt, DIPEA, DMF, N2, 16 h; (k) TFA/CH2Cl2 (1:1), 1 h (39%
overall).
three steps) in a one-pot procedure. 1,4-Butanediamine
was treated with 2-(trimethylsilyl)ethyl p-nitrophenyl
carbonate in MeOH/CH2Cl2 (1:1) to give building block 8
in 93% yield.
Compound 2 was prepared by performing the sequence
shown in Scheme 2 starting from a commercially avail-
able trityl resin preloaded with dicyclohexylmethane-4,4′-
diamine (9). Initially, the resin was elongated with amino
acid building block 5 using diisopropylcarbodiimide (DIC)
and 1-hydroxybenzotriazole (HOBt). The Fmoc group was
removed with piperidine, and introduction of a terminal
Dmt group was achieved with Dmt-Cl to give resin 10.
Borane reduction was performed at 60 °C as previously
described,35-37 and subsequent dissociation of the bo-
rane-polyamine complex was accomplished by treatment
with piperidine at 60 °C, a somewhat milder and faster
treatment as compared to the conditions applied by
Houghten et al. (piperidine, 65 °C, overnight).36 The inner
secondary amine thus formed was Boc protected, and
then the terminal Dmt group was selectively removed
with 0.1 M chloroacetic acid in dichloromethane to give
resin 11. Subsequent attachment of the amino acid (Tyr)
and butanoyl residues was readily performed by pen-
tafluorophenyl (Pfp) ester couplings27 to give resin 12.
Finally, simultaneous deprotection and cleavage from the
resin were achieved with trifluoroacetic acid (TFA/CH2-
Cl2 1:1), and philanthotoxin 2 was isolated by reversed-
phase vacuum liquid chromatography (VLC) as its bis-
(TFA) salt in 39% overall yield.
In sequence B (Scheme 3), the PyBOP/DIPEA reagent
pair was applied in the first step of the total synthesis of
(35) Paikoff, S. J .; Wilson, T. E.; Cho, C. Y.; Schultz, P. G.
Tetrahedron Lett. 1997, 37, 5653-5656.
(36) Ostresh, J . M.; Schoner, C. G.; Hamashin, V. T.; Nefvi, A.;
Meyer, J .-P.; Houghten, R. A. J . Org. Chem. 1998, 63, 8622-8623.
(37) Hall, D. G.; Laplante, C.; Manku, S.; Nagendran, J . J . Org.
Chem. 1999, 64, 698-699.
(38) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J .
Chem. Soc., Chem. Commun. 1994, 201-203.
(39) Poulain, R. F.; Tartar, A. L.; Deprez, B. P. Tetrahedron Lett.
2001, 42, 1495-1498.
(40) Coste, J .; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990,
31, 205-208.
J . Org. Chem, Vol. 69, No. 18, 2004 6151

SCHEME 3. SP S of P h ila n th otoxin 3 a n d 4a
a
Reagents and conditions: (sequence A) (a) 7, DIC, HOBt, DMF, N2, 16 h; (c) TBAF, DMF, 50 °C, 30 min; (d) 5, DIC, HOBt, DMF, N2,
3 h; (e) 20% piperidine in DMF, 2 × 10 min; (f) Dmt-Cl, DIPEA, CH2Cl2, N2, 1.5 h; (g) BH3‚THF, 60 °C, 16 h; (h) piperidine, 60 °C, 3 ×
2 h; (i) Boc2O, DIPEA, CH2Cl2, N2, 16 h; (j) chloroacetic acid (0.1 M in CH2Cl2), 3 × 30 min; (k) (S)-Fmoc-Tyr(tBu)-OPfp, HODhbt, DIPEA,
DMF, N2, 5 h; (l) conditions as in (e); (m) C3H7COOPfp, HODhbt, DIPEA, DMF, N2, 16 h; (n) TFA/CH2Cl2 (1:1), 1 h (43% overall); (sequence
B): (b) 8, PyBOP, DIPEA, DMF, N2, 16 h; (c) to (n) as above (44% overall).
4. The remaining steps from resin 18 to compound 4 were
performed as described above, and the purified philan-
thotoxin 4 was obtained in 44% overall yield. As expected,
the yields of philanthotoxins 3 and 4 were in the same
range, in accordance with the coupling reagent test
results (Table 2). Also, the overall yields were in the same
range as those reported by Wang et al. using the Dde
protection strategy.24
time. Chloroacetic acid in dichloromethane (0.1 M) al-
lowed a selective liberation of Dmt-protected amino
groups of polyamines anchored via an acid-labile trityl
or 2-chlorotrityl linker. Furthermore, Boc groups were
shown to be compatible with these deprotection condi-
tions. Accordingly, this methodology in combination with
borane reduction of resin-bound polyamides constitutes
a general SPS approach to acylpolyamine toxins. More-
over, the presented synthetic methodology described in
this work extends the structural diversity of philantho-
toxins with unique types of polyamines that enable
further exploration of structure-activity relationships on
ionotropic receptors.
As described previously for example by J acobson et
al.,41 tertiary amides such as 2-4 give rise to quite
complex NMR spectra due to rotational isomerism with
a relatively high energy barrier. Hence, NMR spectro-
scopic characterization of the products required a careful
analysis of the 2D homo- and heteronuclear correlation
spectra (COSY, HSQC, and HMBC). Pronounced splitting
was observed for the signals of 1H and 13C nuclei in close
proximity of the tertiary amide bond, as well as for the
Ack n ow led gm en t. We thank the Danish Technical
Research Council (Grant No. 26-00-0312) and the Carls-
berg Foundation (Grant No. ANS-0965/30) for financial
support. We also thank Ms. Uraiwan Ngamrabiab
Adamsen for technical assistance, Ms. Birgitte Simon-
sen for performing HPLC, and Dr. Dan Stærk for
recording the 2D NMR spectra.
1
butanoyl resonances. The assignments of H NMR and
13C NMR resonances are given in detail in the Supporting
Information.
In summary, the use of Dmt as a temporary N-
protecting group in preparative SPS was demonstrated
on polystyrene trityl and 2-chlorotrityl resins for the first
Su p p or tin g In for m a tion Ava ila ble: General procedures,
detailed experimental procedures (5-8), and full characteriza-
tion of compounds 2-5, 7, and 8. This material is available
free of charge via the Internet at http://pubs.acs.org.
(41) J acobson, A. R.; Gintzler, A. R.; Sayre, L. M. J . Med. Chem.
1989, 32, 1708-1717.
J O049278Q
6152 J . Org. Chem., Vol. 69, No. 18, 2004