General Approach for the Liquid-Phase Fragment Synthesis of Orthogonally Protected Naturally Occurring Polyamines and Applications Thereof

Article abstract of DOI:10.1021/acs.joc.9b02066

Orthogonally protected polyamines (PAs) have been synthesized using α,ω-diamines and ω-aminoalcohols as N-C_x-N and N-C_y synthons, respectively, and the Mitsunobu reaction as the key reaction for the assembly of the PA skeleta. The Trt, Dde, and Phth groups have been employed for protecting the primary amino functions and the Ns group for activating the primary amino functions toward alkylation and secondary amino function protection. The approach has been readily extended to accommodate the total synthesis of the spider toxins Agel 416 and HO-416b, incorporating the 3-4-3-3 and the 3-3-3-4 PA skeleton, respectively.

Full text of DOI:10.1021/acs.joc.9b02066

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Article
A general approach for the liquid phase fragment synthesis of orthogonally
protected naturally occurring polyamines and applications thereof
Stefania Kalantzi, Constantinos M Athanassopoulos, Raili
Ruonala, Yrjo Helariutta, and Dionissios Papaioannou
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b02066 • Publication Date (Web): 28 Oct 2019
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A general approach for the liquid phase fragment synthesis of
orthogonally protected naturally occurring polyamines and
applications thereof
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Stefania Kalantzi1, Constantinos M. Athanassopoulos1, Raili Ruonala2,3,
Yrjo Helariutta2,3, Dionissios Papaioannou1,*
1Laboratory of Synthetic Organic Chemistry, Department of Chemistry, University
of Patras, GR-26504 Patras, Greece
2Sainsbury Laboratory, University of Cambridge, Bateman Street, Cambridge CB2
1LR, UK
3Institute of Biotechnology, HiLIFE/Organismal and Evolutionary Biology Research
Programme, Faculty of Biological and Environmental Sciences, Viikki Plant Science
Centre, FI-00014 University of Helsinki, Helsinki, Finland
*Email: dapapaio@upatras.gr, Tel.: +30-2610-962954; Fax: +30-2610-962956
ABSTRACT
Orthogonally protected polyamines (PAs) have been synthesized using α,ω-diamines
and ω-aminoalcohols as N-Cx-N and N-Cy synthons, respectively, and the
Mitsunobou reaction as the key reaction for the assembly of the PA skeleta. The Trt,
Dde and Phth groups have been employed for protecting the primary amino functions
and the Ns group for activating the primary amino functions towards alkylation and
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secondary amino function protection. The approach has been readily extended to
accommodate the total synthesis of the spider toxins Agel 416 and HO-416b
incorporating the 3-4-3-3 and the 3-3-3-4 PA skeleton, respectively.
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Keywords: polyamines, orthogonal protection, fragment synthesis, conjugates,
spider toxins
INTRODUCTION
Polymethylene polyamines (PAs), such as putrescine (Put), spermidine (Spd)
and spermine (Spm), are naturally occurring small molecules found in all leaving
organisms, either in their free polycationic form or conjugated to other biomolecules,
and show interesting biological activities.1-5 Less common PAs, such as the nor (Nsd
and Nsm) and homo (Hsd and Hsm) analogs of Spd (a 3-4 PA) and Spm (a 3-4-3 PA)
as well isomeric PAs, such as thermospermine (Tsm, a 3-3-4 PA) (Figure 1), are
usually found in plant cells,6-8 although the latter was first isolated from the extreme
thermophile Thermus thermophilus.9 Due to their biological significance and the fact
that PAs could form the basis for the development of pharmaceuticals,10,11 there is a
constant interest in the synthesis of PA analogs and conjugates. Interestingly, a
variety of conjugates of PAs with other biomolecules (herein abbreviated as PACs)
with notable biological activities, e.g. PACs from spiders and wasps, bear the other
biomolecule on one of the terminal amino functions of the PA skeleton.2,3 This
imposes interesting synthetic challenges in particular when the PA skeleton is not
symmetrical, as it is the case with the PAs Spd and Tsm.
The synthesis of PA analogs and conjugates has followed two traditional
pathways. The first one involves the selective direct or indirect functionalization of
the desired amino function(s) of a PA skeleton and the second one the assembly of
the PA skeleton from readily available fragments bearing suitable functionalized or
protected amino functionalities.2,3,12-19 We were recently interested in the selective
mono-functionalization of the unsymmetrical Tsm and we therefore decided to
attempt the synthesis of orthogonally protected Tsm using the fragment synthesis
protocol. It should be noted that mono-functionalization of the symmetrical PA Spm
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has been effected by using the N1,N4,N8-Boc3-Spm obtained via the low temperature
selective mono-trifluoroacetylation of Spm.20 Tsm has been first prepared by Ganem
et al from Spd through a five-steps sequence.21 Quite recently, the fragment synthesis
of Tsm as well as Spm and Nsm was reported on a polymer support of the o-
chlorotrityl-type and using the nosyl (Ns) protecting group for amino protection and
activation and the base induced alkylation of the N-nosylated amines by suitable
halides as the key-step for the assembly of the skeleton.22 The Mitsunobu reaction has
been also indicated by the same research group as an alternative for the alkylation of
Ns-activated amines with alcohols23,24 and was used on a solid support for the
synthesis of the spider toxin Agel 416.25
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Based on our long-standing experience in the use of the triphenylmethyl (trityl,
Trt) group for the protection of amino functions of PAs, we reasoned that the Tsm
skeleton incorporating orthogonally protected amino functionalities (7) could be
assembled from readily available N-tritylated building blocks like the N-trityl-γ-
aminobutyric acid (Trt-GABA) or N-tritylputrescine (Trt-Put, 9) as N-C4 and N-C4-
N synthons, respectively, and either 1,3-diaminopropane (Dap) or the corresponding
Dde-protected 3-aminopropanol (10a)25 as N-C3-N or N-C3 synthons, respectively.
The Trt group, as an amino protecting group, offers a number of advantages due to
its high lipophilicity, electron-releasing property, and steric bulk as well as its lability
to mild acids. In particular, the lipophilicity of the Trt group facilitates aqueous work-
up procedures for N-tritylated PAs and derivatives and their subsequent purification
by routine flash column chromatography (FCC).
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Figure 1. Structures of compounds and protecting groups encountered in this work.
On the other hand, the 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde)
group, used to protect selectively primary amino functions, is stable to acids and bases
but is readily removed by a 2% solution of hydrazine in DMF at room temperature.26
Finally, the Ns group was used for N-protection and N-activation and the Mitsunobu
reaction for the assembly of the skeleton. The successful application of this
methodology for the preparation of Tsm derivative 7 lead us to extent it, with some
minor modifications, to the preparation of the orthogonally protected PA derivatives
1-6 depicted in Figure 1. Furthermore, the applicability of the tetra-amine derivatives
4-7 to the synthesis of PACs was exemplified with another syntheses of the spider
toxins Agel 41625 and HO-416b,27 using the derivatives 4 of Spm and 5 of Nsm as
key-intermediates, respectively.
RESULTS AND DISCUSSION
We have reported in 2012 that N-tritylation of GABA, followed by coupling
with Dap and finally LiAlH4-mediated reduction, leads to N8-tritylspermidine (12) in
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52% overall yield.28 Treatment of 12 with NsCl in the presence of diisopropyl
ethylamine (DIPEA) produced the fully protected Spd derivative 13 in 55% yield
which was then alkylated with the Dde-protected 3-aminopropanol (10a), in the
presence of diisopropyl azodicarboxylate (DIAD) and triphenylphosphine (TPP), to
give the orthogonally protected Tsm (3-3-4) derivative 7 in 84% yield (Scheme 1).
Alternatively, compound 7 could be synthesized from Put as follows. Put was
monotritylated according to a published procedure to provide N1-tritylputrescine (Trt-
Put, 9) in 85% yield.29 This compound was subsequently nosylated to give the fully
protected Put derivative 14 in 88% yield. Mitsunobu reaction of compound 14 with
alcohol 10a produced the orthogonally protected Spd derivative 1a. Because this
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compound was co-eluted with the by-product PrO2CNH-NHCO2 Pr (DIADH2), it
was used as such into the next step. Selective removal of the Dde group with 2%
H2NNH2 in DMF, followed by nosylation and a second Mitsunobu reaction with the
same alcohol finally provided the expected Tsm derivative 7 in 66% yield for the last
three steps (Scheme 1). All intermediates and final products were purified by routine
FCC.
We envisaged that intermediate 14 could be also useful for the preparation of
the corresponding orthogonally protected Hsd (4-4) and Hsm (4-4-4) derivatives with
a minor modification. Because the Dde protected 4-aminobutan-1-ol (10b) does not
efficiently condense with N-nosylated functionalities under Mitsunobu reaction
conditions (see below), we used instead the corresponding N-phthalyl protected 4-
aminobutanol (11) as an N-C4 synthon. That way, the expected orthogonally protected
Hsd derivative 3 was obtained in 80% yield. From this intermediate, the Phth group
was selectively removed with H2NNH2 in refluxing EtOH, the primary amino
function thus exposed was nosylated and the resulting new intermediate 15 was
finally condensed with a second molecule of compound 11 to give access to the
orthogonally protected Hsm derivative 6 in 65% yield for the last three steps.
Compound 6 contained DIADH2 in the ratio 1:0.25 (see experimental).
Scheme 1. Synthesis of orthogonally protected Spd (1a), Tsm (7), Hsd (3) and
Hsm (6)α
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αReagents and conditions: (i) (a) Me3SiCl, (b) Et3N, TrtCl, (c) MeOH, CH2Cl2/MeCN
(5:1), 85%; (ii) Dap, PyBrOP, CH2Cl2, 0 ºC, 30 min, 81%; (iii) LiAlH4, THF, 66 ºC,
overnight, 75%; (iv) NsCl, DIPEA or Et3N, CH2Cl2, 0 ºC for 30 min then 25 ºC for 2
h, 55%-90%; (v) TPP, DIAD, THF, 25 ºC, overnight, 77%-84%; (vi) TrtCl, CH2Cl2,
25 ºC, 2 h, 85%; (vii) 2% H2NNH2 in DMF, 25 ºC, 30 min, 87%; (viii) TPP, DIAD,
THF, 25ºC-40ºC, overnight, 87%; (ix) H2NNH2, EtOH, 78 ºC, 2 h, 93%.
On the other hand, N-tritylation of 1,3-diaminopropane (Dap) in the way
described for Trt-Put provided the N1-tritylamino-3-aminopropane (Trt-Dap, 8) in
85% yield. This compound was nosylated and the thus obtained fully protected Dap
derivative 16 was condensed with alcohol 10a under Mitsunobu reaction conditions
to give the orthogonally protected Nsd derivative 2 in 74% overall yield (Scheme 2).
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Dde removal and subsequent nosylation of the resulting primary amino function
provided the dinosylated intermediate 17 in 60% yield. This was then reacted with a
second molecule of alcohol 10a in the presence of DIAD and TPP to provide the
orthogonally protected Nsm derivative 5. Due to the co-elution of derivative 5 with
DIADH2, alkylation of intermediate 17 with bromide 10c was alternatively used.
Nevertheless, derivative 5 containing DIADH2 can be subjected to removal of the
Dde group, as the thus obtained partially deprotected PA derivative can be obtained
free of DIADH2 by routine FCC (see below).
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Scheme 2. Synthesis of orthogonally protected Spd (1b), Spm (4), Nsd (2) and
Nsm (5) α
αReagents and conditions: (i) TrtCl, CH2Cl2, 25 ºC, 2 h, 85%; (ii) NsCl, Et3N, CH2Cl2,
0 ºC for 30 min then 25 ºC for 2 h, 66-84%; (iii) TPP, DIAD, THF, 25 ºC, overnight,
89%; (iv) 2% H2NNH2 in DMF, 25 ºC, 30 min, 72%; (v) TPP, DIAD, THF, 25 ºC-40
ºC, overnight, 82%; (vi) H2NNH2, EtOH, 78 ºC, 2 h, 94%; (vii) K2CO3, DMF, 60 ºC,
2.5 h, 76-88%.
Compound 16 was also considered as a key-intermediate for the step-wise
assembly of the Spm skeleton. However, attempted Mitsunobu reaction of compound
16 with alcohol 10b25 was sluggish and produced the expected Spd derivative in only
25% even after repeated additions of alcohol, DIAD and TPP. Τherefore,we decided
to use the corresponding 4-phthalimidobutan-1-ol (11), which cannot undergo
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intramolecular alkylation, instead of alcohol 10b. Indeed, using compound 11 as the
alcohol component in the Mitsunobu reaction with intermediate 16, we obtained the
expected orthogonally protected Spd derivative 1b in 82% yield (Scheme 2).
Selective Phth-group removal and nosylation of the unmasked primary amino
function produced the dinosylated intermediate 18 in 62% yield. Coupling compound
of 18 with alcohol 10a in the presence of DIAD and TPP produced the orthogonally
protected Spm derivative 4. This compound co-eluted with DIADH2 and the side-
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product iPrO2CNH-N(R)CO2 Pr, where R is the alkyl group of alcohol 10a. However,
selective removal of the Dde group produces N4,N9-dinosyl-N1-tritylspermine which
can be readily purified and actually was used in the synthesis of toxin Agel-416 (see
Scheme 4). Alternatively, the pure orthogonally protected Spm derivative 4 can be
prepared in 88% yield by alkylation of intermediate 18 with bromide 10c.
The applicability of the orthogonally protected PA derivatives 1-7 to the
synthesis of PACs was exemplified with alternative syntheses of spider toxin HO-
416b (a 4-3-3-3 PAC) using the Nsm derivative 5 (a 3-3-3 PA derivative) as key-
intermediate and the spider toxin Agel 416 (a 3-3-4-3 PAC) using the Spm derivative
4 (a 3-4-3 PA derivative) as key-intermediate. These syntheses could be effected by
either a convergent or a linear way. We used the former way in the synthesis of the
HO-416b toxin and the latter in the synthesis of the Agel 416 toxin.
Accordingly, activation of (indol-3-yl)acetic acid (IAA, 20a) with N-
hydroxysuccinimide (HOSu) and N,N’-dicyclohexylcarbodiimide (DCC) and
coupling with 4-aminobutanol provided the N-(4-hydroxybutyl)-2-(1H-indol-3-
yl)acetamide (21a) in 65% yield. On the other hand Dde deprotection of intermediate
5, followed by nosylation, gave the trinosylated Nsm derivative 22 in 76% yield
(Scheme 3). Unfortunately, alcohol 21a could not be coupled directly with compound
22 under Mitsunobu reaction conditions obviously due to the unprotected NH
function of the indole nucleus. We therefore decided to protect the NH functionality
of IAA (20a) with the acid-labile tert-butoxycarbonyl (Boc) group prior to coupling
with 4-aminobutanol. Attempted introduction of the Boc group in IAA (20a) in a
manner similar to the one-pot sequence described by our research group for the
synthesis N-tritylamino acids,30 involving silylation of the carboxyl function, tert-
butoxycarbonylation in the presence of 4-dimethylaminopyridine (DMAP) and
finally methanolysis of the resulting silyl ester, failed to produce the expected Nind-
Boc-IAA (20b).
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Scheme 3. Synthesis of the spider toxin HO-416b. α
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αReagents and conditions: (i) (a) HOSu, DCC (b) 4-aminobutanol, DMF, 25 ºC, 2 h,
65-72%; (ii) (a) PhCOCH2Br, Et3N, DMF, 0 ºC for 30 min then 25 ºC for 2 h, 81%,
(b) Boc2O, Et3N, DMAP, CH2Cl2, 25 ºC, 3 h, 91%, (c) PhSH, Na2CO3, DMF, 60 ºC,
2 h, 84%; (iii) (a) 2% H2NNH2 in DMF, 25 ºC, 30 min, 94%, (b) NsCl, Et3N, CH2Cl2,
0 ºC for 30 min then 25 ºC for 1.5 h, 81%; (iv) TPP, DIAD, THF, 40 ºC, overnight;
(v) PhSH, Na2CO3, DMF, 25 ºC, overnight; (vi) 10% TFA in CH2Cl2 and PhSH, 25
ºC, 1 h, 70%.
Literature procedures for the preparation of Nind-Boc-IAA (20b) use a three-
steps sequence, involving protection of the carboxyl function as an isolable methyl25
or allyl31 ester, followed by reaction with Boc2O in the presence of DMAP and finally
carboxyl group deprotection. We used instead the phenacyl group for the protection
of the carboxyl function of IAA (20a). Thus, reaction of IAA (20a) with phenacyl
bromide in the presence of Et3N produced the corresponding phenacyl ester in 81%
yield. Reaction of this ester with Boc2O in the presence of DMAP and Et3N resulted
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in N-protection and the thus obtained, in 91% yield, fully protected IAA was treated
with thiophenol and Na2CO3 to produce Nind-Boc-IAA (20b) in 84% yield. Activation
of the thus obtained acid Nind-Boc-IAA (20b) with HOSu and DCC and coupling with
4-aminobutanol produced the Nind-Boc protected alcohol 21b in 72% yield. This
alcohol was then coupled directly with the trinosylated Nsm derivative 22 under
Mitsunobu reaction conditions to give the expected fully protected crude HO-416b
(23a) which contained triphenylphosphine oxide (TPPO), DIADH2 and a side-
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product PrO2CNH-N(R')CO2 Pr, where R' is the alkyl group of alcohol 21b.
Treatment of crude intermediate 23a with PhSH and Na2CO3 in DMF resulted in the
deprotection of the three secondary amino functions to give pure intermediate 23b in
57% yield for the last two steps. From this compound, toxin HO-416b was readily
obtained, as the corresponding penta-trifluoroacetate salt, in 70% yield and 97%
purity according to HPLC analysis, upon deprotection with 10% trifluoroacetic acid
(TFA) in CH2Cl2 in the presence of the cation scavenger PhSH.
For the synthesis of the Agel 416 toxin we decided to explore the viability of
the linear way, which involves first the extension of the PA chain of Spm to the
desired length. Thus, the Dde protecting group of Spm derivative 4 was selectively
removed with 2% H2NNH2 in DMF and the resulting free primary amino function
was nosylated to give the trinosylated Spm derivative 25 in 80%. This intermediate
was then directly coupled with alcohol 10a under Mitsunobu reaction conditions to
produce the fully protected pentaamine 26a which co-eluted with TPPO. The thus
obtained crude 26a was subjected to Dde group removal to give pure N4,N8,N13-
trinosyl-N16-trityl-4,8,13-triazahexadeka-1,16-diamine following routine FCC.
Alternatively, derivative 25 was alkylated with bromide 10c to provide pure
intermediate 26a in 72% yield. From this intermediate, the Dde group was selectively
removed and the resulting free primary amino function was acylated with IAA (20a),
which had been preactivated with HOSu and N,N’-diisopropylcarbodiimide (DIC).
That way, the partially protected Agel 416 (27a) was obtained, containing N,N’-
diisopropylurea (DICU). Crude intermediate 27a was then selectively deprotected at
the secondary amino functions with PhSH and Na2CO3 in DMF to give pure
intermediate 27b in 49% yield for the two steps. Finally, trityl group removal with
10% TFA in CH2Cl2 in the presence PhSH, provided the expected toxin Agel 416, as
the corresponding penta-trifluoroacetate salt, in 73% yield and 93% purity according
to HPLC analysis.
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Scheme 4. Synthesis of the spider toxin Agel 416. α
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αReagents and conditions: (i) 2% H2NNH2 in DMF, 25 ºC, 30 min, 83%-91%; (ii)
NsCl, Et3N, CH2Cl2, 0 ºC for 30 min then 25 ºC for 1.5 h, 87%; (iii) TPP, DIAD,
THF, 25 ºC, overnight; (iv) K2CO3, DMF, 60 ºC, 2.5 h, 72%; (v) HOSu, DIC, DMF;
(vi) PhSH, Na2CO3, DMF, 25 ºC, overnight; (vii) 10% TFA in CH2Cl2 and PhSH, 25
ºC, 1 h, 73%.
CONCLUSIONS
Τhe mono-N-tritylated putrescine and propane-1,3-diamine were used as N-
C4-N and N-C3-N synthons, respectively and the N-Dde or N-phthaloyl-protected 3-
aminopropan-1-ol (or the corresponding bromide) and 4-aminobutan-1-ol were used
as N-C3 and N-C4 synthons, respectively, in the liquid phase fragment synthesis of
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the naturally occurring PAs spermidine, spermine and thermospermine and their
lower and higher homologs nor- and homo-spermidine and -spermine. Key-figures in
the assembly of PA chains in the required length were the use of the lipophilic trityl
group, which facilitates aqueous work-up and purification by routine flash column
chromatography, for N-protection and the nosyl group for the protection of secondary
amino functions and activation of primary amino functions towards alkylation with
alcohols in the presence of TPP and DIAD (Mitsunobu reaction) or the corresponding
bromides in the presence of K2CO3.
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The resulting orthogonally protected triamines and tetra-amines are well-
suited, for example, for the preparation of unsymmetrically bisalkylated PA analogs
and for the selective monoacylation of PAs, in particular asymmetric ones, leading
to biologically interesting PACs. In addition, they can be extended to any direction
by the selective removal of any of the protecting groups giving rise to higher order
PAs. This extension may also provide access to a series of PA conjugates, such as the
PA-type toxins from spiders and wasps. Indeed, this possibility was exemplified in
the present work with alternative syntheses of the spider toxins Agel 416 and HO-
416b, incorporating a pentaamine skeleton (3-4-3-3 and 3-3-3-4, respectively) and
the lipophilic carboxylic acid IAA (20a). From the two alternative routes examined,
the convergent one involving acylation of the ω-aminoalcohol with the in situ
generated succinimidyl ester of N-Boc-protected IAA, followed by a Mitsunobu
reaction gave purer final product than the linear one, involving first extension of the
PA chain using the Mitsunobu reaction, followed by coupling with the succinimidyl
ester of unprotected IAA. Both syntheses were finalized by routine PhSNa-mediated
removal of the Ns groups, followed by TFA-mediated acidolysis of Boc and/or Trt
groups. The present methodology can be also applied to the synthesis of a variety of
PA analogs and PACs with changes in the aliphatic region of the PA skeleton by
selecting the required α,ω-diaminoalkanes and ω-aminoalcohols, as N-Cn-N and N-
Cn synthons, respectively. We are currently investigating the application of the
present methodology to the synthesis of other PA analogs and conjugates and their
biological evaluation as potential anticancer agents.
EXPERIMENTAL SECTION
General information. Melting points were determined with an Electrothermal
apparatus and are uncorrected. 1H-NMR and 13C-NMR spectra were obtained at 600
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and 150 MHz, respectively, on Bruker AvanceIII HD spectrometer. Chemical shifts
(δ) are reported for CDCl3 solutions in parts per million (ppm) downfield from
tetramethylsilane (TMS), used as internal standard, or for (CD3)2SO solutions.
The HRMS-analysis for oily or foamy samples, using LC-MS grade MeOH as
solvent, was performed using an ESI-LTQ-ORBITRAP XL unit (Thermo Scientific,
Bremen, Germany). The Orbitrap Unit was operated in positive mode, with a spray
voltage of 3.2 kV, while the sheath gas (N2) flow rate and auxiliary gas (N2) flow rate
were adjusted to 12 and 2 arbitrary units, respectively. The capillary voltage and the
tube lens voltage were set to 10 and 110 V, respectively. The scan ranged from m/z
150 up to 2000. Electron-spray ionization (ESI) mass spectra were recorded at 30
eV, on a Waters Micromass ZQ spectrometer using HPLC grade MeOH or MeCN as
solvent, or MeCN/H2O as solvent system.
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Analytical reversed phase high-performance liquid chromatography was
performed on Agilent Technologies, 1260 Affinity, Quaternary Pump VL system
equipped with a photodiode array detector. The purity of the final conjugates was
determined using UV detection (λ=254 nm). The chromatographic method employed
the following: column, LeChrosorb RP18 (25 cm x 4.6 mm, 5 μm); mobile phase I,
0.08% TFA in water; mobile phase II, acetonitrile with 0.08% TFA; flow rate, 1.0
mL/min; elution profile, gradient elution from 5 to 50% II over 30 min. Elemental
analyses for solid compounds were determined on a Carlo Erba EA 1108 CHNS
elemental analyzer.
Flash column chromatography (FCC) was performed on Acros Organics silica
gel 0.035-0.070 mm, 60 Å and TLC on Merck silica gel 60 F254 films (0.2 mm)
precoated on aluminium foil. Spots were visualized with UV light at 254 nm and by
spraying with a ninhydrine solution (0.3 g ninhydrin, 3 mL gl. acetic acid, 97 mL 1-
butanol). The solvent systems used for the development of TLC or FCC are the
following: (A) CHCl3/MeOH (98:2), (B) CHCl3/MeOH (95:5), (C) CHCl3/MeOH
(9:1), (D) CHCl3/MeOH/NH3 (99:1:0.1), (E) CHCl3/MeOH/NH3 (98:2:0.2), (F)
CHCl3/MeOH/NH3 (97:3:0.3), (G) CHCl3/MeOH/NH3 (95:5:0.5), (H)
CHCl3/MeOH/NH3 (9:1:0.1), (I) CHCl3/MeOH/NH3 (8.5:1.5:0.15), (J)
CHCl3/MeOH/NH3
(8:2:0.2),
(K)
CHCl3/MeOH/NH3
(7:3:0.3),
(L)
CHCl3/MeOH/NH3 (6:4:0.4), (M) PhMe/EtOAc (93:7), (N) PhMe/EtOAc (9:1), (O)
PhMe/EtOAc (8:2), (P) PhMe/EtOAc (7:3), (Q) PhMe/EtOAc (6:4), (R)
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PhMe/EtOAc (1:1), (S) EtOAc/PhMe (7:3), (T) EtOAc/PhMe (8:2), (U) Hex/EtOAc
(8:2), (V) Hex/EtOAc (7:3), (W) Hex/EtOAc (1:1).
All solvents were dried and/or purified according to standard procedures prior
to use. Solvents were routinely removed at ca. 40 ºC under reduced pressure on Bücki
Rotavapor RE 111 apparatus. Air-sensitive reagents were handled under inert
atmosphere (Ar). All reagents employed in the present work were purchased from
either Sigma-Aldrich or Alfa Aesar or Merck or Acros Organics or Fluorochem and
were used without further purification. DIAD was used in the context of this work as
a commercially available safer alternative to DEAD.
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For the needs of the present work, the N8-tritylspermidine (12) was prepared
according to a published procedure.28
General procedure for the preparation of N1-tritylated diamines
The N1-tritylputrescine (Τrt-Put, 9) was prepared according to a published
procedure.29 Using identical procedure, the preparation of N1-trityl-1,3-
diaminopropane (Trt-Dap, 8) was realized.
N1-Trityl-1,3-diaminopropane (8). Colorless oil (1.29 g, 85%); Rf (G): 0.32;1H NMR
(600 MHz, CDCl3) δ 7.47 (d, J = 7.2 Hz, 6H), 7.27 (t, J = 7.2 Hz, 6H), 7.18 (t, J =
7.2 Hz, 3H), 2.78 (t, J = 6.6 Hz, 2H), 2.20 (t, J = 6.6 Hz, 2H), 1.63 (quint., J = 6.6
Hz, 2H), 1.46 (br. s, 3H) ppm; 13C{1H} NMR (150 MHz, CDCl3) δ 146.1 (3C), 128.6
(6C), 127.8 (6C), 126.2 (3C), 70.9, 41.4, 40.6, 34.7 ppm; MS (ESI, 30 eV): m/z 317.11
[M+H]+, 243.17 [Trt]+.
General procedure for the protection of ω-aminoalcohols with the Dde
group
In general, the procedure described in the literature25 was employed for the N-
protection of 3-aminopropan-1-ol and 4-aminobutan-1-ol with the Dde group as
follows:
To a stirred solution of Dde-OH (1.36 g, 7.46 mmol) in EtOH (3 mL), the ω-
aminoalkanol (7.46 mmol) was added. The resulting mixture was heated under reflux
for 30 min. Then it was left to attain room temperature and the solvent was evaporated
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under reduced pressure to leave a crystalline residue. Recrystallisation from EtOAc
gave the expected N-protected compound.
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N-1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl-3-aminopropan-1-ol (10a).
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White crystals (1.55 g, 87%); mp 106-108 C; Rf (B): 0.21; 1H NMR (600 MHz,
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CDCl3) δ 13.45 (br. s, 1H), 3.78 (t, J = 6 Hz, 2H), 3.56 (q, J = 6 Hz, 2H), 2.57 (s, 3H),
2.35 (s, 4H), 2.00 (br. s, 1H), 1.92 (quint., J = 6 Hz, 2H), 1.02 (s, 6H) ppm; 13C{1H}
NMR (150 MHz, CDCl3) δ 197.8 (2C), 173.7, 107.9, 59.4, 52.9 (2C), 40.2 (2C), 31.6,
30.1 (2C), 28.3 (2C), 17.9 ppm; MS (ESI, 30 eV): m/z 517.44 [2M+K]+, 262.48
[M+Na]+, 240.52 [M+H]+.
N-1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl-4-aminobutan-1-ol (10b).
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Beige crystals (1.15 g, 91%); mp 105-107 C; Rf (B): 0.35; H NMR (600 MHz,
CHCl3) δ 13.45 (br. s, 1H), 3.70 (t, J = 6 Hz, 2H), 3.45 (q, J = 6 Hz, 2H), 2.56 (s, 3H),
2.35 (s, 4H), 1.79 (quint., J = 6 Hz, 2H), 1.74 (br. s, 1H), 1.68 (quint., J = 6 Hz, 2H)
1.02 (s, 6H) ppm; C{1H} NMR (150 MHz, CDCl3) δ 197.7 (2C), 173.5, 107.9, 62.0,
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52.9 (2C), 43.3, 30.1, 29.8, 28.3 (2C), 25.7, 17.9 ppm; MS (ESI, 30 eV): m/z 529.26
[2M+Na]+, 276.20 [M+Na]+, 254.26 [M+H]+.
Preparation of 1-bromo-3-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-
aminopropane (10c).
Triphenylphosphine (0.82 g, 3.12 mmol) was added portion-wise during a period of
2 h to a well-stirred solution of 10a (0.66 g, 2.78 mmol) and carbon tetrabromide
(1.38 g, 4.17 mmol) in dry CH2Cl2 (1.5 mL) at room temperature. After an additional
1 h of stirring the reaction mixture was applied on the top of a chromatography
column and subjected to FCC, using EtOAc as eluant, to give bromide 10c.
White solid (0.74 g, 88%); mp 63-66 C; Rf (EtOAc): 0.41; 1H NMR (600 MHz,
o
CDCl3) δ 13.56 (br. s, 1H), 3.60 (q, J = 6.6 Hz, 2H), 3.49 (t, J = 6.6 Hz, 2H), 2.59 (s,
3H), 2.37 (s, 4H), 2.21 (quint., J = 6.6 Hz, 2H), 1.03 (s, 6H) ppm; 13C{1Η} NMR
(150 MHz, CDCl3) δ 198.3 (2C), 173.9, 108.1, 52.9 (2C), 41.4, 31.7, 30.1, 29.5, 28.3
(2C), 17.8 ppm; Anal. Calcd (%) for C13H20BrNO2: C, 51.67; H, 6.67; N, 4.63. Found:
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C, 51.87; H, 6.55; N, 4.48; MS (ESI, 30 eV): m/z 326.48 and 324.44 [M+Na]+, 304.49
and 302.45 [M+Η]+.
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Preparation of 4-phthalimidobutan-1-ol (11).
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A solution of 4-aminobutan-1-ol (0.5 mL, 5.5 mmol) in THF (2.5 mL) was added
during a period of 20 min to a stirred slurry of N-(ethoxycarbonyl)phthalimide (1.1 g,
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5 mmol) in THF (2.5 mL) at 0 C. The mixture was further stirred for 2 h at room
temperature. The completion of reaction was confirmed by TLC, using solvent system
R. After removal of the solvent under reduced pressure, the residue diluted with
CH2Cl2 and washed twice with H2O and once with brine. The organic layer was dried
over Na2SO4 and evaporated to dryness to leave a thick oil. Even after purification
with FCC, using solvent system R for elution, the product 11 was found (by 1ΝΜR)
to contain ethyl carbamate in the molar ratio 11:ethyl carbamate = 1.4:1 and used as
such into the following alkylation reactions.
Thick colorless oil (0.81 g, 67%); Rf (R): 0.30; 1H NMR (600 MHz, CDCl3) δ 7.84-
7.83 (m, 2H), 7.72-7.70 (m, 2H), 3.74 (t, J = 7.2 Hz, 2H), 3.69 (t, J = 7.2 Hz, 2H),
1.97 (br. s, 1H), 1.78 (quint., J = 7.2 Hz, 2H), 1.62 (quint., J = 7.2 Hz, 2H) ppm;
13C{1H} NMR (150 MHz, CDCl3) δ 168.5 (2C), 133.9 (2C), 132.2 (2C), 123.2 (2C),
62.2, 37.7, 29.8, 25.1 ppm.
General procedure for the nosylation of N-tritylated polyamines
A typical procedure for the mononosylation of N-tritylated PAs is the following: To
an ice-cold solution of N1-tritylputrescine (9) (1.18 g, 3.50 mmol) in dry CH2Cl2 (14
mL) and Et3N (1.1 mL, 7.4 mmol), 2-nitrobenzenesulfonyl chloride (0.82 g, 3.70
mmol) was added portion-wise over 1.5 h under an inert atmosphere. The mixture
was kept at 0 oC for further 30 min and then allowed to attain room temperature. The
completion of the reaction was confirmed by TLC using the solvent system H. The
reaction mixture was then diluted with CH2Cl2 and washed twice with 5% aqueous
solution of Na2CO3, twice with H2O and once with brine. The organic layer was dried
over Na2SO4 and evaporated to dryness. The product 14 was obtained pure following
FCC purification using solvent system N for elution.
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In the case of the preparation of N1,N4-dinosyl-N8-tritylspermidine (13) from N8-
tritylspermidine (12) were double nosylation was required, 4.4 equiv of DIPEA and
2.2 equiv of NsCl were employed.
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N1,N4-Dinosyl-N8-tritylspermidine (13). Reaction time: 2 h; slightly yellow oil (0.15
g, 55%); Rf (O): 0.28; 1H NMR (600 MHz, CDCl3) δ 8.11-8.09 (m, 1H), 7.98 (dd, J
= 7.8 and 1.2 Hz, 1H), 7.85-7.83 (m, 1H), 7.73-7.69 and 7.69-7.63 (two m, 4H), 7.60
(dd, J = 7.2 and 1.2 Hz, 1H), 7.44-7.41 (m, 6H), 7.29-7.23 (m, 6H), 7.20-7.15 (m,
3H), 5.59 (unresolv. t, 1H), 3.36 (t, J = 6.6 Hz, 2H), 3.24 (t, J = 7.2 Hz, 2H), 3.16 (q,
J = 6.6 Hz, 2H), 2.07 (unresolv. t, 1H), 1.82 (quint., J = 6.6 Hz, 2H), 1.53 (quint., J =
7.2 Hz, 4H), 1.40 (quint., J = 7.2 Hz, 2H) ppm; 13C{1H} NMR (150 MHz, CDCl3) δ
148.1, 148.0, 146.1 (3C), 133.7, 133.6, 133.5, 133.2, 132.8, 131.8, 131.0, 130.9, 128.6
(6C), 127.8 (6C), 126.3 (3C), 125.3, 124.2, 70.8, 47.9, 44.7, 43.1, 40.7, 28.9, 27.8,
26.2 ppm; HRMS (Orbitrap-ESI) m/z calcd for C38H40N5O8S2 [M+ H]+ 758.2318,
found 758.2335.
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N1-Nosyl-N4-tritylputrescine (14). Reaction time: 2 h; slightly yellow solid; (1.59 g,
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88%); mp 149-152 C; Rf (N): 0.36; H NMR (600 MHz, CDCl3) δ 8.14-8.10 (m,
1H), 7.87-7.83 (m, 1H), 7.74-7.70 (m, 2H), 7.44-7.41 (m, 6H), 7.28-7.24 (m, 6H),
7.20-7.16 (m, 3H), 5.29 (t, J = 6.6 Hz, 1H), 3.08 (q, J = 6.6 Hz, 2H), 2.11 (t, J = 6.6
Hz, 2H) 1.61 (quint., J = 6.6 Hz, 2H), 1.55 (br. s, 1H), 1.48 (quint., J = 6.6 Hz, 2H)
13
ppm; C{1H} NMR (150 MHz, CDCl3) δ 148.1, 146.0 (3C), 133.9, 133.5, 132.7,
131.1, 128.6 (6C), 127.8 (6C), 126.3 (3C), 125.4, 70.9, 43.9, 43.0, 27.8, 27.6 ppm;
Anal. Calcd (%) for C29H29N3O4S: C, 67.55; H, 5.67; N, 8.15. Found: C, 67.84; H,
5.38; N, 8.09; MS (ESI, 30 eV): m/z 554.47 [M+K]+, 538.60 [M+Na]+, 516.62
[M+H]+, 243.66 [Trt]+.
N1,N5-Dinosyl-N9-tritylhomospermidine (15). Reaction time: 2 h; yellow oil (0.3 g,
80%); Rf (O): 0.35; 1H NMR (600 MHz, CDCl3) δ 8.12-8.10 (m, 1H), 7.96 (d, J = 7.8
Hz, 1H), 7.85-7.82 (m, 1H), 7.74-7.71 (m, 2H), 7.69-7.62 (m, 2H), 7.59 (d, J = 7.8
Hz, 1H), 7.45-7.41 (m, 6H), 7.29-7.24 (m, 6H), 7.20-7.16 (m, 3H), 5.27 (t, J = 6 Hz,
1H), 3.28 (t, J = 7.2 Hz, 2H), 3.24 (t, J = 6 Hz, 2H), 3.10 (q, J = 6 Hz, 2H), 2.09 (t, J
= 6.9 Hz, 2H), 1.61-1.51 (m, 7H), 1.42 (quint., J = 6.9 Hz, 2H) ppm; 13C{1H} NMR
(150 MHz, CDCl3) δ 148.1, 148.0, 146.1 (3C), 133.63, 133.59, 133.57, 133.5, 132.9,
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131.7, 131.1, 130.7, 128.6 (6C), 127.8 (6C), 126.3 (3C), 125.4, 124.2, 70.8, 47.3,
46.5, 43.12, 43.09, 27.8, 26.6, 26.0, 25.0 ppm; HRMS (Orbitrap-ESI) m/z calcd for
C39H42N5O8S2 [M+H]+ 772.2475, found 772.2439.
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N1-Nosyl-N3-trityl-1,3-diaminopropane (16). Reaction time: 2 h; Slightly yellow
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solid (1.89 g, 83%); mp 185-189 C; Rf (O): 0.29; H NMR (600 MHz, CDCl3) δ
8.15-8.11 (m, 1H), 7.84-7.80 (m, 1H), 7.75-7.69 (m, 2H), 7.44-7.40 (m, 6H), 7.29-
7.24 (m, 6H), 7.21-7.16 (m, 3H), 5.72 (br. s, 1H), 3.26 (t, J = 6.6 Hz, 2H), 2.19 (t, J
= 6.6 Hz, 2H), 1.68 (quint., J = 6.6 Hz, 2H), 1.55 (br. s, 1H) ppm; 13C{1H} NMR (150
MHz, CDCl3) δ 148.1, 145.7 (3C), 133.9, 133.4, 132.6, 131.1, 128.6 (6C), 127.9 (6C),
126.6 (3C), 125.3, 71.1, 42.6, 41.4, 30.6 ppm; Anal. Calcd (%) for C28H27N3O4S: C,
67.05; H, 5.43; N, 8.38. Found: C, 66.85; H, 5.58; N, 8.64; MS (ESI, 30 eV): m/z
540.57 [M+K]+, 524.45 [M+Na]+, 243.59 [Trt]+.
N1,N4-Dinosyl-N7-tritylnorspermidine (17). Reaction time: 2 h; Pale yellow solid
(1.62 g, 84%); mp 166.0-168.4 oC; Rf (N): 0.25; 1H NMR (600 MHz, CDCl3) δ 8.11
(dd, J = 7.2 and 1.7 Hz, 1H), 7.99 (dd, J = 7.8 and 0.9 Hz, 1H), 7.84 (dd, J = 7.2 and
1.7 Hz, 1H), 7.75-7.70 and 7.70-7.63 (two m, 4H), 7.59 (dd, J = 7.2 and 0.7 Hz, 1H),
7.43-7.39 (m, 6H), 7.28-7.24 (m, 6H), 7.20-7.16 (m, 3H), 5.60 (unresolv. t, 1H), 3.39-
3.34 (m, 4H), 3.14 (q, J = 6.6 Hz, 2H), 2.08 (unresolv. t, 2H), 1.81 (quint., J = 6.6 Hz,
2H), 1.66 (unresolv. quint., 2H), 1.55 (br. s, 1H) ppm; C{1H} NMR (150 MHz,
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CDCl3) δ 148.1, 148.0, 145.8 (3C), 133.7, 133.63, 133.60, 133.0, 132.8, 131.7,
130.90, 130.89, 128.6 (6C), 127.9 (6C), 126.4 (3C), 125.5, 124.3, 70.9, 46.2, 44.8,
40.9, 40.6, 29.6, 29.0 ppm; Anal. Calcd for C37H37N5O8S2: C, 59.74; H, 5.01; N, 9.42.
Found: C, 59.93; H, 4.89; N, 9.21; MS (ESI, 30 eV): m/z 782.42 [M+K]+, 766.31
[M+Na]+, 744.60 [M+H]+, 243.54 [Trt]+.
N4,N8-Dinosyl-N1-tritylspermidine (18). Reaction time: 2 h; Pale yellow solid (0.86
g, 66%); mp 178-181 oC; Rf (O): 0.28; 1H NMR (600 MHz, (CD3)2SO) δ 8.08 (t, J =
6.6 Hz, 1H), 7.99-7.96 (m, 1H), 7.96-7.92 (m, 3H), 7.88-7.85 and 7.85-7.82 (two m,
3H), 7.79 (td, J = 7.2 and 0.9 Hz, 1H), 7.36-7.33 (m, 6H), 7.29-7.24 (m, 6H), 7.18-
7.14 (m, 3H), 3.25 (t, J = 7.2 Hz, 2H), 3.15 (t, J = 6.6 Hz, 2H), 2.87 (q, J = 6.6 Hz,
2H), 2.65 (t, J = 7.2 Hz, 1H), 1.91 (q, J = 6.6 Hz, 2H), 1.61 (quint., J = 6.6 Hz, 2H),
1.44 (quint., J = 6.6 Hz, 2H), 1.37 (quint., J = 7.2 Hz, 2H) ppm; 13C{1H} NMR (150
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MHz, (CD3)2SO) δ 148.2, 148.0, 146.5 (3C), 134.8, 134.4, 133.2, 133.1, 132.8, 132.2,
130.1, 129.9, 128.8 (6C), 128.1 (6C), 126.5 (3C), 124.8, 124.7, 70.9, 47.2, 46.1, 42.7,
41.2, 29.2, 26.7, 25.5 ppm; Anal. Calcd for C38H39N5O8S2: C, 60.22; H, 5.19; N, 9.24.
Found: C, 60.35; H, 5.01; N, 9.1; MS (ESI, 30 eV): m/z 796.25 [M+K]+, 780.32
[M+Na]+, 243.52 [Trt]+.
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N1,N4,N8-Trinosyl-N11-tritylnorspermine (22). Reaction time: 1.5 h; Yellow foam
(1.49 g, 81%); Rf (O): 0.17; 1H NMR (600 MHz, CDCl3) δ 8.11-8.07 (m, 1H), 8.01
(unresolv. dd, 1H), 7.97 (dd, J = 7.2 and 0.9 Hz, 1H), 7.86-7.82 (m, 1H), 7.74-7.70
and 7.70-7.64 (two m, 6H), 7.59 (dd, J = 7.8 and 1 Hz, 1H), 7.56 (dd, J = 7.2 and
0.8 Hz, 1H), 7.45-7.39 (m, 6H), 7.29-7.23 (m, 6H), 7.21-7.16 (m, 3H), 5.61 (t, J =
6.6 Hz, 1H), 3.40-3.35 (m, 4H), 3.31-3.24 (m, 4H), 3.14 (q, J = 6.6 Hz, 2H), 2.09
(t, J = 6.6 Hz, 2H), 1.83 (quint., J = 6.6 Hz, 4H), 1.66 (quint., J = 6.6 Hz, 2H), 1.60
(br. s, 1H) ppm; 13C{1H} NMR (150 MHz, CDCl3) δ 148.1, 148.0 (2C), 145.9 (3C),
133.9, 133.7, 133.6, 133.5, 133.0, 132.9, 132.6, 132.1, 131.8, 130.94, 130.92, 130.7,
128.6 (6C), 127.8 (6C), 126.3 (3C), 125.4, 124.3, 124.2, 70.9, 46.0, 45.5, 45.2, 44.9,
40.8, 40.7, 29.3, 28.8, 27.6 ppm; HRMS (Orbitrap-ESI) m/z calcd for
C46H48N7O12S3 [M+H]+ 986.2523, found 986.2530.
N1,N4,N8-Trinosyl-N12-tritylspermine (25). Reaction time: 1.5 h; Yellow foam (0.93
1
g, 87%); Rf (O): 0.18; H NMR (600 MHz, CDCl3) δ 8.11-8.08 (m, 1H), 8.01-7.97
(m, 2H), 7.85-7.81 (m, 1H), 7.74-7.70, 7.70-7.67 and 7.67-7.62 (three m, 6H), 7.60-
7.57 (m, 2H), 7.42-7.38 (m, 6H), 7.28-7.24 (m, 6H), 7.20-7.14 (m, 3H), 5.60 (t, J = 6
Hz, 1H), 3.37-3.31 (m, 4H), 3.30-3.26 (m, 4H), 3.13 (q, J = 6 Hz, 2H), 2.06 (t, J = 6
Hz, 2H), 1.83 (quint., J = 6 Hz, 2H), 1.64 (quint., J = 7.2 Hz, 2H), 1.54-1.50 (m, 5H)
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ppm; C{1H} NMR (150 MHz, CDCl3), δ 148.1, 148.0, 147.9, 145.9 (3C), 133.7,
133.6, 133.55, 133.50, 133.3, 132.9, 132.8, 131.9, 131.7, 1310, 130.83, 130.80, 128.6
(6C), 127.8 (6C), 126.3 (3C), 125.4, 124.3, 124.2, 70.9, 47.3, 46.6, 46.5, 44.8, 40.9,
40.8, 29.3, 28.8, 25.04, 24.99 ppm; HRMS (Orbitrap-ESI) m/z calcd for
C47H50N7O12S3 [M+H]+ 1000.2680, found 1000.2568.
General procedure for the Mitsunobu reaction between nosylated PAs
and N-protected ω-aminoalcohols
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A typical procedure for the Mitsunobu reaction of N-nosylated PAs and N-protected
ω-aminoalcohols is the following:
To an ice-cold solution of Spd derivative 13 (0.76 g, 1 mmol), alcohol 10a (0.31 g,
1.30 mmol) and TPP (0.34 g, 1.30 mmol) in dry THF (2.9 mL), DIAD (0.26 mL, 1.30
mmol) was added under an inert atmosphere. The reaction mixture was allowed to
attain room temperature where it was stirred for additional 2 h. The progress of the
reaction was monitored by TLC using the solvent system P. Then, additional TPP
(118 mg, 0.45 mmol) and DIAD (118 mg, 0.45 mmol) were added. The reaction
mixture was further stirred for 2 h at room temperature and the completion of the
reaction was confirmed by TLC. The solvent was evaporated and the product 7 was
obtained pure following FCC purification using EtOAc as eluant.
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The same procedure was used for the synthesis of all compounds tabulated below
using the indicated for each case (a) different ratios of PA derivatives, alcohols, TPP
and DIAD, (b) reaction time and (c) reaction temperature.
N1-Dde-N4-nosyl-N8-tritylspermidine (1a). Quantities of reagents: 14 (1.03 g, 2.00
mmol), 10a (0.62 g, 2.60 mmol), TPP (0.90 g, 3.33 mmol), DIAD (0.66 mL, 3.33
o
mmol); Reaction time: overnight; Reaction temperature: 25 C; Yellow oil (1.13 g,
77%); Rf (Q): 0.18; 1H NMR (600 MHz, CDCl3) δ 13.48 (unresolv. t, 1H), 8.00 (d, J
= 7.8 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.62 (t, J =7.8 Hz, 1H), 7.60 (d, J = 7.8 Hz,
1H), 7.45-7.41 (m, 6H), 7.29-7.23 (m, 6H), 7.20-7.14 (m, 3H), 3.43-3.38 (m, 4H),
3.28 (t, J = 7.5 Hz, 2H), 2.50 (s, 3H), 2.34 (s, 4H), 2.08 (t, J = 6.6 Hz, 2H), 1.92
(quint., J = 7.5 Hz, 2H), 1.65 (br. s, 1H), 1.57 (quint., J = 6.6 Hz, 2H), 1.43 (quint., J
= 6.6 Hz, 2H), 1.01 (s, 6H) ppm; 13C{1H} NMR (150 MHz, CDCl3) δ 173.7, 148.0,
146.1 (3C), 133.6, 133.2, 131.7, 131.0, 128.6 (6C), 127.8 (6C), 126.3 (3C), 124.2,
108.0, 70.8, 47.6, 44.6, 43.1, 40.6, 30.1, 28.3 (2C), 27.9, 27.8, 25.9, 17.9 ppm; MS
(ESI, 30 eV): m/z 775.22 [M+K]+, 759.47 [M+Na]+, 737.55 [M+H]+, 243.53 [Trt]+.
N4-Nosyl-N8-phthaloyl-N1-tritylspermidine (1b). Quantities of reagents: 16 (0.90 g,
1.80 mmol), 11 (0.51 g, 2.34 mmol), TPP (1.20 g, 4.50 mmol), DIAD (0.89 mL, 4.50
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mmol); Reaction temperature and time: 25 C, overnight then 40 C, 2 h; Slightly
yellow oil (1.04 g, 82%); Rf (N): 0.20; 1H NMR (600 MHz, CDCl3) δ 8.00-7.97 (m,
1H), 7.83-7.79 (m, 2H), 7.73-7.69 (m, 2H), 7.65-7.60 (m, 2H), 7.56-7.54 (m, 1H),
7.46-7.40 (m, 6H), 7.30-7.25 (m, 6H), 7.21-7.16 (m, 3H), 3.68 (t, J = 6.6 Hz, 2H),
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3.40 (t, J = 7.8 Hz, 2H), 3.33 (t, J = 7.8 Hz, 2H), 2.11 (t, J = 6.6 Hz, 2H), 1.74-1.65
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(m, 4H), 1.60-1.55 (m, 3H) ppm; C{1H} NMR (150 MHz, CDCl3) δ 168.3 (2C),
148.0, 145.9 (3C), 134.0 (2C), 133.6, 133.3, 132.1 (2C), 131.5, 130.8, 128.6 (6C),
127.8 (6C), 126.3 (3C), 124.1, 123.3 (2C), 70.9, 46.8, 45.8, 40.9, 37.2, 29.6, 25.7,
25.5 ppm; HRMS (Orbitrap-ESI) m/z calcd for C40H39N4O6S [M+ H]+ 703.2590,
found 703.2612.
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N1-Dde-N4-nosyl-N7-tritylnorspermidine (2). Quantities of reagents: 16 (1.30 g, 2.60
mmol), 10a (0.81 g, 3.38 mmol), TPP (1.13 g, 4.18 mmol), DIAD (0.82 mL, 4.18
o
mmol); Reaction time: overnight; Reaction temperature: 25 C; Yellow oil (1.67 g,
89%); Rf (Q): 0.25; 1H NMR (600 MHz, CDCl3) δ 13.50 (unresolv. t, 1H), 8.01 (dd,
J = 7.2 and 0.6 Hz, 1H), 7.67 (td, J = 7.2 and 0.6 Hz, 1H), 7.63 (td, J = 7.8 and 0.6
Hz, 1H), 7.59 (dd, J = 7.8 and 0.6 Hz, 1H), 7.46-7.37 (m, 6H), 7.29-7.23 (m, 6H),
7.20-7.14 (m, 3H), 3.44-3.37 (m, 6H), 3.52 (s, 3H), 3.31 (s, 4H), 2.11 (t, J = 6.6 Hz,
2H), 1.93 (quint., J = 7.2 Hz, 2H), 1.72 (quint., J = 6.6 Hz, 2H), 1.64 (br. s, 1H), 1.00
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(s, 6H) ppm; C{1H} NMR (150 MHz, CDCl3) δ 199.1 (2C), 173.8, 148.0, 145.9
(3C), 133.6, 133.0, 131.7, 131.0, 128.6 (6C), 127.8 (6C), 126.3 (3C), 124.2, 108.0,
70.9, 53.6, 52.2, 46.1, 44.8, 40.9, 40.6, 30.1, 29.4, 28.2 (2C), 28.0, 17.9 ppm; HRMS
(Orbitrap-ESI) m/z calcd for C41H47N4O6S [M+H]+ 723.3216, found 723.3201.
N5-Nosyl-N1-phthaloyl-N9-tritylhomospermidine (3). Quantities of reagents: 14 (263
mg, 0.51 mmol), 11 (145 mg, 0.66 mmol), TPP (345 mg, 1.28 mmol), DIAD (0.25
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mL, 1.28 mmol); Reaction temperature and time: 25 C, overnight then 40 C, 2 h;
Slightly yellow oil (0.29 g, 80%); Rf (N): 0.20; 1H NMR (600 MHz, CDCl3) δ 7.99-
7.95 (m, 1H), 7.83-7.80 (m, 2H), 7.72-7.67 (m, 2H), 7.60-7.57 (m, 2H), 7.56-7.52 (m,
1H), 7.46-7.41 (m, 6H), 7.29-7.23 (m, 6H), 7.20-7.14 (m, 3H), 3.64 (t, J = 7.2 Hz,
2H), 3.31 (t, J = 7.2 Hz, 2H), 3.26 (t, J = 7.2 Hz, 2H), 2.71 (t, J = 6.6 Hz, 2H), 1.64
(quint., J = 7.2 Hz, 2H), 1.59-1.51 (m, 5H), 1.42 (quint., J = 6.6 Hz, 2H) ppm; 13C{1H}
NMR (150 MHz, CDCl3) δ 168.3 (2C), 148.0, 146.2 (3C), 134.0 (2C), 133.8, 133.3,
132.1 (2C), 131.5, 130.8, 128.6 (6C), 127.8 (6C), 126.2 (3C), 124.1, 123.3 (2C), 70.8,
42.5, 46.7, 43.1, 37.2, 27.9, 26.1, 25.7, 25.4 ppm; HRMS (Orbitrap-ESI) m/z calcd
for C41H41N4O6S [M+ H]+ 717.2747, found 717.2722.
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N1-Dde-N4,N8-dinosyl-N12-tritylspermine (4). Quantities of reagents: 18 (811 mg,
1.07 mmol), 10a (333 mg, 1.39 mmol), TPP (468 mg, 1.74 mmol), DIAD (0.34 mL,
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1.74 mmol); Reaction time: overnight; Reaction temperature: 25 C; Yellow oil; Rf
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(R): 0.17; MS (ESI, 30 eV): m/z 1001.60 [M+Na]+, 979.01 [M+H]+, 243.57 [Trt]+.
The NMR and HRMS spectra of compound 4 are provided below.
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N1-Dde- N4,N8-dinosyl-N11-tritylnorspermine (5). Quantities of reagents: 17 (185 mg,
0.25 mmol), 10a (77 mg, 0.32 mmol), TPP (172 mg, 0.64 mmol), DIAD (0.13 mL,
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0.64 mmol); Reaction time: overnight; Reaction temperature: 25 C; Yellow oil; Rf
(Q): 0.15; MS (ESI, 30 eV): m/z 1003.51 [M+K]+, 965.22 [M+H]+.
The NMR and HRMS spectra of compound 5 are provided below.
N5,N10-Dinosyl-N1-phthaloyl-N14-tritylhomospermine (6). Quantities of reagents: 15
(295 mg, 0.38 mmol), 11 (107 mg, 0.49 mmol), TPP (226 mg, 0.84 mmol), DIAD
1
(0.16 mL, 0.84 mmol). Compound 6 contained DIADH2 in the ratio 1:0.25 (by H-
NMR); Reaction temperature and time: 25 oC, 2 h then 40 oC, 1.5 h and finally 25 oC,
1
overnight; Yellow oil (0.32 g, 87%); Rf (O): 0.28; H NMR (600 MHz, CDCl3) δ
7.99-7.94 (m, 2H), 7.85-7.81 (m, 2H), 7.72-7.69 (m, 2H), 7.68-7.58 (m, 5H), 7.54-
7.51 (m, 1H), 7.46-7.41 (m, 6H), 7.29-7.23 (m, 6H), 7.20-7.14 (m, 3H), 3.64 (t, J =
6.6 Hz, 2H), 3.30-3.25 (m, 6H), 3.23 (t, J = 7.2 Hz, 2H), 2.08 (t, J = 6.6 Hz, 2H), 1.62
(quint., J = 7.2 Hz, 2H), 1.58-1.45 (m, 9H), 1.41 (quint., J = 6.6 Hz, 2H) ppm; 13C{1H}
NMR (150 MHz, CDCl3) δ 168.3 (2C), 148.0 (2C), 146.1 (3C), 134.0 (2C), 133.7,
133.5, 133.4, 133.3, 132.1 (2C), 131.7, 131.6, 130.7, 130.6, 128.6 (6C), 127.8 (6C),
126.3 (3C), 124.14, 124.10, 123.2 (2C), 70.8, 47.2, 46.8, 46.7, 46.5, 43.2, 37.2, 27.9,
26.0, 25.7, 25.4, 25.1, 25.0 ppm; MS (ESI, 30 eV): m/z 995.68 [M+Na]+, 973.80
[M+H]+, 243.74 [Trt]+.
N1-Dde- N4,N8-dinosyl-N12-tritylthermospermine (7). Quantities of reagents: 13 (0.76
g, 1 mmol), 10a (0.31 mg, 1.30 mmol), TPP (0.47 mg, 1.75 mmol), DIAD (0.34 mL,
1.75 mmol); Reaction time: overnight; Reaction temperature: 25 oC; Yellow oil (0.82
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g, 84%); Rf (EtOAc): 0.38; H NMR (600 MHz, CDCl3) δ 13.47 (unresolv. t, 1H),
8.05-8.01 (m, 1H), 7.96 (dd, J = 7.2 and 1.2 Hz, 1H), 7.69-7.61 (m, 4H), 7.59 (dd, J
= 7.8 and 1.2 Hz, 1H), 7.58-7.55 (m, 1H), 7.45-7.41 (m, 6H), 7.29-7.24 (m, 6H), 7.20-
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7.15 (m, 3H), 3.40 (t, J = 7.2 Hz, 2H), 3.37 (q, J = 7.2 Hz, 2H), 3.30 (t, J = 7.2 Hz,
2H), 3.27 and 3.24 (two t, J = 7.2 Hz, 4H), 2.48 (s, 3H), 2.35 (br. s, 4H), 2.06 (t, J =
7.2 Hz, 2H), 1.91 and 1.86 (two quint., J = 7.2 Hz, 4H), 1.61 (br. s, 1H), 1.53 (quint.,
J = 7.2 Hz, 1H), 1.40 (quint., J = 7.2 Hz, 2H), 1.02 (s, 6H) ppm; 13C{1H} NMR (150
MHz, CDCl3) δ 173.8, 148.0, 147.9, 146.1 (3C), 133.8, 133.6, 133.2, 132.7, 132.0,
131.7, 131.2, 130.8, 128.6 (6C), 127.8 (6C), 126.3 (3C), 124.2, 124.1, 108.1, 70.8,
47.8, 45.2, 45.1, 44.9, 43.1, 40.4, 30.0, 28.3 (2C), 27.9, 27.8, 27.5, 26.0, 17.9 ppm;
HRMS (Orbitrap-ESI) m/z calcd for C51H59N6O10S2 [M+H]+ 979.3734, found
979.3718.
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N1-Dde-N4,N8,N13-trinosyl-N16-trityl-4,8,13-triazahexadecane-1,16-diamine (26a).
Quantities of reagents: 23 (101 mg, 0.10 mmol), 10a (31 mg, 0.13 mmol), TPP (105
mg, 0.40 mmol), DIAD (0.08 mL, 0.40 mmol); Reaction temperature and time: 25
oC, overnight then 40 oC, 2 h; Yellow oil; Rf (R): 0.18.
The NMR and HRMS spectra of compound 26a are provided below.
Preparation of 2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)acetic acid (20b)
A. Protection of the carboxyl function of IAA with the phenacyl group
To an ice-cold solution of IAA (0.175 g, 1 mmol) and dry Et3N (0.20 mL, 1.34 mmol)
in dry DMF (3 mL), 2-bromoacetophenone (0.23 g, 1.16 mmol) was added under an
inert atmosphere and the reaction was stirred at 0 oC for 30 min. The reaction mixture
was then allowed to reach room temperature and further stirred for 2 h. The
completion of the reaction was confirmed by TLC using the solvent system C. The
reaction mixture was diluted with H2O and extracted thrice with EtOAc. The organic
layer washed twice with H2O and once with brine and then was dried over Na2SO4
and evaporated to dryness. The desired product, that is phenacyl 2-(1H-indol-3-
yl)acetate, was obtained pure following FCC and using solvent system V as eluant.
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White solid (0.24 g, 81%); mp 87.8-90.0 C; Rf (W): 0.44; H NMR (600 MHz,
CDCl3) δ 8.14 (br. s, 1H), 7.89 (dd, J = 8.1 and 1.3 Hz, 2H), 7.66 (d, J = 8.4 Hz, 1H),
7.59 (tt, J = 8.1 and 1.3 Hz, 1H), 7.46 (t, J = 8.1 Hz, 2H), 7.36 (d, J = 7.8 Hz, 1H),
7.26 (br. s, 1H), 7.22-7.19 (m, 1H), 7.16-7.14 (m, 1H), 5.36 (s, 2H), 3.98 (unresolv.
d, 2H) ppm; 13C{1H} NMR (150 MHz, CDCl3) δ 192.7, 171.9, 136.5, 134.7, 134.3,
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129.2 (2C), 128.2 (2C), 127.7, 123.7, 122.6, 120.1, 119.3, 111.6, 108.4, 66.7, 31.3
ppm; MS (ESI, 30 eV): m/z 332.34 [M+Κ]+, 316.40 [M+Na]+.
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B. Protection of the amino function of phenacyl 2-(1H-indol-3-yl)acetate with the
Boc-group
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To an ice-cold solution of phenacyl 2-(1H-indol-3-yl)acetate (0.24 g, 0.81 mmol), dry
Et3N (0.12 mL, 0.89 mmol) and DMAP (10 mg, 0.08 mmol) in dry CH2Cl2 (2 mL),
Boc2O (0.19 g, 0.89 mmol) was added under an inert atmosphere. The reaction was
kept for 3 h at room temperature. The completion of the reaction was confirmed by
TLC using the solvent system V. Then, the reaction mixture was diluted with CH2Cl2
and washed twice with H2O and once with brine. The organic layer was dried over
Na2SO4 and evaporated to dryness. The desired product, that is phenacyl 2-(1-(tert-
butoxycarbonyl)-1H-indol-3-yl)acetate, was obtained pure following purification by
FCC and using the solvent system U as eluant.
1
Yellow oil (0.29 g, 91%); Rf (U): 0.20; H NMR (600 MHz, CDCl3) δ 8.15 (br. s,
1H), 7.89 (dd, J = 8.4 and 1.2 Hz, 2H), 7.67 (br. s, 1H), 7.61-7.58 (m, 2H), 7.49-7.46
(m, 2H), 7.35-7.32 (m, 1H), 7.28-7.25 (m, 1H), 5.38 (s, 2H), 3.92 (unresolv. d, 2H),
13
1.67 (s, 9H) ppm; C{1H} NMR (150 MHz, CDCl3) δ 192.4, 170.9, 150.0, 135.9,
134.6, 134.3, 130.5, 129.3 (2C), 128.2 (2C), 125.1, 125.0, 123.1, 119.5, 115.7, 113.1,
84.0, 66.9, 31.1, 28.6 (3C) ppm; MS (ESI, 30 eV): m/z 809.13 [2M+Na]+, 416.15
[M+Na]+.
C. Deprotection of the carboxyl function of phenacyl 2-(1-(tert-butoxycarbonyl)-1H-
indol-3-yl)acetate
To a solution of phenacyl 2-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)acetate (0.29 g,
0.74 mmol) in DMF (7 mL), Na2CO3 (0.41 g, 3.91 mmol) first and then PhSH (0.30
mL, 2.96 mmol) were added under an inert atmosphere. The reaction mixture was
kept at 60 ºC for 2 h. The completion of the reaction was confirmed by TLC using
the solvent system U. The reaction mixture was then diluted with 5% citric acid and
extracted with EtOAc. The organic layer washed twice with H2O and once with brine,
dried over Na2SO4 and evaporated to dryness. The product Nind-Boc-IAA (20b) was
obtained pure following FCC purification and using solvent system R as eluant.
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White solid (0.17 g, 84%); mp 117-120 oC; Rf (R): 0.17; 1H NMR (600 MHz, CDCl3)
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δ 8.14 (br. s, 1H), 7.58 (br. s, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H),
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7.25 (t, J = 7.8 Hz, 1H), 3.75 (s, 2H), 1.66 (s, 9H) ppm; C{1H} NMR (150 MHz,
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CDCl3) δ 176.9, 150.0, 135.8, 130.3, 129,4, 125.0, 123.1, 119.4, 115.7, 112.9, 84.2,
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31.2, 28.6 (3C) ppm.
General procedure for coupling IAA (20a) or N-Boc-IAA (20b) with 4-
aminobutan-1-ol
To an ice-cold solution of IAA or N-Boc-IAA (1 mmol) and HOSu (0.23 g, 2 mmol)
in DMF (4 mL), DCC (0.23 g, 1.1 mmol) was added under an inert atmosphere. After
15 min at 0 ºC, the reaction mixture was further stirred at room temperature for 2 h.
Completion of the activation reaction was confirmed by TLC using solvent system C.
Then, 4-aminobutan-1-ol (0.3 mL, 3 mmol) was added and the reaction was
completed within a few minutes. A few drops of H2O and one drop of gl. acetic acid
were added to the reaction mixture in order to destroy excess of DCC. The reaction
mixture was then diluted with EtOAc and the precipitated urea was removed by
filtration. The filtrate was diluted with EtOAc and washed twice with 5% NaHCO3
and twice with H2O, dried over Na2SO4 and evaporated to dryness. The product was
obtained pure following FCC purification and using solvent system C as eluant.
N-(4-Hydroxybutyl)-2-(1H-indol-3-yl)acetamide (21a). Orange solid (0.16 g, 65%);
mp 80.0-83.6 oC; Rf (C): 0.23; 1H NMR (600 MHz, CDCl3) δ 8.55 (br. s, 1H), 7.55
(d, J = 7.8 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.23 (td, J = 8.4 and 0.6 Hz, 1H), 7.15
(ddd, J = 8.4, 7.8 and 0.6 Hz, 1H), 7.12 (d, J = 2.4 Hz, 1H), 5.89 (unresolv. t, 1H),
3.73 (s, 2H), 3.52 (t, J = 6 Hz, 2H), 3.20 (q, J = 6.6 Hz, 2H), 1.48-1.40 (m, 5H) ppm;
13C{1H} NMR (150 MHz, CDCl3) δ 171.8, 136.5, 127.1, 123.9, 122.6, 120.0, 118.7,
111.5, 109.0, 62.3, 39.3, 33.4, 29.6, 26.0 ppm; Anal. Calcd (%) for C14H18N2O2: C,
68.27; H, 7.37; N, 11.37. Found: C, 68.48; H, 7.52; N, 11.48.
tert-Butyl 3-(2-((4-hydroxybutyl)amino)-2-oxoethyl)-1H-indole-1-carboxylate (21b).
Beige solid (0.13 g, 72%); mp 122-126 oC; Rf (C): 0.43; 1H NMR (600 MHz, CDCl3)
δ 8.15 (d, J = 6.6 Hz, 1H), 7.54 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.35 (td, J = 6.6 and
0.6 Hz, 1H), 7.26 (ddd, J = 7.8, 6.6 and 0.6 Hz, 1H), 5.78 (unresolv. t, 1H), 3.66 (s,
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2H), 3.56 (t, J = 6 Hz, 2H), 3.23 (q, J = 6 Hz, 2H), 1.68 (s, 9H), 1.51-1.45 (m, 5H)
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ppm; C{1H} NMR (150 MHz, CDCl3) δ 170.3 (2C), 149.5, 129.8, 125.0, 124.9,
122.9, 119.0, 115.4, 113.9, 84.1, 62.3, 39.4, 33.3, 29.6, 28.2 (3C), 26.1 ppm; Anal.
Calcd (%) for C19H26N2O4: C, 65.87; H, 7.56; N, 8.09. Found: C, 66.12; H, 7.38; N,
7.95.
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Coupling of Nsm derivative 22 with alcohol 21b under Mitsunobu
reaction conditions - Synthesis of the crude fully protected toxin HO-
416b (23a). Quantities of reagents: 22 (197 mg, 0.20 mmol), 21b (90 mg, 0.26
mmol), TPP (140 mg, 0.52 mg), DIAD (0.1 mL, 0.52 mmol), dry THF (0.6 mL);
o
Reaction time: overnight; Reaction temperature: 40 C; Slightly yellow oil; Rf (S):
0.29; 1H NMR (600 MHz, CDCl3) δ 8.16 (unresolv. d, 1H), 8.00 (dd, J = 7.8 and 0.6
Hz, 1H), 7.97-7.93 (m, 2H), 7.72-7.63 (m, 4H), 7.60-7.54 (m, 5H), 7.53-7.48 (m, 2H),
7.44-7.40 (m, 6H), 7.33 (t, J = 7.8 Hz, 1H), 7.30-7.23 (m, 7H), 7.21-7.15 (m, 3H),
5.80 (t, J = 6.6 Hz, 1H), 3.64 (s, 2H), 3.37 (t, J = 7.2 Hz, 2H), 3.39-3.24 and 3.24-
3.20 (two m, 10H), 3.18 (q, J = 6.6 Hz, 2H), 2.09 (t, J = 6.6 Hz, 2H), 1.86-1.77 (m,
4H), 1.68 (s, 9H), 1.61 (br. s, 1H), 1.51-1.45 (m, 4H), 1.39 (quint., J = 7.2 Hz, 2H)
ppm; MS (ESI, 30 eV): m/z 1314.32 [M+H]+, 243.18 [Trt]+.
General procedure for the alkylation of N-nosylated PAs with bromide
10c
A typical procedure for the alkylation of N-nosylated PAs with bromide 10c is the
following:
To a solution of dinosylated derivative 17 (1.31 g, 1.76 mmol) and bromide 10c (0.59
g, 1.94 mmol) in dry DMF (3 mL), K2CO3 (0.53 g, 3.70 mmol) was added under an
inert atmosphere. The reaction mixture was stirred at 60 oC for 2.5 h. The progress of
the reaction was monitored by TLC using the solvent system R. The reaction mixture
was then diluted with H2O and extracted once with EtOAc. The organic layer was
washed twice with H2O and once with brine. It was then dried over Na2SO4 and it
was evaporated to dryness. The product 5 was obtained pure following FCC
purification using solvent system Q as eluant.
The same procedure was used for the synthesis of Spm derivative 4 and the penta-
amine derivative 26a.
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N1-Dde- N4,N8-dinosyl-N12-tritylspermine (4). Yellow oil (0.43 g, 88%); Rf (R): 0.17;
1H-NMR (600 MHz, CDCl3) δ 13.48 (unresolv. t, 1H), 8.04 (dd, J = 7.8 and 1.2 Hz,
1H), 7.99 (dd, J = 7.8 and 1.2 Hz, 1H), 7.71-7.63 (m, 4H), 7.62-7.58 (m, 2H), 7.45-
7.39 (m, 6H), 7.31-7.25 (m, 6H), 7.22-7.16 (m, 3H), 3.44-3.38 (m, 4H), 3.37-3.33 (m,
4H), 3.31 (t, J = 6.6 Hz, 2H), 2.51 (s, 3H), 2.37 (s, 4H), 2.09 (t, J = 6.6 Hz, 2H), 1.94
(quint., J = 6.6 Hz, 2H), 1.67 (quint., J = 6.6 Hz, 2H), 1.62-1.51 (m, 5H), 1.04 (s, 6H)
ppm; 13C{1H} NMR (150 MHz, CDCl3) δ 173.7, 147.99, 147.96, 145.9 (3C), 133.7,
133.5, 133.4, 133.0, 131.9, 131.6, 131.0, 130.8, 128.6 (6C), 127.8 (6C), 126.3 (3C),
124.2 (2C), 108.0, 70.9, 53.5, 52.3, 47.0, 46.6, 45.6, 44.7, 40.9, 40.5, 30.1, 29.4, 28.3
(2C), 27.9, 25.0, 24.8, 17.8 ppm; HRMS (Orbitrap-ESI) m/z calcd for C51H59N6O10S2
[M+H]+ 979.3734, found 979.3729.
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N1-Dde- N4,N8-dinosyl-N11-tritylnorspermine (5). Yellow oil; (1.29 g, 76%); Rf (Q):
0.15; 1H NMR (600 MHz, CDCl3) δ 13.49 (unresolv. t, 1H), 8.07-8.03 (m, 1H), 7.98
(dd, J = 7.8 and 0.6 Hz, 1H), 7.70-7.63 (m, 4H), 7.59 (dd, J = 7.8 and 1.2 Hz, 1H),
7.58-7.55 (m, 1H), 7.43-7.38 (m, 6H), 7.29-7.23 (m, 6H), 7.20-7.14 (m, 3H), 3.44-
3.39 (m, 4H), 3.39-3.35 (m, 2H), 3.31 (t, J = 7.2 Hz, 2H), 3.27 (t, J = 7.2 Hz, 2H),
2.50 (s, 3H), 2.36 (s, 4H), 2.08 (t, J = 6.6 Hz, 2H), 1.93 (quint., J = 7.2 Hz, 2H), 1.86
(quint., J = 7.2 Hz, 2H), 1.66 (quint., J = 6.6 Hz, 2H), 1.57 (br. s, 1H), 1.03 (s, 6H)
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ppm; C{1H} NMR (150 MHz, CDCl3) δ 173.8, 148.0, 147.9, 145.7 (3C), 133.8,
133.6, 133.0, 132.7, 132.0, 131.7, 131.2, 130.8, 128.6 (6C), 127.9 (6C), 126.3 (3C),
124.22, 124.21, 108.1, 70.9, 46.0, 45.3, 45.1, 44.9, 40.9, 40.5, 30.1, 29.4, 28.3 (2C),
27.9, 27.5, 17.9 ppm; HRMS (Orbitrap-ESI) m/z calcd for C50H57N6O10S2 [M+H]+
965.3578, found 965.3541.
N1-Dde-N4,N8,N13-trinosyl-N16-trityl-4,8,13-triazahexadecane-1,16-diamine (26a).
Reaction time: 2.5 h; Reaction temperature: 60 oC; Yellow oil (0.85 g, 72%); Rf (R):
0.18; 1H NMR (600 MHz, CDCl3) δ 13.45 (unresolv. t, 1H), 8.05-8.02 (m, 1H), 7.99-
7.95 (m, 2H), 7.71-7.65 (m, 5H), 7.64 (td, J = 7.2 and 1.2 Hz, 1H), 7.61-7.56 (m, 3H),
7.42-7.38 (m, 6H), 7.28-7.23 (m, 6H), 7.20-7.14 (m, 3H), 3.41 (t, J = 7.2 Hz, 2H),
3.38 (q, J = 6.6 Hz, 2H), 3.33 (t, J = 7.8 Hz, 2H), 3.21-3.27 (m, 6H), 3.24 (t, J = 7.2
Hz, 2H), 2.48 (s, 3H), 2.36 (s, 4H), 2.06 (t, J = 6.6 Hz, 2H), 1.91 (quint., J = 7.8 Hz,
2H), 1.86 (quint., J = 6.6 Hz, 2H), 1.64 (quint., J = 7.2 Hz, 2H), 1.54-1.50 (m, 5H),
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1.03 (s, 6H) ppm; 13C{1H} NMR (150 MHz, CDCl3) δ 173.8, 148.0 (2C), 147.9, 145.9
(3C), 133.9, 133.7, 133.5, 133.3, 133.0, 132.7, 132.0, 131.9, 131.7, 131.1, 130.8,
130.7, 128.6 (6C), 127.8 (6C), 126.3 (3C), 124.24, 124.20 (2C), 108.0, 70.9, 47.2,
46.6, 45.6, 45.1, 45.0 (2C), 40.9, 40.5, 30.1, 29.4, 28.3 (2C), 27.8, 27.3, 25.0, 24.9,
17.9 ppm; HRMS (Orbitrap-ESI) m/z calcd for C60H69N8O14S3 [M+H]+ 1221.4095,
found 1221.4077.
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General procedure for the selective removal of the Dde group
A typical procedure for the selective removal of the Dde group from fully protected
PAs and conjugates is the following:
To a solution of N1-Dde-N4-nosyl-N8-tritylspermidine (1a) (0.85 g, 1.16 mmol) in
DMF (15 mL), H2NNH2∙H2O (0.30 mL, 6.15 mmol) was added. The reaction mixture
was stirred at room temperature for 30 min. The progress of the reaction was
monitored by TLC using EtOAc as eluant. The reaction mixture was then diluted with
CH2Cl2 and washed twice with H2O and once with brine. The organic layer was dried
over Na2SO4 and evaporated to dryness. The product was obtained pure following
FCC purification using initially solvent system E, to remove non-polar fractions, and
then solvent system H as eluants.
The same procedure was used for the preparation of the partially protected PAs
tabulated below. The solvent systems used for the purification of the following
compounds are also provided.
N4-Nosyl-N8-tritylspermidine. Slightly yellow oil (0.58 g, 87%); Rf (E): 0.10; Solvent
systems used for FCC purification: initially E, then H; 1H NMR (600 MHz, CDCl3)
δ 7.98 (dd, J = 7.8 and 1.8 Hz, 1H), 7.66-7.60 (m, 2H), 7.59 (dd, J = 7.8 and 1.8 Hz,
1H), 7.45-7.42 (m, 6H), 7.28-7.24 (m, 6H), 7.20-7.16 (m, 3H), 3.37 (t, J = 7.2 Hz,
2H), 3.27 (t, J = 7.2 Hz, 2H), 2.72 (t, J = 6.6 Hz, 2H), 2.08 (t, J = 6.6 Hz, 2H), 1.68
(quint., J = 6.6 Hz, 2H), 1.59-1.54 (m, 5H), 1.42 (quint., J = 7.2 Hz, 2H) ppm; 13C{1H}
NMR (150 MHz, CDCl3) δ 148.1, 146.1 (3C), 133.7, 133.3, 131.6, 130.7, 128.6 (6C),
127.8 (6C), 126.2 (3C), 124.1, 70.8, 47.5, 45.0, 43.1, 40.0, 31.6, 27.9, 26.0 ppm;
HRMS (Orbitrap-ESI) m/z calcd for C32H37N4O4S [M+H]+ 573.2536, found
573.2522.
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N4-Nosyl-N1-tritylnorspermidine. Yellow oil (1.05 g, 72%); Rf (E): 0.15; Solvent
systems used for FCC purification: initially E, then H; 1H NMR (600 MHz, CDCl3)
δ 7.99 (unresolv. dd, 1H), 7.67-7.60 (m, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.43-7.39 (m,
6H), 7.28-7.24 (m, 6H), 7.20-7.16 (m, 3H), 3.39 and 3.36 (two t, J = 7.2 Hz, 4H),
2.71 (t, J = 6.6 Hz, 2H), 2.09 (t, J = 6.6 Hz, 2H), 1.73-1.64 (m, 4H), 1.51 (br. s, 3H)
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ppm; C{1H} NMR (150 MHz, CDCl3) δ 148.1, 145.9 (3C), 133.5, 133.3, 131.5,
130.8, 128.6 (6C), 127.8 (6C), 126.3 (3C), 124.2, 70.9, 45.7, 45.1, 40.9, 39.0, 31.7,
24.5 ppm; HRMS (Orbitrap-ESI) m/z calcd for C31H35N4O4S [M+H]+ 559.2379,
found 559.2402.
N4,N9-Dinosyl-N1-tritylspermine. Yellow oil (0.79 g, 91%); Rf (G): 0.19; Solvent
systems used for FCC purification: initially G, then H; 1H NMR (600 MHz, CDCl3)
δ 8.00-7.96 (m, 2H), 7.68-7.62 (m, 4H), 7.59-7.56 (m, 2H), 7.43-7.39 (m, 6H), 7.28-
7.24 (m, 6H), 7.20-7.16 (m, 3H), 3.36-3.32 and 3.32-3.26 (two m, 8H), 2.69 (t, J =
6.6 Hz, 2H), 2.07 (t, J = 6.6 Hz, 2H), 1.70-1.63 (m, 4H), 1.53 (unresolv. m, 7H) ppm;
13C{1H} NMR (150 MHz, CDCl3) δ 148.44, 148.39, 146.3 (3C), 133.9, 133.8, 132.1,
132.0, 131.12, 131.08, 129.0 (6C), 128.2 (6C), 126.7 (3C), 124.6, 124.5, 71.3, 47.3,
47.1, 46.0, 45.5, 41.3, 39.4, 32.1, 29.8, 25.5, 25.4 ppm; HRMS (Orbitrap-ESI) m/z
calcd for C41H47N6O8S2 [M+H]+ 815.2897, found 815.2868.
N4,N8-Dinosyl-N1-tritylnorspermine. Yellow oil (1.51 g, 94%); Rf (G): 0.18; Solvent
systems used for FCC purification: initially G then J; 1H NMR (600 MHz, CDCl3) δ
8.02-7.97 (m, 2H), 7.69-7.63 (m, 4H), 7.60-7.57 and 7.57-7.54 (two m, 2H), 7.44-
7.38 (m, 6H), 7.29-7.24 (m, 6H), 7.21-7.16 (m, 3H), 3.37 (t, J = 7.2 Hz, 4H), 3.29
and 3.27 (two t, J = 7.2 Hz, 4H), 2.70 (t, J = 6.6 Hz, 2H), 2.08 (t, J = 6.6 Hz, 2H),
1.86 (quint., J = 7.2 Hz, 2H), 1.70-1.63 (m, 4H), 1.44 (br. s, 3H) ppm; 13C{1H} NMR
(150 MHz, CDCl3) δ 148.44, 148.40, 146.3 (3C), 134.0, 133.9, 133.53, 133.47,
132.16, 132.09, 131.23, 131.22, 129.0 (6C), 128.2 (6C), 126.7 (3C), 124.6, 124.6,
71.3, 46.4, 45.9, 45.5, 45.4, 41.3, 39.3, 32.1, 29.8, 27.9 ppm; HRMS (Orbitrap-ESI)
m/z calcd for C40H45N6O8S2 [M+H]+ 801.2740, found 801.2752.
N4,N8,N13-Trinosyl-N16-trityl-4,8,13-triazahexadecane-1,16-diamine (26b). Orange
oil; (0.85 g, 83%); Rf (G): 0.11; Solvent systems used for FCC purification: initially
G, then H; 1H NMR (600 MHz, CDCl3) δ 8.00-7.96 (m, 3H), 7.69-7.63 (m, 6H), 7.59-
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