Synthesis and in vitro biological properties of novel cationic derivatives of amphotericin B

Article abstract of DOI:10.1002/chem.200701237

Novel cationic amphotericin B derivatives as highly potent antifungal agents are reported. These semisynthetic derivatives of amphotericin B were elaborated through a series of modifications both on the nitrogen atom of the mycosamine and on the Cl-16 carboxylic acid moiety. The antifungal activity of the new conjugates was tested against Saccharomyces cerevisiae and also against nine different strains of Candida albicans and Candida glabrata, including an amphotericin resistant strain. High potency was observed in the case of polyamine derivatives bearing two 3-aminopropyl chains on the mycosamine. The evaluation of the biological properties also included the determination of the hemolytic activity of the compounds by measuring the EH₅₀ values.

Full text of DOI:10.1002/chem.200701237

FULL PAPER
DOI: 10.1002/chem.200701237
Synthesis and In Vitro Biological Properties ofNovel Cationic Derivatives of
Amphotericin B
ValØrie Paquet, Astrid A. Volmer, and Erick M. Carreira*[a]
Abstract: Novel cationic amphotericin
B derivatives as highly potent antifun-
gal agents are reported. These semi-
synthetic derivatives of amphotericin B
were elaborated through a series of
modifications both on the nitrogen
atom of the mycosamine and on the C-
16 carboxylic acid moiety. The antifun-
gal activity of the new conjugates was
tested against Saccharomyces cerevisiae
and also against nine different strains
of Candida albicans and Candida glab-
rata, including an amphotericin resist-
ant strain. High potency was observed
in the case of polyamine derivatives
bearing two 3-aminopropyl chains on
the mycosamine. The evaluation of the
biological properties also included the
determination of the hemolytic activity
of the compounds by measuring the
EH50 values.
Keywords:
antifungal agents
chemistry natural products
total synthesis
amphotericin
B
·
·
bioorganic
·
·
Introduction
Therefore this drug is poorly absorbed orally and intrave-
nous administration is essential to ensure effectiveness. A
great deal of effort has been devoted to reduce the toxicity
of AmB and some improvement was achieved by modifying
the formulations used to administer AmB, such as liposomal
product AmBisome.[6–9] In this study, structural modifica-
tions of the molecule have been made in order to improve
the therapeutic index. In addition, different strategies were
tested to alter the physical, chemical and pharmacological
properties of AmB.
In recent decades, the incidence of nocosomial fungal infec-
tions clearly has risen to a point where some mycoses are
real therapeutic challenges. In the case of blood infections
caused by Candida species, the incidence has increased by
almost 500% throughout the 1980s. This was mainly attrib-
uted to aggravating factors such as immunosuppressive ther-
apy, an increasing number of AIDS patients and chemother-
apy.[1]
Fungal species, typically Candida and Aspergillus, account
for up to 25% of all hospital-acquired blood infections but
the emergence of other fungal species is now changing the
spectrum of disease.[2] Amphotericin B (AmB, 1) is the most
effective antifungal agent available for the treatment of sys-
temic mycoses. With the broadest spectrum of activity, AmB
is often referred to as the “golden standard” of antifungal
agents.[3–5] However, some drawbacks are normally associat-
ed with treatment, especially severe side effects, which in-
clude nephrotoxicity. Furthermore, AmB is only available as
a deoxycholate suspension (Fungizone) since the amphiphil-
ic nature of this molecule makes it insoluble in water.
To date the majority of the structure–activity relationship
studies relied on chemical modification of natural AmB[10–15]
To a lesser extent, genetic engineering has also been used to
access other derivatives.[16–18] Previous studies revealed that
the presence of a positively charged nitrogen atom on the
mycosamine is essential for antifungal activity.[10] Further-
more, results indicate that the lack of a negative charge on
the carboxylic acid reduces the hemotoxicity of the mole-
cule. Even though biological activity is not always affected
by synthetic alteration, it is clear that the overall charge of
the molecule is crucial. Following these observations, the
zwitterionic character of AmB appears as a decisive factor
in the conformation and the aggregation state of the mole-
cule which is ultimately reflected on the activity. Therefore
the design of new derivatives should mainly focus on the
ionizable groups of AmB. Since good biological activity is
exhibited by compounds in which the nitrogen atom is posi-
tively charged at physiological pH, cationic derivatives of
AmB are potentially highly active analogues. The presence
of a net charge on the molecule should hinder self-associa-
[a] Dr. V. Paquet, A. A. Volmer, Prof. Dr. E. M. Carreira
Laboratorium für Organische Chemie, ETH Zürich, HCI H335
8093 Zürich (Switzerland)
Fax : (+41)44-632-1328
E-mail: carreira@org.chem.ethz.ch
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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tion thus favoring the existence of monomeric AmB with re-
duced toxicity. At the same time, cationic AmB derivatives
could increase hydrogen-binding ability essential according
to the proposed ion channel model.[19] Moreover, the pres-
ence of polar functional groups may give rise to the forma-
tion of water soluble salts.
ers.[29] Even though the mycosamine has been previously
modified, it was reported that N-alkylation of polyene mac-
rolides is rather difficult.[30] However, from our previous
work on AmB,[31] we have demonstrated that selective alky-
lation of the primary amine by reductive alkylation can be
efficiently accomplished using unprotected AmB (1). We
also employed this reductive alkylation approach for the
synthesis of new conjugates of pimaricin and nystatin.[32]
Using this alkylation strategy, the synthesis of polyamines
analogues of AmB was carried out. The mycosamine of
AmB was reductively alkylated twice using a variety of ami-
noaldehydes and NaBH3CN as reducing agent (Scheme 1).
The natural polyamines putrescine, spermidine and sper-
mine are found in living organisms where they interact with
a wide variety of cellular targets. Even though these polycat-
ionic molecules have increased binding affinity for several
biomolecules, their specific function is still obscure.[20] Since
they are protonated at physiological pH, polyamines effi-
ciently interact with negatively charged phospholipids in the
biological membrane. In the case of yeast cells, exogenous
polyamines are specifically internalized by a membrane
uptake system.[21] Consequently, these strong interactions
between polyamines and polar head-groups might influence
not only the membrane functions but also various properties
such as permeability.[22] Recently, polyamine conjugates and
synthetic analogues were found to be involved in many bio-
logical processes just as the naturally occurring poly-
amines.[23] In fact, research on polyamine-based compounds
is a very active field especially for pharmaceutical oriented
applications.[24,25] Since the membrane is suggested to be to
be the primary target of AmB, polyamine conjugates of
AmB have the potential of displaying increased potency.
Herein, we report novel cationic derivatives of AmB as
highly potent antifungal agents. Some of the semi-synthetic
derivatives were elaborated by anchoring a polyamine
moiety to the mycosamine of AmB using a reductive alkyla-
tion reaction. Further chemical elaboration at the C-16 posi-
tion was performed through various amide coupling reac-
tions. The antifungal activity of the new derivatives was
tested against Saccharomyces cerevisiae and several Candida
strains. The hemolytic activity was ranked on the basis of
measured EH50 value, which can be related to the toxicity of
the new AmB derivatives.
Scheme 1. a) FmocNH
ACHRTNEU(G CH2)nCHO (n=1–3) or FmocNH2ACHTREUNG(CH2)4-
AHCTREUNG
The reported yields (22–76%) are the combined yields for
the double alkylation reaction and the removal of the 9-flu-
orenylmethoxycarbonyl (Fmoc) protecting groups on the
amine functionalities. Various chain lengths between the ni-
trogen atoms were chosen in order to screen their effect on
potency. Furthermore, compound 5 bearing four primary
amine groups might eventually give some indications on the
number of protonable nitrogen atoms optimal for good bio-
logical activity.
After the synthesis of symmetrically substituted poly-
amines derivatives of AmB, monoalkylation reaction was
performed (Scheme 2) using reaction conditions previously
described.[33]
Results and Discussion
Design and synthesis: In order to increase the biological ac-
tivity of AmB, we mainly focused our research on modifying
both the position and the number of amine functions on the
mycosamine moiety. The rationale behind these structural
alterations was that the primary amine group on AmB is be-
lieved to play an important role in membrane interac-
tions.[26] This approach was also supported by the presence
of a positive charge that modifies the conformation and the
aggregation state of the molecule which are important fac-
tors for activity.[27,28]
In addition to primary amine functions, amino acid and
polyamine structures also introduce basic functionalities
protonated at physiological pH thereby giving access to cat-
ionic derivatives of AmB. In the same way, the guanidine
group has the ability to promote hydrogen-bonding interac-
tions even with the phosphate group present in lipid bilay-
The monoalkylated derivative 6 can be directly deprotect-
ed using piperidine to afford 7. Alternately, 6 can be further
alkylated using a different aldehyde moiety thus generating
asymmetrically substituted mycosamine. The derivative 8
was isolated in 26% yield after a sequence of three steps
from the native AmB.
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Natural Products
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various biomolecules to AmB, such as fluorescein.[31] This
linker has the advantage of being selectively attached
through a double reductive alkylation reaction and at the
same time preserving the basicity of the nitrogen on the my-
cosamine. After the assembly of the linker with AmB, the
intermediate can be easily deprotected following a known
procedure (Scheme 3).[35]
The 6-amino-1-hexanoyl-(piperazinyl)-AmB derivative 12
was then ready for an amide coupling reaction using N-pro-
tected lysine activated as the hydroxysuccinimide ester
(Succ). After removal of the Fmoc group, the desired lysine
derivative 13 was isolated in 28% yield. Subsequently, the
arginine derivative 14 can be synthesized in one step in
20% yield using reagent 9.
Scheme 2. a) FmocNHCH2CH2CHO, NaBH3CN, DMF; b) piperidine,
DMSO, 7%; c) FmocNHCH2CHO, NaBH3CN, DMF; d) piperidine,
DMSO, 26%.
Nitrogen-based AmB derivatives were also elaborated by
the introduction of guanidine functionality. The guanidine
group can be directly attached to the mycosamine of AmB
by using 1H-pyrazolecarboxamidine hydrochloride (9) as re-
[34]
agent [Eq. (1)].
The derivative 10 was then isolated in
97% yield in only one step.
The introduction of the guanidine functionality is also
possible on the side chains already attached to the mycosa-
mine. The bisguanidine derivative 11 was synthesized by re-
acting the bis-2-aminoethyl derivative 2 with an excess of
1H-pyrazolecarboxamidine hydrochloride (9) [Eq. (2)]. Fi-
nally, 11 was isolated in 55% yield after a double guanidina-
tion reaction.
In addition to the polyamine structures, there are several
accessible amino acids containing multiple amines that can
be linked to the mycosamine. Although peptide coupling re-
action could be used to link amino acids to the mycosamine,
only very few examples are known. However, our group has
previously reported the synthesis of a readily accessible pi-
perazine linker as a synthetic anchor for the conjugation of
Scheme 3. a) FmocNHACTHER(NGU CH2)4CHCAHTRE(UNG NHFmoc)CO2Suc, DIPEA, DMF; b)
piperidine, DMSO, 28%; c) 9, DIPEA, 20%.
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E. M. Carreira et al.
Many of AmBs toxic side effects are a direct result of the
high dosage that have to be administered to patients. Its low
solubility is a major problem in the field. One of the possi-
bilities to increase the solubility of a compound is to attach
a carbohydrate.[36] A common, but limited method to attach
carbohydrates to amines is via the Amadori rearrange-
ment.[37–39] A glycoconjugation method, which is not only
limited to the anomeric position and based on sulfonyl
esters, was developed in our group.[40] It gives the possibility
to introduce conjugation at any position at the sugar unit. In
addition, it is possible to conjugate to a linker and vary the
chain length of this linker, making it a versatile method. In
this study, this strategy was used to link carbohydrates to
the piperazine linker (Scheme 4). Glucose and glucosamine
were both attached to the mycosamine to examine the cu-
mulative effect of the carbohydrate and the influence of the
extra amine of glucosamine on the activity of AmB.
Employing similar standard methods methyl 3,4-di-O-acetyl-
2-deoxy-a-d-glucopyranoside (16) was synthesized.[39,44–47]
Both compounds were subjected to the same reaction condi-
tions, which through displacement of the mesylate and oxi-
dation of the corresponding sulfonyl acetate lead to com-
pounds 17 and 18 in a yield of 79 and 60%, respectively,
over three steps. Generation of the sulfonyl chloride under
standard conditions,[48] followed by coupling to [6-(2,5-dihy-
dro-1H-pyrrol-1-yl)-6-oxohexyl]amine (24)[31] and dihydrox-
ylation of the pyrroline double bond afforded compounds 19
and 20 (24 and 46% yield) in three steps. Protection group
exchange of 20 to 21 in 26% yield makes both compounds
ready for oxidative cleavage to subject this to AmB in a
double reductive alkylation reaction. 22 and 23 (47 and
39% yield) were isolated after a sequence of two sequential
deprotection steps.
Cognizant of the fact that the overall charge of the mole-
cule will be an important aspect of these new cationic deriv-
atives of AmB, we also prepared other conjugates. In order
to retain good hydrogen-bonding ability, we surveyed deriv-
atives that contained alcohol and carbonyl functionality on
the side chains (Scheme 5).
Methyl 2,3,4-tri-O-acetyl-a-d-glucopyranoside (15) was
prepared according to known literature procedures.[41–43]
Scheme 5. a) i) TBSOCH2CH2CHO, NaBH3CN, DMF, ii) HF/pyridine,
28% for 25; b) MeO2CCH2CH2CHO, NaBH3CN, DMF, 45% for 26; c)
LiOH, THF/H2O, 43%.
We first prepared diol derivative 25 using the correspond-
ing protected aldehyde for the alkylation reaction followed
by the deprotection of the silyl groups using HF/pyridine.
The two steps sequence allowed the isolation of 25 in 28%
yield. In the case of the diester 26, the double alkylation
was carried out using methyl-4-oxobutanoate, thus affording
directly the desired compound in 45% yield. Finally, after
the hydrolysis of the two esters on 26, the diacid derivative
27 was isolated in 43% yield.
The incorporation of several basic amino groups, especial-
ly polyamine structures on the mycosamine is believed to
enhance the potency of AmB. These structural modifications
will most likely also have a positive influence on the solubil-
ity properties of the new derivatives. To achieve our goal of
improving the pharmacological properties of AmB, we
sought to address toxicity as manifest by hemolysis because
this represents a serious issue. The hemolytic property of
AmB is among one of the major problems associated with
the clinical use of this antibiotic. However, substitution of
Scheme 4. a) MsCl, Et3N, CH2Cl2; b) KSAc, DMF; c) H2O2, KOAc,
AcOH, 79% for 17, 60% for 18 over three steps; d) SO2Cl2, PPh3,
CH2Cl2; e) Et3N, 24; f) K2OsO4, NMO, acetone/H2O/tBuOH, 24% for 19,
46% for 20 over three steps; g) H2, Pd/C; h) FmocSuc, Na2CO3, H2O/di-
oxane 26% over two steps; i) NaIO4, SiO2, H2O/CHCl3; j) AmB,
NaBH3CN, DMF; k) K2CO3, piperidine, H2O/MeOH, 59% for 22, 66%
for 23 over three steps.
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Natural Products
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the carboxylic acid at C16 has been shown to reduce its he-
motoxicity. Esterification reactions were reported early on
for the introduction of various substituents at that posi-
tion.[49] More recently, some polyene compounds underwent
chemical modification or isolation from a recombinant
strain of Streptomyces diastaticus var. 108 leading to partri-
cin A,[50] CE-108 and rimocidin derivatives.[51] For AmB,
amide derivatives were also generated in order to introduce
diversity.[52–54] Although a variety of amide derivatives have
been synthesized over the years, only limited improvement
in therapeutic index has been noted. The majority of the
compounds had lower antifungal activity over AmB and the
enhancement is often attributed to greater water solubility
or reduced toxicity. Unfortunately, no direct correlation can
be draw between the structure of the amide moiety and the
effect on the toxicity of the compound. For example, the re-
lationship between self-association and the toxicity of some
derivatives was studied because it was demonstrated for
AmB that monomers and non-water-soluble aggregates are
less toxic then water-soluble aggregates.[55] However, it was
shown by UV spectroscopy that the toxicity was independ-
ent of the ratio monomer/aggregate in the case of amide de-
rivatives of AmB.[56] Despite very little indication on the
nature of the best side chain, amide derivatives were synthe-
sized using peptide coupling reagents. And even if the effect
on the toxicity seemed unpredictable, several amine struc-
tures were used in the reaction in order to generate ana-
logues with very low hemotoxicity (Scheme 6).
should allow improvements in yields. For comparison pur-
pose, the corresponding methyl ester derivative 37 was syn-
thesized in 59% yield using trimethylsilyldiazomethane in
methanol (Scheme 7).
Scheme 7. a) TMSCHN2, MeOH, 59%.
In this study, reductive alkylation was used in order to in-
troduce several nitrogen-based functionalities specifically on
the mycosamine of AmB. Further chemical elaboration at
C16 was also possible using amide coupling reaction condi-
tions. A series of new semi-synthetic derivatives of AmB are
now accessible through these simple transformations.
Biological assays: The antifungal activity of the semi-syn-
thetic derivatives of AmB was tested following the NCCLS
protocol by measuring the minimal inhibitory concentrations
(MICs) required to completely inhibit growth of yeast. This
assay served as the basis for the comparison between the
novel compounds and natural AmB.
First, the effect on the activity of the new derivatives as a
result of the chemical modification on the mycosamine was
evaluated. As shown in Table 1, the amino derivatives were
assayed against Saccharomyces cerevisiae in order to com-
pare their antifungal activity. We expected that the introduc-
tion of several basic amino groups on the mycosamine, espe-
cially polyamine derived structures, would improve the po-
The amide coupling reaction was performed on the poly-
amine derivative 28 where the mycosamine side chains were
protected as Fmoc groups. Primary and secondary amines
were used in the reaction in combination with (benzotriazol-
1-yloxy)-tripyrrolidinophosphonium
hexafluorophosphate
(PyBOP) and 1-hydroxybenzotriazole (HOBt). The low
yields (7 to 49%) obtained over the two steps are mostly at-
tributable to the partial deprotection of the Fmoc groups
and difficulties in the purification of these compounds. Fur-
ther optimization with respect to the protecting group
Scheme 6. a) PyBOP, HOBt, DIPEA, RNH2; b) piperidine, DMSO.
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E. M. Carreira et al.
Table 1. Minimum inhibitory concentration for compounds bearing
modified mycosamine.
gal activity. The modification of the mycosamine by incorpo-
ration of a guanidine group considerably improved the po-
tency of compound 10 (entry 9). But the substitution of the
two amino groups by guanidines in compound 11 had only
limited effect on the MIC value with a three-fold increase in
antifungal activity (entry 10). Conjugates containing differ-
ent functional groups that could also participate in hydro-
gen-bonding interactions were also surveyed. Diol derivative
25 showed no significant gain in activity (entry 11). In the
case of the diester derivative 26, the biological activity is di-
minished to a MIC value of 2.0 mm which was six times less
then with natural AmB (entries 12 vs 1). In compound 27
the presence of negatively charge functional groups on the
mycosamine was detrimental and the diacid derivative was
completely inactive (entry 13). The screening of various
amino derivatives of AmB was pursued through the incor-
poration of an amino acid moiety. Lysine and arginine were
chosen because they include nitrogen-based functionalities.
Derivatives 13 and 14 were tested and a MIC value of
0.10 mm was obtained in both cases revealing a three-fold in-
crease in activity. Without a primary amine in carbohydrate
derivative 22 a seven-fold loss in activity is found (entry 16).
With the reintroduction of an amino group as a glucosamine
moiety the original activity was restored for compound 23
(entry 17) with respect to natural AmB.
From the first screening on the mycosamine, we found
that the introduction of bisaminopropylene moiety on the
mycosamine of AmB improves significantly the antifungal
activity. Although the incorporation of a guanidine on the
mycosamine enhanced the potency, the observed effect was
limited to AmB. The other polyene macrolides modified
with the guanidine moiety did not benefit from this modifi-
cation.[32] Since the bisaminopropylene motif on the mycosa-
mine lead to superior results, its use was continued in the
amide derivative in order to capitalize on the improved po-
tency. Amide analogues 29–36 were first tested against S.
cerevisiae to compare their activity with the reference com-
pound 3 and natural AmB (Table 2). Most compounds ex-
hibit similar antifungal activity to AmB where the MIC
values ranged between 0.20 and 0.60 mm. However, the com-
Entry
Compound
MIC [mm][a]
1
2
3
4
5
6
7
8
1
2
7
8
3
4
28
5
10
11
25
26
27
13
14
22
23
0.30
0.25
0.10
0.090
0.020
>10
0.50
0.080
0.020
0.10
0.50
2.0
9
10
11
12
13
14
15
16
17
>10
0.10
0.10
2.0
0.30
[a] Assayed with S. cerevisiae wild type (BY4741) following the NCCLS
protocol; see Experimental Section for details.
tency. Two aminoethylene side chains had no beneficial
effect on the MIC value of compound 2 (entry 2). However,
the presence of only one aminopropylene side chain (7) led
to improved activity with a MIC value of 0.10 mm compared
with 0.30 mm for AmB (entries 1 vs 3). A similar antifungal
activity is obtained after the introduction of an aminoethy-
lene group on the mycosamine in derivative 8 with a MIC
value of 0.090 mm. Moreover, the bis-aminopropyl derivative
3 with a MIC value of 0.020 mm is 15 times more potent than
the native drug.
In addition, the slightly decreased activity of 4 (entry 6)
suggests that the length of the spacer between the amino
groups is a very important factor for activity. Furthermore,
the basicity of the amino groups placed on the side chains is
crucial because masking of the primary amine as carbamate
groups completely inhibits the activity (28, entry 7). Howev-
er, the reduced potency of derivative 5 compared to 3 (en-
tries 8 vs 5) gives some indications that a large number of
primary amino groups are not necessary for optimal antifun-
Table 2. Minimum inhibitory concentration against various yeast strains.
Entry Compound S. cerevisiae
C. albicans
C. glabrata
DSY562 DSY565
BY4741
CAF2-1
DSY294
DSY296
DSY654
DSY1751
DSY1764
[mm]
[mm]
[mm]
[mm]
[mm]
[mm]
[mm]
[mm]
[mm]
1
2
3
4
5
6
7
8
9
1
7
3
0.30
0.10
0.020
0.10
0.040
0.20
0.40
0.40
0.060
0.60
0.60
0.040
0.30
0.10
0.10
0.50
2.0
0.30
0.50
0.25
0.30
1.4
0.40
0.10
0.20
0.25
1.0
0.30
0.50
0.25
0.30
1.0
0.40
0.20
0.10
0.10
0.75
0.30
0.30
0.50
0.10
1.0
0.50
0.25
0.10
0.25
2.0
0.40
0.50
0.25
0.20
1.5
0.30
0.10
0.10
0.50
2.0
0.40
1.0
1.0
50
0.50
0.10
0.20
0.25
0.50
0.30
0.30
0.50
0.080
0.70
0.40
0.040
0.50
0.20
0.20
0.25
0.50
0.20
0.30
0.25
0.070
1.0
2.0
1.0
3.0
3.0
0.50
1.0
0.50
1.5
4.0
1.0
1.0
37
29
30
31
32
33
34
35
36
0.50
1.5
1.5
10
11
12
0.50
0.040
0.50
0.040
0.50
0.020
1.0
0.060
1.0
0.040
0.080
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Table 3. Hemotoxicity of semi-synthetic derivatives of AmB.
parison of azole derivative 36 with AmB or even with the
ester analogue 37 shows a clear improvement in the potency.
Interestingly, derivatives 29 and 33 both containing a pri-
mary amine in the amide side chain were also much more
active with MIC values of 0.040 and 0.060 mm.
Entry
Compound
EH50 [mm]
1
2
1
3
4.0
10
50
3
4
5
6
7
8
9
10
11
37
29
30
31
32
33
34
35
36
30
Screening was pursued in order to explore further the
scope of the antifungal activity. The novel derivatives were
tested against several Candida strains which include wild
type strain as well as different clinical isolates and mutant
strains (Table 2). The majority of the derivatives were as
potent as AmB against the wild type Candida strain (CAF2-
1) with the exception of 29 and 34 where the MIC values
were higher (1.4 and 2.0 mm, respectively). Against the C. al-
bicans clinical isolates DSY294 and DSY296, the MIC
values observed for the various amide analogues were in the
same range as AmB (0.10–1.0 mm vs 0.40 mm). As with the
wild type, the strains appear to be very sensitive to the azole
derivative 36 with MIC values of 0.040 and 0.020 mm.
Mutant strains DSY654 (Dcdr1/cdr2) and DSY1751 (Derg3)
were very sensitive to only a few compounds (3, 7 and 36)
while most amide derivatives exhibit limited antifungal ac-
tivity (0.20–2.0 mm) when compared to AmB. The com-
pounds were next screened against an AmB-resistant strain
(DSY1764, Derg3/erg11). AmB has a MIC value of 50 mm
against this strain, and it is well worth noting that such high
MIC value can be categorized as resistant strain. In the
assay, all the compounds (3, 7 and 29--37) displayed a dra-
matic increase in activity over AmB with MIC values rang-
ing between 0.50 and 4.0 mm. In some cases (30 and 32), this
represented a 100-fold increase, which is the highest increase
in potency observed in this study. Finally, antifungal tests
were carried out using clinical isolated Candida glabrata
strains (DSY562 and DSY565). With the exception of 34
and 35, the antifungal activity of the derivatives was gener-
ally superior to AmB. This was especially true in the case of
azole 36 which was at least 10 times more active than AmB
(0.040 vs 0.50 mm). Overall, the derivative 36 displays the
best improvement in antifungal activity to AmB. Through-
out the various yeast strains tested, this conjugate was typi-
cally 10 times more active and even more so with the resist-
ant strain.
200
200
200
120
100
150
75
seems to play an important role in the resulting toxicity. For
example, derivatives 29 and 33 both incorporate a primary
amino group on the side chain but their corresponding toxic-
ity is completely different. The length of the side chain ap-
pears to make a notable effect because 29 has an EH50 value
of 30 mm while 33 is four times less toxic with an EH50 value
of 120 mm (entries 3 and 8). Interestingly, derivative 36 was
almost 20 times less toxic than AmB with an EH50 value of
75 mm, even more when the observed enhanced antifungal
activity is also taken in to consideration.
The substitution patterns present on the mycosamine
have a tremendous impact on the biological properties of
the AmB derivatives. It appears that superior potency arises
from the presence of a specific polyamine motif on the my-
cosamine, namely a bisaminopropylene pattern. The ob-
served effect on the antifungal activity strongly depends on
the mechanism operating in that case. Although the exact
mechanism of action is still under debate, AmB is believed
to interact with the yeast membrane and forms a channel,
leading to K+ efflux and ultimately to cell death. The trans-
membrane pore model is based on the formation a barrel-
stave structure with up to eight molecules of AmB interdigi-
tated with eight molecules of sterol.[57] However, several
other mechanisms have been postulated for AmB and very
few studies have been conducted on semi-synthetic deriva-
tives of AmB.[58]
The question of the toxicity of the novel AmB derivatives
had to be addressed in order to complete this comparative
study. All the amide and ester derivatives were tested in a
hemolysis assay in order to evaluate their toxicity toward
human erythrocytes. The EH50 value which is the concentra-
tion of an agent that causes 50% hemoglobin release can be
measured and compared to natural AmB (Table 3. As ex-
pected, AmB and derivative 3 displayed the highest toxicity
since the free carboxylic acid was unmodified (entries 1 and
2). The methyl ester derivative 37 was 5 times less toxic
than AmB with an EH50 value of 50 mm, a result consistent
with previous observations involving ester analogues. Signifi-
cantly, compounds containing an amide functionality at C16
exhibited a very low toxicity with an EH50 values up to
200 mm in the case of compounds 30, 31 and 32 (entries 5 to
7). The chemical structure of the side chain on the amide
Taking into account the activity enhancement observed
for polyamine derivative 3, a different mechanistic hypothe-
sis might be invoked in order to explain the improved anti-
fungal properties. It is difficult to explain the superior anti-
fungal activity of 3, only with the increase solubility since
highly soluble derivatives have been made and their potency
was equivalent to the natural AmB.[13] Furthermore, the spe-
cificity of the bisaminopropylene motif cannot be explained
solely by proton-donor properties or improved ion channel
formation. Because the derivatives have similar structure to
natural polyamines, these analogues might efficiently use
the same transport system present in yeast cell. Moreover,
internalization of AmB has been postulated as the origin of
the toxicity towards mammalian cells. This polyamine motif
on the mycosamine seems to confer internalization capacity
to the derivatives. It was established that internalization of
Chem. Eur. J. 2008, 14, 2465 – 2481
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2471

E. M. Carreira et al.
AmB leads to a 10-fold increase in activity which is similar
to the enhancement we observed in the case of derivative
3.[59] Furthermore, cationic derivatives of AmB are used as
tools to promote the internalization of oligonucleotides.[60–62]
These similar cationic derivatives have the ability to transi-
ently permeabilize the membrane thus allowing large mole-
cules to penetrate inside the cell. Moreover, the fact that
the polyamine analogues were highly active against the
AmB resistant strain also tends to confirm that an alterna-
tive mechanism is operating in those cases. Finally, the supe-
rior activity displayed by derivative 36 could be attributed
to the synergistic combination of the bisaminopropylene
moiety on the mycosamine and the presence of a triazole
motif on the side chain of the amide functionality. The anti-
fungal properties of the triazole portion were predicted be-
cause azoles constitute a large, successful class of antifungal
agents. Any biological activity derived from the triazole im-
plies some degree of internalization since the molecular
target of those compounds is the cytochrome P450.[63] Even
though these new cationic derivatives might operate through
discrete mechanisms, we speculate their behavior differs
from unmodified AmB. Their cationic nature seems to
confer to the compounds improved permeabilizing proper-
ties. It is still not clear, if the improvement is the result of
internalization or those compounds are acting just as cation-
ic surfactants similarly to cationic peptides.[64]
Experimental Section
Antifungal activity: The minimum inhibitory concentration (MIC) values
were determined for the various strains (Table 4) using an assay inspired
Table 4. Yeast strains used in this study.
Strains
Genotype
Reference
[66]
[67]
[68]
[68]
[69]
[69]
[70]
BY4741
CAF2-1
MATa his3D1 leu2D0 lys2D0 ura3D0
Dura3:imm434/URA3
DSY294 azole-susceptible clinical strain
DSY296 azole-resistant clinical strain
DSY562 azole-susceptible clinical strain
DSY565 azole-resistant clinical strain
DSY654 Dcdr1:hisG/Dcdr1:hisG Dcdr2:hisG-URA3-hisG/
Dcdr2:hisG
DSY1751 Derg3A:hisG/Derg3B:hisG-URA3-hisG
Derg11:hisG/ERG11
DSY1764 Derg3A:hisG/Derg3B:hisG Derg11:hisG/
Derg11:hisG-URA3-hisG
[71]
[71]
by the standard protocol approved by the National Committee of Clinical
Laboratory Standards (NCCLS) but using YEPD liquid medium instead
of the RPMI-1640 medium.[65] The strains were cultivated overnight in
5 mL YEPD liquid medium at 308C (378C for the strain DSY1764) with
constant shaking. The saturated cultures were diluted to an OD600 of 0.1
(3”107 cellsmLꢀ1). Using 24 wells plate, each well was prepared by
adding 1% (12 mL) of DMSO solution of the tested compound with 1%
(12 mL) yeast cells solution and completed with YEPD (1.176 mL). The
plates were sealed with Parafilm and then incubated for 18 h at 308C
(36 h at 378C for the strain DSY1764). The optical density was read at
600 nm using 1.5 mL cuvettes. The MIC value was defined as the drug
concentration needed to inhibit growth, less than 5% compared to a
drug-free culture.
Conclusion
Hemolytic activity: Human blood, anticoagulated with citrate or EDTA,
was centrifuged (2000”g) at 48C for 10 min. The pellets were washed
three times with PBS buffer (pH 7.2 with 2 gLꢀ1 glucose) and then dilut-
ed to a concentration of 4% (4”108 cellsmLꢀ1). All experiments were
done in triplo and the total volume for the hemolysis tubes was 1.5 mL.
The solutions were prepared by adding 1% (14 mL) of DMSO solution of
the tested compound with 736 mL of PBS buffer and completed with
750 mL of 4% erythrocytes. After one hour incubation at 378C, the sam-
ples were centrifuged (1500”g) at 48C for 5 min and the absorbance of
the supernatant was measured at 560 nm. The concentration that led to
50% hemolysis (EH50) was intrapolated graphically. The value for the
100% hemolysis was obtained by the treatment with 100 mm of AmB.
As a member of the polyene macrolide family, AmB is
highly insoluble in water and also causes severe side effects.
Many attempts to overcome those drawbacks were under-
taken through the chemical modification of its structure. As
previously mentioned, the chemical alteration of the carbox-
ylate and the mycosamine moieties have been reported thus
changing the zwitterionic character of AmB.
In this study, the biological properties of a series of cat-
ionic derivatives of AmB were investigated in order to im-
prove the therapeutic index of the natural drug. Based on
the comparison of several mycosamine analogues, we found
that the incorporation of a specific polyamine motif resulted
in a dramatic increase in antifungal activity. Derivative 3
was 15 times more active than AmB with a MIC value of
0.020 mm. Because the presence of a bisaminopropylene
moiety led to an enhancement of activity, amide derivatives
were synthesized in order to optimize the biological activity.
Amide 36 bearing a triazole functionality displayed the best
antifungal activity compare to all the compounds. The 10-
fold increase in activity was observed not only with S. cerevi-
siae but also with several Candida strains including C. albi-
cans and C. glabrata. Even against an AmB resistant strain,
the various cationic derivatives were much more active then
AmB. Finally, the replacement of the carboxylic acid for an
amide group had a positive effect since the compounds were
significantly less hemotoxic.
Chemistry: All reactions were carried out in oven-dried glassware under
an atmosphere of argon. Amphotericin B (AmB) was purchased from
Apollo Scientifics (90% HPLC purity). All the other compounds were
purchased from Fluka, Senn and Aldrich and used without further purifi-
cation. Dimethylformamide was purified by distillation and methanol
was distilled over magnesium oxide. Diisopropylethylamine and piperi-
dine were distilled from KOH under nitrogen. The reactions were moni-
tored by thin layer chromatography using Merck silica gel 60 FB254B
plates and visualized using UV, aqueous ceric ammonium molybdate
stain and 10% sulfuric acid in methanol. Flash chromatography was per-
formed using E. Merck silica gel 60 (230–400 mesh). Analytical high-per-
formance liquid chromatography (HLPC) was performed on a Merck Hi-
tachi Chromatography System (Interface D-7000, UV detector L-7400,
Pump L-7100, degasser). The flow rate was 1.0 mLminꢀ1 and the detector
wavelength was fixed at l=405 nm. Column: LiChrospher 100 RP-18
(5 mm) (LiChroCART (250”4 mm), (solvent A: MeOH, solvent B: 5 mm
NH4OAc). All chromatograms were taken at ambient temperature. The
NMR spectra were recorded on Varian Gemini (300 MHz) and a Bruker
DRX-500 spectrometer (500 MHz). Chemical shifts (d) are reported in
ppm with the tetramethylsilane resonance as the internal standard (d
1
0.00) or the solvent signal for H (d 7.26 or 3.31) and 13C (d 77.0 or 49.0).
2472
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 2465 – 2481

Natural Products
FULL PAPER
The data are reported as follows: (s=singlet, d=doublet, t=triplet, q=
quartet, m=multiplet or unresolved, br=broad signal, coupling constant
in Hz, integration). 13C NMR spectra were recorded with complete
74.2, 73.9, 73.5, 70.3, 69.2, 68.7, 68.2, 67.8, 66.2, 65.5, 65.2, 46.5, 44.7, 44.2,
42.5, 42.4, 41.9, 35.1, 29.0, 18.4, 18.2, 16.7, 12.0 ppm; MALDI-FT: m/z:
calcd for C55H91N3O17: 1066.3209; found: 1066.6421 [M+H]+.
1
proton decoupling. H and 13C NMR spectra are included in the Support-
2,6-Bis-{amino-(9-fluorenylmethoxy-carbonyl)}hexanal: To a solution of
2,6-bis-{amino-(9-fluorenylmethoxycarbonyl)}-hexanol[74]
(500 mg,
ing Information. Mass spectrometric measurements were performed by
the mass spectrometry service of the LOC at the ETHZ on a Ion Spec
Ultima 4.7 spectrometer for MALDI-FT: m/z: using 2,5-dihydroxy ben-
zoic acid as matrix (20 kV) and ESI-FT.
0.860 mmol) in CH2Cl2 (50.0 mL) was added Dess–Martin periodinane
(730 mg, 1.72 mmol). After 2 h at room temperature, EtOAc (100 mL)
was added to the reaction mixture which was then washed with a saturat-
ed aqueous solution Na2S2O5 (2”100 mL). The organic layer was dried
over MgSO4 and the solvent was removed under reduced pressure. The
crude aldehyde was purified by flash chromatography (EtOAc/hexane
1:1) providing the desired aldehyde as a white solid (300 mg, 60%). Rf =
0.4 (EtOAc/hexane 1:1); 1H NMR (500 MHz, [D6]DMSO): d = 9.44 (s,
1H), 7.88 (d, J=7 Hz, 4H), 7.74 (d, J=8 Hz, 2H), 7.67 (d, J=8 Hz, 2H),
7.41 (t, J=7 Hz, 4H), 7.32 (t, J=7 Hz, 4H), 4.36 (d, J=7 Hz, 2H), 4.30
(d, J=7 Hz, 2H), 4.26–4.18 (m, 2H), 3.87 (m, 1H), 2.97 (q, J=6 Hz,
2H), 1.71 (brs, 1H), 1.47–1.25 ppm (m, 6H); 13C NMR (125 MHz,
[D6]DMSO): d = 201.7, 156.4, 156.1, 144.0, 143.8, 140.8, 127.7, 127.6,
127.1, 125.2, 120.2, 65.6, 65.2, 59.7, 46.8, 29.1, 27.3, 22.5 ppm; MALDI-
FT: m/z: calcd for C36H34N2O5: 597.2365; found: 597.2360 [M+Na]+.
N,N-Di-(2-aminoethyl)-AmB (2): To a solution of N-(9-fluorenylmethoxy-
carbonyl)-2-aminoacetaldehyde[72] (290 mg, 1.03 mmol) and AmB (1)
(320 mg, 0.340 mmol) in DMF (3.00 mL) was added NaBH3CN (65.0 mg,
1.03 mmol) followed by a drop of conc. HCl. After 16 h at room tempera-
ture, Amberlite IRA-743 (500 mg) was added and the mixture was stirred
for an hour. After filtration, the solution was concentrated down and
added dropwise to diethyl ether (250 mL). The yellow precipitate was fil-
tered and purified by flash chromatography (CHCl3/MeOH/H2O 40:8:1).
The isolated yellow solid was dissolved in DMSO (5.00 mL) and piperi-
dine (0.200 mL, 2.10 mmol) was added. After 2 h at room temperature,
the solution was added dropwise to diethyl ether (250 mL). The yellow
precipitate was filtered and washed with diethyl ether (2”100 mL) pro-
viding the desired compound 2 as a yellow solid (70 mg, 74%). 1H NMR
(500 MHz, [D6]DMSO): d = 6.46–6.01 (m, 13H), 5.42 (dd, J=15, 10 Hz,
1H), 5.23 (brs, 1H), 4.85–4.57 (m, 3H), 4.30–4.24 (m, 3H), 4.06–3.45 (m,
15H), 3.09 (m, 2H), 2.35–2.26 (m, 2H), 2.16 (d, J=6 Hz, 1H), 1.82–1.18
(m, 21H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92 ppm (d, J=
7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d = 176.8, 170.5, 136.6,
134.0, 133.6, 133.4, 133.1, 132.7, 132.4, 132.1, 131.8, 131.2, 128.2, 96.8,
77.2, 73.9, 73.5, 73.2, 72.1, 69.1, 68.7, 67.9, 66.1, 66.0, 51.4, 46.5, 44.7, 44.3,
42.5, 42.2, 41.9, 35.1, 30.6, 29.0, 28.3, 18.4, 18.2, 16.8, 12.0 ppm; MALDI-
FT: m/z: calcd for C51H83N3O17: 1010.5801; found: 1010.5795 [M+H]+.
N,N-Di-(2,6-diaminohexyl)-AmB (5): To a solution of AmB (1) (92 mg,
0.100 mmol) and 2,6-bis-{amino-(9-fluorenylmethoxycarbonyl)}hexanal
(290 mg, 0.500 mmol) in DMF (5.00 mL) was added NaBH3CN (31.0 mg,
0.500 mmol) followed by a drop of conc. HCl. After 16 h at room temper-
ature, Amberlite IRA-743 (500 mg) was added and the mixture was
stirred for an hour. After filtration, the solution was concentrated and
added dropwise to diethyl ether (250 mL). The yellow precipitate was fil-
tered and purified by flash chromatography (CHCl3/MeOH/H2O 40:8:1).
The isolated yellow solid was dissolved in DMSO (2.00 mL) and piperi-
dine (0.100 mL, 1.06 mmol) was added. After 2 h at room temperature,
the solution was added dropwise to diethyl ether (250 mL). The yellow
precipitate was filtered and washed with diethyl ether (2”100 mL) pro-
viding the desired compound 5 as a yellow solid (10 mg, 22%). 1H NMR
(500 MHz, [D6]DMSO): d = 6.40–5.86 (m, 13H), 5.47–5.41 (m, 1H), 5.36
(dd, J=15, 10 Hz, 1H), 5.14 (brs, 1H), 4.75–4.60 (m, 3H), 4.46–4.44 (m,
1H), 4.27–4.12 (m, 3H), 4.00–3.96 (m, 1H), 3.32 (brs, OH), 3.06–3.01 (m,
3H), 2.87–2.80 (m, 3H), 2.60–2.50 (m, 1H), 2.24–2.18 (m, 1H), 2.09 (d,
J=6 Hz, 1H), 1.73–1.08 (m, 24H), 1.04 (d, J=6 Hz, 3H), 0.96 (d, J=
6 Hz, 3H), 0.84 ppm (d, J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO):
d = 177.4, 170.5, 137.5, 135.2, 134.0, 133.8, 133.6, 133.2, 132.3, 132.1,
131.9, 131.2, 128.8, 96.7, 77.0, 74.2, 73.9, 73.5, 73.2, 70.9, 69.3, 69.1, 68.8,
68.5, 67.8, 66.5, 66.3, 66.1, 65.5, 65.4, 52.6, 40.4, 35.0, 33.2, 29.0, 22.8, 18.4,
18.1, 18.0, 17.0, 16.9, 12.0 ppm; MALDI-FT: m/z: calcd for C59H101N5O17:
1152.7271; found: 1152.7265 [M+H]+.
N,N-Di-(3-aminopropyl)-AmB (3): Piperidine (0.100 mL, 1.06 mmol) was
added to a solution of 28[32] (200 mg, 0.135 mmol) in DMSO (3.00 mL).
After 2 h at room temperature, the solution was added dropwise to dieth-
yl ether (250 mL). The yellow precipitate was filtered and washed with
diethyl ether (2”100 mL) providing the desired compound 3 as a yellow
solid (135 mg, 95%). Rf = 0.10 (CHCl3/MeOH/H2O 10:6:1); 1H NMR
(500 MHz, [D6]DMSO): d = 6.48–5.97 (m, 13H), 5.42 (dd, J=15, 10 Hz,
1H), 5.23 (brs, 1H), 4.82–4.66 (m, 3H), 4.46–4.40 (m, 1H), 4.28–4.21 (m,
2H), 4.07–4.02 (m, 1H), 3.94–3.88 (m, 1H), 3.34 (brs, OH), 3.13–3.07 (m,
3H), 2.82–2.64 (m, 3H), 2.31–2.26 (m, 1H), 2.16 (d, J=6 Hz, 1H), 1.83–
1.24 (m, 18H), 1.18 (d, J=6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=
6 Hz, 3H), 0.92 ppm (d, J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO):
d = 177.5, 170.4, 136.7, 134.0, 133.8, 133.7, 133.1, 132.4, 132.1, 131.9,
131.2, 128.1, 96.5, 77.2, 74.2, 73.9, 73.4, 70.7, 69.1, 68.7, 68.6, 67.8, 66.1,
65.6, 65.5, 65.4, 46.5, 44.7, 44.3, 42.5, 41.9, 38.6, 35.1, 29.0, 18.4, 18.2, 17.1,
16.9, 12.0 ppm; MALDI-FT: m/z: calcd for C53H87N3O17: 1038.6114;
found: 1038.6108 [M+H]+.
N-(3-Aminopropyl)-AmB (7):[33] To AmB (1) (300 mg, 0.324 mmol) in
DMF/MeOH (6:4, 30 mL) was added N-(9-fluorenyl-methoxycarbonyl)-
3-aminopropanal[72] (96 mg, 0.324 mmol) and NaBH3CN (122 mg,
1.95 mmol). After 18 h the reaction mixture was added dropwise to dieth-
yl ether (1 L). The yellow precipitate was filtered and washed with dieth-
yl ether (2”10 mL) and purified by flash chromatography (CHCl3/
MeOH/H2O 40:8:1) affording N-(9-fluorenylmethoxycarbonyl)-3-amino-
propyl-AmB (6) (73.7 mg, 61.2 mmol). The resulting yellow solid was dis-
solved in DMSO (0.720 mL) and piperidine (72 mL) was added. After 1 h
the reaction mixture was added dropwise to diethyl ether (100 mL). Fil-
tration and washing (2”5 mL) afforded 7 (20 mg, 6.8%) as an orange
N,N-Di-(4-aminobutyl)-AmB (4): To a solution of 2,2,2-trifluoro-N-(4-oxo-
butyl)acetamide[73] (160 mg, 0.860 mmol) and AmB (1) (200 mg,
0.216 mmol) in DMF (2.00 mL) was added NaBH3CN (54.0 mg,
0.860 mmol) followed by a drop of conc. HCl. After 16 h at room temper-
ature, Amberlite IRA-743 (500 mg) was added and the mixture was
stirred for an hour. After filtration, the solution was concentrated and
added dropwise to diethyl ether (250 mL). The yellow precipitate was fil-
tered and purified by flash chromatography (CHCl3/MeOH/H2O 40:8:1).
The isolated yellow solid was dissolved in THF (5.00 mL) and 1m LiOH
solution (2.00 mL, 2.00 mmol) was added at 08C. After 2 h, the solution
was acidified dropwise to pH 4 with diluted HCl. The mixture was con-
centrated and added slowly to diethyl ether (250 mL). The yellow precip-
itate was filtered and washed with diethyl ether (2”100 mL) providing
the desired compound 4 as a yellow solid (155 mg, 69%). 1H NMR
(500 MHz, [D6]DMSO): d = 6.48–5.99 (m, 13H), 5.42 (dd, J=15, 10 Hz,
1H), 5.23 (brs, 1H), 4.87–4.57 (m, 4H), 4.48–4.38 (m, 2H), 4.30–4.21 (m,
4H), 4.09–4.03 (m, 3H), 3.98–3.92 (m, 4H), 3.49 (brs, OH), 3.16–3.04 (m,
4H), 2.35–2.26 (m, 2H), 2.16 (d, J=6 Hz, 1H), 1.85–1.19 (m, 23H), 1.11
(d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92 pppm (d, J=7 Hz, 3H);
13C NMR (125 MHz, [D6]DMSO): d = 177.4, 170.5, 136.7, 133.8, 133.6,
133.3, 133.1, 132.4, 132.1, 131.8, 131.2, 128.2, 98.4, 96.8, 96.7, 77.1, 76.6,
1
solid. H NMR (500 MHz, [D6]DMSO): d = 6.46 (dd, J=14, 11 Hz, 1H),
6.42–6.22 (m, 8H), 6.18 (s, 1H), 6.16 (d, J=4 Hz, 1H), 6.12–6.06 (m,
1H), 6.05 (d, J=10 Hz, 1H), 6.00 (dd, J=15, 8 Hz, 1H), 5.74 (brs, 1H),
5.43 (dd, J=15, 10 Hz, 1H), 5.38 (brs, 1H), 5.26–5.19 (m, 1H), 4.85–4.58
(m, 7H), 4.45 (s, 2H), 4.43–4.35 (m, 2H), 4.30–4.22 (m, 4H), 4.09–4.03
(m, 2H), 4.02–3.95 (m, 2H), 3.82 (s, 2H), 3.93–3.22 (m, 14H), 3.15 (dd,
J=9, 6 Hz, 1H), 3.13–3.05 (m, 3H), 3.03–2.91 (m, 3H), 2.89–2.71 (m,
4H), 2.38–2.32 (m, 1H), 2.32–2.20 (m, 2H), 2.16 (d, J=6 Hz, 2H), 1.86–
1.76 (m, 2H), 1.75–1.66 (m, 2H), 1.66–1.21 (m, 12H), 1.14 (d, J=6 Hz,
3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92 ppm (d, J=7 Hz,
3H); 13C NMR (125 MHz, [D6]DMSO): d = 177.0, 170.5, 137.0, 136.6,
133.8, 133.6, 133.4, 133.1, 132.8, 132.4, 132.1, 131.8, 131.7, 131.2, 127.9,
100.1, 96.8, 77.9, 77.1, 73.8, 73.5, 70.3, 69.1, 68.7, 67.8, 67.4, 66.1, 65.6,
Chem. Eur. J. 2008, 14, 2465 – 2481
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2473

E. M. Carreira et al.
62.4, 58.5, 46.5, 44.7, 44.6, 44.3, 42.4, 41.9, 37.8, 35.0, 29.0, 26.7, 18.4, 18.1,
17.0, 16.8, 12.0 ppm; MALDI-FT: m/z: calcd for C50H80N2O17: 981.5530;
found: 981.5512 [M+H]+.
3H), 0.92 ppm (d, J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d =
177.6, 170.5, 157.1, 142.1, 136.7, 133.8, 133.6, 133.4, 133.1, 132.4, 132.1,
131.8, 131.2, 128.5, 97.6, 96.9, 77.1, 74.1, 73.9, 73.5, 69.1, 68.7, 68.4, 67.9,
66.1, 65.5, 65.1, 64.8, 63.6, 50.0, 45.9, 44.7, 42.7, 41.9, 41.0, 40.4, 35.7, 30.7,
29.0, 24.9, 23.1, 18.6, 18.4, 18.3, 18.0, 16.8, 12.0 ppm; MALDI-FT: m/z:
calcd for C53H88N7O17: 1094.6237; found: 1094.6231 [M+H]+.
N-(3-Aminopropyl)-N-(2-aminoethyl)-AmB (8): To a solution of N-(9-
fluorenylmethoxycarbonyl)-3-aminopropanal[72] (0.320 mg, 1.08 mmol) in
DMF (5.00 mL) and MeOH (3.00 mL) was added AmB (1) (1.00 g,
1.08 mmol). After 5 h, NaBH3CN (200 mg, 3.32 mmol) was added to the
mixture. After 16 h at room temperature, Amberlite IRA-743 (2.00 g)
was added and the mixture was stirred for an hour. After filtration, the
solution was concentrated down and added dropwise to diethyl ether
(250 mL). The yellow precipitate was filtered and purified by flash chro-
matography (CHCl3/MeOH/H2O 40:8:1) providing N-(9-fluorenylme-
thoxycarbonyl)-3-aminopropyl-AmB (6) as a yellow solid. To a solution
2,6-Diamino-N-[6-oxo-6-(piperazin-1-yl)-hexyl]-hexanamide-AmB (13):
To a solution of 6-amino-1-hexanoyl-(piperazinyl)-AmB[31] (12) (110 mg,
0.100 mmol) in DMF (2.00 mL) was added 2,6-di-[N-(9-fluorenylmethoxy-
carbonyl)]-lysine hydroxysuccinimide ester[76] (380 mg, 0.500 mmol) and
diisopropylethylamine (0.100 mL, 0.500 mmol). After 18 h at room tem-
perature, the solution was concentrated down and added dropwise to di-
ethyl ether (250 mL). The yellow precipitate was filtered and purified by
flash chromatography (CHCl3/MeOH/H2O 40:8:1). The isolated yellow
solid was dissolved in DMSO (3.00 mL) and piperidine (0.100 mL,
1.00 mmol) was added. After 2 h at room temperature, the solution was
added dropwise to diethyl ether (250 mL). The yellow precipitate was fil-
tered and washed with diethyl ether (2”100 mL) providing the desired
of
0.250 mmol) in DMF (3.00 mL) and MeOH (3.00 mL) was added to the
previously isolated (100 mg, 0.083 mmol). After 3 h, NaBH3CN
N-(9-fluorenylmethoxycarbonyl)-2-aminoacetaldehyde[75]
(70 mg,
6
(16.0 mg, 0.250 mmol) was added to the mixture. After 16 h at room tem-
perature, Amberlite IRA-743 (500 mg) was added and the mixture was
stirred for an hour. After filtration, the solution was concentrated and
added dropwise to diethyl ether (250 mL). The yellow precipitate was fil-
tered and purified by flash chromatography (CHCl3/MeOH/H2O 40:8:1).
The isolated yellow solid was dissolved in DMSO (5.00 mL) and piperi-
dine (0.200 mL, 2.10 mmol) was added. After 2 h at room temperature,
the solution was added dropwise to diethyl ether (250 mL). The yellow
precipitate was filtered and washed with diethyl ether (2”100 mL) pro-
compound 13 as
a
yellow solid (10 mg, 27%). 1H NMR (500 MHz,
[D6]DMSO): d = 7.78 (brs, 1H), 6.46–5.95 (m, 13H), 5.51 (dd, J=15,
10 Hz, 1H), 5.44 (dd, J=15, 10 Hz, 1H), 5.20 (brs, 1H), 5.13–5.11 (m,
1H), 4.79–4.70 (m, 3H), 4.48 (s, 1H), 4.29–4.14 (m, 4H), 4.06 (s, 2H),
3.90–3.79 (m, 2H), 3.38 (s, OH), 3.14–3.04 (m, 2H), 2.72–2.58 (m, 2H),
2.38–2.21 (m, 3H), 2.16 (d, J=6 Hz, 1H), 1.74–1.24 (m, 21H), 1.17 (d,
J=6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.91 ppm (d,
J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d = 174.9, 173.8, 170.4,
170.2, 137.5, 136.6, 133.9, 133.7, 133.6, 133.5, 133.2, 132.7, 132.3, 132.0,
131.8, 131.4, 128.3, 96.7, 77.1, 73.8, 73.5, 72.2, 69.3, 69.2, 68.8, 67.7, 67.6,
67.5, 67.3, 66.2, 66.1, 65.9, 65.5, 64.8, 54.6, 44.6, 44.3, 42.3, 42.0, 41.9, 35.0,
34.9, 30.7, 28.9, 26.1, 26.0, 25.6, 24.5, 22.3, 18.4, 18.2, 17.1, 16.9, 12.1,
12.0 ppm; MALDI-FT: m/z: calcd for C63H103N5O19: 1256.7145; found:
1256.7140 [M+Na]+.
viding the desired compound
8 as a yellow solid (22.0 mg, 26%).
1H NMR (500 MHz, [D6]DMSO): d = 6.48–5.97 (m, 13H), 5.70–5.50 (m,
1H), 5.46–5.40 (m, 1H), 5.23 (brs, 1H), 4.91–4.54 (m, 5H), 4.45–4.18 (m,
6H), 4.07–3.77 (m, 6H), 3.60–3.36 (m, 7H), 3.23–3.01 (m, 5H), 2.94–2.74
(m, 5H), 2.35–2.22 (m, 2H), 2.16 (d, J=6 Hz, 1H), 1.81–1.14 (m, 22H),
1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.91 ppm (d, J=7 Hz, 3H);
13C NMR (125 MHz, [D6]DMSO): d = 177.5, 170.5, 136.7, 133.9, 133.6,
133.1, 132.4, 132.3, 132.1, 132.0, 131.8, 131.2, 128.5, 96.8, 77.1, 74.2, 73.9,
73.8, 73.5, 73.4, 70.4, 69.3, 69.1, 68.8, 68.7, 68.6, 67.9, 67.8, 66.1, 65.6, 46.5,
45.6, 44.7, 44.4, 44.3, 42.5, 42.4, 41.9, 35.1, 29.0, 25.1, 23.8, 18.4, 18.2, 16.9,
12.0 ppm; MALDI-FT: m/z: calcd for C52H85N3O17: 1024.5930; found:
1024.5950 [M+H]+.
2-Amino-6-guanidine-N-[6-oxo-6-(piperazin-1-yl)-hexyl]hexanamide-
AmB (14): To a solution of 13 (50.0 mg, 0.0400 mmol) in DMF (2.00 mL)
was added
9
(8.00 mg, 0.0500 mmol) and diisopropylethylamine
(0.0170 mL, 0.100 mmol). After 18 h at room temperature, the solution
was concentrated down and added dropwise to diethyl ether (250 mL).
The yellow precipitate was filtered and washed with diethyl ether (2”
100 mL) providing the desired compound 14 as a yellow solid (10.0 mg,
20%). 1H NMR (500 MHz, [D6]DMSO): d = 7.60 (brs, 1H), 6.49–5.91
(m, 14H), 5.44 (dd, J=15, 10 Hz, 1H), 5.37 (brs, 1H), 5.21 (brs, 1H),
4.81–4.64 (m, 3H), 4.44–4.31 (m, 1H), 4.28–4.16 (m, 2H), 4.07–4.04 (m,
1H), 4.00–3.96 (m, 1H), 3.83–3.79 (m, 2H), 3.34 (s, OH), 3.09–3.06 (m,
2H), 2.32–2.24 (m, 3H), 2.16 (d, J=6 Hz, 1H), 1.85–1.22 (m, 18H), 1.17
(d, J=6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.91 ppm
(d, J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d = 174.7, 173.5,
170.5, 170.2, 157.1, 136.7, 136.1, 133.8, 133.6, 133.4, 133.1, 132.7, 132.4,
132.1, 131.8, 131.5, 131.1, 128.6, 96.9, 77.0, 74.4, 73.8, 73.6, 73.5, 69.2, 69.1,
68.7, 67.7, 67.4, 67.2, 66.1, 65.5, 65.4, 65.3, 64.9, 53.4, 46.1, 46.0, 44.7, 44.3,
43.6, 42.0, 35.0, 28.9, 28.5, 28.3, 25.1, 24.8, 24.5, 22.2, 22.0, 18.4, 18.1, 16.9,
13.8, 12.0 ppm; MALDI-FT: m/z: calcd for C64H105N7O19: 1298.7363;
found: 1298.7358 [M+Na]+.
Guanidine-AmB (10): To a solution of AmB (1) (295 mg, 0.320 mmol) in
DMF (3.00 mL) was added 1H-pyrazole-1-carboxamidine monohydro-
chloride (9) (50.0 mg, 0.340 mmol) and diisopropylethylamine (0.230 mL,
1.30 mmol). After 16 h at room temperature, the solution was concentrat-
ed down and added dropwise to diethyl ether (250 mL). The yellow pre-
cipitate was filtered and washed with diethyl ether (2”100 mL) providing
the desired compound 10 as a yellow solid (300 mg, 97%). 1H NMR
(500 MHz, [D6]DMSO): d = 6.47–6.06 (m, 12H), 6.00 (dd, J=14, 8 Hz,
1H), 5.77 (brs, 1H), 5.44 (dd, J=15, 10 Hz, 1H), 5.22 (brs, 1H), 4.76–
4.50 (m, 4H), 4.41–4.37 (m, 1H), 4.26 (brs, 1H), 4.19–4.15 (m, 1H), 4.07–
4.04 (m, 1H), 4.01–3.96 (m, 1H), 3.53 (brs, OH), 3.24–3.21 (m, 1H),
3.17–3.14 (m, 1H), 3.11–3.08 (m, 2H), 2.79–2.77 (m, 1H), 2.31–2.26 (m,
1H),2.17 (d, J=6 Hz, 1H), 1.85–1.26 (m, 18H), 1.18 (d, J=6 Hz, 3H),
1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92 ppm (d, J=7 Hz, 3H);
13C NMR (125 MHz, [D6]DMSO): d = 176.3, 170.5, 157.9, 136.7, 133.8,
133.6, 133.4, 133.1, 132.4, 132.1, 131.8, 131.2, 128.6, 97.0, 95.7, 77.1, 74.2,
73.9, 73.5, 72.7, 71.0, 70.1, 69.1, 68.8, 68.1, 67.7, 66.1, 65.4, 65.2, 56.1, 46.3,
44.7, 44.3, 42.3, 42.0, 40.4, 35.0, 29.0, 18.4, 17.9, 16.9, 12.0 ppm; MALDI-
FT: m/z: calcd for C48H75N3O17: 966.5175; found: 966.5169 [M+H]+.
Methyl 2,3,4-tri-O-acetyl-a-d-glucopyranoside (15): The following com-
pound was synthesized according to known procedures[41–43] starting from
a-methyl d-glucopyranoside affording compound 15 as white solid in
55% yield over three steps. M.p. 1098C; Rf = 0.42 (hexane/EtOAc 4:5);
[a]2D7 = +132.68 (c = 1.07, CHCl3); 1H NMR (300 MHz, CDCl3): d =
5.41 (dd, J=10, 10 Hz, 1H), 4.93 (dd, J=10, 10 Hz, 1H), 4.86 (d, J=
4 Hz, 1H), 4.76 (dd, J=10, 4 Hz, 1H), 3.69 (ddd, J=10, 4, 2 Hz, 1H),
3.60 (dd, J=13, 2 Hz, 1H), 3.49 (dd, J=13, 4 Hz, 1H), 3.31 (s, 3H), 2.74
(brs, 1H), 1.96 (s, 3H), 1.94 (s, 3H), 1.91 ppm (s, 3H); 13C NMR (CDCl3,
75 MHz): d = 170.1, 169.9, 169.8, 96.5, 70.7, 69.7, 69.2, 68.6, 68.5, 60.1,
55.1, 20.7, 20.4 ppm; IR (film): n˜ =3512, 2941, 1750, 1432, 1370, 1224,
1168, 1128, 1038, 929, 772, 600, 488 cmꢀ1; MALDI-FT: m/z: calcd for
C13H20O9: 343.1000; found: 343.1006 [M+Na]+; elemental analysis calcd
(%) for C13H20O9: C 48.75, H 6.29; found: C 48.75, H 6.20.
N,N-Di-(2-ethylguanidine)-AmB (11): To a solution of N,N-di-(2-amino-
ethyl)-AmB (2) (100 mg, 0.100 mmol) in DMF (2.00 mL) was added 9
(36.0 mg, 0.250 mmol) and diisopropylethylamine (0.350 mL, 2.00 mmol).
After 48 h at room temperature, the solution was concentrated down and
added dropwise to diethyl ether (250 mL). The yellow precipitate was fil-
tered and washed with diethyl ether (2”100 mL) providing the desired
compound 11 as a yellow solid (60.0 mg, 55%). 1H NMR (500 MHz,
[D6]DMSO): d = 7.60 (brs, 1H), 7.42 (brs, 2H), 6.52–6.02 (m, 13H),
5.43 (dd, J=15, 10 Hz, 1H), 5.23 (brs, 1H), 4.80–4.72 (m, 3H), 4.64 (s,
1H), 4.45–4.22 (m, 3H), 4.08–3.98 (m, 2H), 3.53 (m, 18H, OH), 3.15–
3.08 (m, 1H), 2.32–2.26 (m, 1H), 2.17 (d, J=6 Hz, 1H), 1.83–1.23 (m,
18H), 1.19 (d, J=6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz,
Methyl 3,4-di-O-acetyl-2-deoxy-2-[(benzyloxycarbonyl)amino]-a-d-gluco-
pyranoside (16): The following compound was synthesized starting from
2474
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 2465 – 2481

Natural Products
FULL PAPER
methyl 2-deoxy-2-[(benzyloxycarbonyl)amino]-a-d-glucopyranoside ac-
cording to known procedures[39,44–47] affording compound 16 in 60% yield
in three steps as white solid. M.p. 111–1138C; Rf = 0.26 (hexane/EtOAc
1:2); [a]2D4 = +101.28 (c = 1.05, CHCl3); 1H NMR (300 MHz, CDCl3):
d=7.36–7.29 (m, 5H), 5.26 (dd, J=11, 9 Hz, 1H), 5.13 (d, J=12 Hz,
1H), 5.07–4.96 (m, 3H), 4.76 (d, J=4 Hz, 1H), 4.02 (ddd, J=11, 10,
4 Hz, 1H), 3.76–3.70 (m, OH), 3.68 (dd, J=8, 3 Hz, 1H), 3.58 (ddd, J=
13, 6, 4 Hz, 1H), 3.38 (s, 3H), 2.40 (dd, J=8, 6 Hz, 1H), 2.05 (s, 3H),
1.90 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d = 170.8, 170.3, 155.6,
136.1, 128.4, 128.1, 128.0, 98.5, 71.0, 69.7, 68.7, 66.9, 61.1, 55.5, 53.8, 20.8,
20.7 ppm; IR (film): n˜ =3500, 3400, 3350, 2941, 1747, 1520, 1456, 1368,
1239, 1156, 1122, 1035 cmꢀ1; MALDI-FT: m/z: calcd for C19H25NO9:
434.1422; found: 434.1415 [M+Na]+; elemental analysis calcd (%) for
C19H25NO9: C 55.47, H 6.30, N 3.40; found: C 55.35, H 6.30, N 3.42.
C13H19KO11S: 429.0438; found 429.0430 [M+Na]+; elemental analysis
calcd (%) for C 36.96, H 4.53; found: C 36.76, H 4.62.
Methyl 6-methylsulfonyl-3,4-di-O-acetyl-2-deoxy-2-[(benzyloxycarbonyl)-
amino]-a-d-glucopyranoside: Triethylamine (6.20 mL, 44.7 mmol) and
methylsulfonyl chloride (3.50 mL, 44.7 mmol) was added at room temper-
ature to a solution of 16 (16.7 g, 40.6 mmol) in CH2Cl2 (400 mL). After
stirring for 3 h the reaction volume was concentrated to half the volume.
The concentrated reaction mixture was washed with water (2”200 mL).
The combined organic layers were washed with saturated NaCl solution
(400 mL), dried over MgSO4 and concentrated. The isolated mesylate
(19.9 g, quant.) was obtained as a white solid. The NMR showed that it
was pure enough for the next reaction. An analytical sample was purified
by flash chromatography (hexane/EtOAc 2:1). M.p. 113–1148C; Rf
=
0.50 (hexane/EtOAc 1:2); [a]2D2 = +95.48 (c = 0.97, CHCl3); 1H NMR
(300 MHz, CDCl3): d = 7.36–7.30 (m, 5H), 5.21 (dd, J=11, 9 Hz, 1H),
5.12 (d, J=12 Hz, 1H), 5.03 (d, J=12 Hz, 1H), 5.03 (dd, J=10, 9 Hz,
1H), 4.98 (brs, 1H), 4.76 (d, J=4 Hz, 1H), 4.27 (d, J=4 Hz, 2H), 4.05
(dd, J=11, 4 Hz, 1H), 4.00 (dd, J=4, 4 Hz, 1H), 3.41 (s, 3H), 3.05 (s,
3H), 2.04 (s, 3H), 1.90 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d =
170.6, 169.3, 155.5, 128.4, 127.9, 128.1, 98.4, 71.0, 136.0, 68.1, 67.5, 67.0,
55.7, 53.6, 37.7, 20.6, 20.7 ppm; IR (film): n˜ =3358, 2941, 1749, 1522,
1456, 1360, 1235, 1176, 1043, 1010, 944 cmꢀ1; MS MALDI-FT: m/z: calcd
for C20H27NO11S: 512.1197; found 512.1192 [M+Na]+; elemental analysis
calcd (%) for C20H27NO11S: C 49.07, H 5.56, N 2.86; found: C 48.84, H
5.64, N 2.82.
Methyl 6-methylsulfonyl-2,3,4-tri-O-acetyl-a-d-glucopyranoside: To a so-
lution of 15 (834 mg, 2.60 mmol) in CH2Cl2 (26 mL) was added pyridine
(3.15 mL, 39.0 mmol) and methylsulfonyl chloride (503 mL, 6.50 mmol)
and was stirred at 08C for 1 h. After another 3 h stirring at room temper-
1
ature the reaction mixture was concentrated to = of the volume, saturat-
3
ed NaHCO3 solution (10 mL) was added, and the water layer was ex-
tracted with EtOAc (4”20 mL). The combined organic layers were
washed with saturated NaCl solution (20 mL) and dried over MgSO4.
The organic layer was concentrated and flash chromatography (hexane/
EtOAc 1:1) afforded the desired mesylate (983 mg, 95%) as a white
solid. M.p. 1138C; Rf = 0.26 (hexane/EtOAc 1:1); [a]2D5 = +129.38 (c =
0.91, CHCl3); 1H NMR (300 MHz, CDCl3): d = 5.47 (dd, J=10, 10 Hz,
1H), 5.02 (dd, J=10, 10 Hz, 1H), 4.95 (d, J=4 Hz, 1H), 4.86 (dd, J=10,
4 Hz, 1H), 4.27 (d, J=4 Hz, 2H), 4.05 (ddd, J=7, 4, 4 Hz, 1H), 3.41 (s,
3H), 3.05 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.00 ppm (s, 3H); 13C NMR
(75 MHz, CDCl3): d = 169.9, 169.8, 169.5, 96.7, 70.6, 69.8, 68.3, 67.0,
66.8, 55.7, 37.7, 20.8, 20.7 ppm; IR (film): n˜ =2942, 1750, 1360, 1223,
1175, 1035, 969, 942, 829, 751 cmꢀ1; ESI-FT: m/z: calcd for C14H22O11S:
421.0775; found: 421.0769 [M+Na]+; elemental analysis calcd (%) for
C14H22O11S: C 42.21, H 5.57; found: C 42.02, H 5.61.
Methyl
6-acetylthio-3,4-di-O-acetyl-2-deoxy-2-[(benzyloxycarbonyl)-
amino]-a-d-glucopyranoside: Potassium thioacetate (13.9 g, 122 mmol)
was added at room temperature to a solution of the mesylate (19.9 g,
40.6 mmol) in DMF. After stirring at 558C for 18 h DMF was distilled off
under high vacuum until a volume of 50 mL was obtained. To the mixture
was added CH2Cl2 (500 mL) and this was washed with water (3”500 mL)
and with saturated NaCl (400 mL) and dried over MgSO4. The organic
phase was concentrated and flash chromatography (hexanes/EtOAc 2:1)
and recrystallization from ethanol afforded the thioacetate (14.5 g, 76%)
as light orange needles. M.p. 978C; Rf = 0.27 (hexane/EtOAc 1:1); [a]D28
=
Methyl 6-acetylthio-2,3,4-tri-O-acetyl-a-d-glucopyranoside: To a solution
of the mesylate (938 mg, 2.35 mmol) in ethanol was added potassium thio-
acetate. This was stirred for 3 h at reflux. After cooling to room tempera-
ture, water (75 mL) was added. The water layer was extracted with
EtOAc (4”50 mL), the combined organic layers were washed with satu-
rated NaCl solution (50 mL) and dried over MgSO4. The organic layer
was concentrated and flash chromatography (hexane/EtOAc 2:1) afford-
ed the thioacetate (881 mg, 99%) as a green oil. Rf = 0.40 (hexane/
EtOAc 1:1); [a]2D6 = +109.58 (c = 0.84, CHCl3); 1H NMR (300 MHz,
CDCl3): d = 5.42 (dd, J=10, 9 Hz, 1H), 4.94 (dd, J=10, 10 Hz, 1H),
4.87 (d, J=4 Hz, 1H), 4.83 (dd, J=10, 4 Hz, 1H), 3.90 (ddd, J=10, 7,
3 Hz, 1H), 3.38 (s, 3H), 3.19 (dd, J=14, 3 Hz, 1H), 3.06 (dd, J=14, 7 Hz,
1H), 2.33 (s, 3H), 2.05 (s, 3H), 1.98 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3): d = 194.4, 170.0, 169.9, 169.8, 96.5, 70.9, 70.8, 70.0, 68,2, 55.4,
30.5, 30.1, 20.8, 10.8 ppm; IR (film): n˜ =2360, 2341, 1750, 1697, 1369,
1225, 1044, 668 cmꢀ1; ESI-FT: m/z: calcd for C15H22O9S: 401.0877; found:
401.0871 [M+Na]+; elemental analysis calcd (%) for C15H22O9S C 47.61,
H 5.86; found: C 47.54, H 5.87.
+85.88 (c = 0.94, CHCl3); 1H NMR (300 MHz, CDCl3): d = 7.39–7.28
(m, 5H), 5.15 (dd, J=11, 9 Hz, 1H), 5.12 (d, J=12 Hz, 1H), 5.03 (d, J=
12 Hz, 1H), 4.97 (brs, 1H), 4.96 (dd, J=10, 9 Hz, 1H), 4.68 (d, J=10 Hz,
1H), 4.00 (ddd, J=10, 10, 4 Hz, 1H), 3.84 (ddd, J=10, 8, 3 Hz, 1H), 3.37
(s, 3H), 3.21 (dd, J=14, 3 Hz, 1H), 3.02 (dd, J=14, 8 Hz, 1H), 2.34 (s,
3H), 2.07 (s, 3H), 1.89 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d =
194.3, 170.7, 169.5, 155.5, 136.0, 128.3, 128.0, 127.9, 98.2, 71.1, 70.7, 68.7,
66.9, 55.3, 53.7, 30.4, 30.2, 20.7, 20.6 ppm; IR (film): n˜ =3343, 2950, 1748,
1697, 1519, 1366, 1238, 1119, 1044, 747, 699, 627 cmꢀ1; HRMS MALDI:
m/z: calcd for C21H27NO9S: 492.1299; found: 492.1299 [M+Na]+; elemen-
tal analysis calcd (%) for C21H27NO9S: C 53.72, H 5.80, N 2.98; found:
C 53.72, H 5.79, N 3.09.
Methyl 3,4-di-O-acetyl-2-deoxy-2-[(benzyloxycarbonyl)amino]-a-d-gluco-
pyranoside-6-sulfonate potassium salt (18): To a solution of the thioace-
tate (6.80 g, 14.5 mmol) in acetic acid (73.0 mL) was added potassium
acetate (1.70 g, 17.4 mmol) and dropwise hydrogen peroxide (20.0 mL,
174 mmol) and the reaction mixture was stirred for 24 h at 408C. Then
potassium sulfite (30.0 g, 191 mmol) was added to quench the hydrogen
peroxide. Filtration, concentration and flash chromatography afforded
the desired compound (18) (5.90 g, 79%) as a white solid. M.p. 63–648C;
Methyl 2,3,4-tri-O-acetyl-a-d-glucopyranoside-6-sulfate potassium salt
(17): 30% H2O2 (2.16 mL, 19.1 mmol) and potassium acetate (187 mg,
1
1.91 mmol) were added to
a
solution of the thioacetate (602 mg,
Rf = 0.26 (CH2Cl2/MeOH 5:1); H NMR (300 MHz, CD3OD): d = 7.41–
7.27 (m, 5H), 7.03 (d, J=10 Hz, NH), 5.20 (dd, J=11, 9 Hz, 1H), 5.13 (d,
J=13 Hz, 1H), 5.00 (d, J=13 Hz, 1H), 4.81 (d, J=10 Hz, 1H), 4.71 (d,
J=4 Hz, 1H), 4.31 (ddd, J=10, 5, 5 Hz, 1H), 3.95 (ddd, J=11, 10, 6 Hz,
1H), 3.49 (s, 3H), 2.97 (dd, J=4, 4 Hz, 2H), 2.01 (s, 3H), 1.81 ppm (s,
3H); 13C NMR (75 MHz, CD3OD): d = 172.0, 171.8, 158.4, 138.3, 129.5,
129.1, 128.8, 99.6, 73.1, 72.9, 67.6, 67.4, 56.3, 55.1, 53.6, 20.7, 20.6 ppm; IR
(neat): n˜ = 3347, 2947, 2843, 1732, 1529, 1451, 1372, 1217, 1031, 929, 742,
697 cmꢀ1; MALDI-FT: m/z: calcd for C19H24KNO11S: 536.0599; found:
536.0589 [M+Na]+.
1.59 mmol) in glacial acetic acid (8.00 mL). After 9 h at 508C the reaction
mixture was quenched by the addition of potassium sulfite (319 mg,
17.5 mmol). The reaction was concentrated and flash chromatography
(CH2Cl2/MeOH 6:1) afforded the desired compound 17 (563 mg, 84%
mmol) as a white solid. M.p. 1228C; Rf = 0.43 (CH2Cl2/MeOH 5:1);
1H NMR (300 MHz, CDCl3): d = 5.43 (dd, J=10, 10 Hz, 1H), 5.01 (dd,
J=10, 9 Hz, 1H), 4.99 (s, 1H), 4.92 (dd, J=10, 4 Hz, 1H), 4.38–4.20 (m,
1H), 3.44 (s, 3H), 3.32–3.08 (m, 2H), 2.07 (s, 3H), 2.05 (s, 3H), 1.99 ppm
(s, 3H); 13C NMR (75 MHz, CDCl3): d = 170.8, 170.3, 169.8, 96.5, 70.9,
70.6, 70.3, 65.7, 55.8, 50.8, 20.9, 20.8 ppm; IR (KBr): n˜ = 3487, 2950,
2849, 1752, 16.46, 1574, 1374, 1224, 1123, 1037, 931, 907, 882, 824, 792,
Methyl 6-(chlorosulfonyl)-2,3,4-tri-O-acetyl-a-d-glucopyranoside: Tri-
phenyl phosphine (1.55 g, 5.92 mmol) and sulfuryl chloride (475 mL,
5.92 mmol) were added at 08C to a solution of 17 (500 mg, 1.18 mmol) in
754, 631, 598, 567, 535, 489, 470 cmꢀ1
; ESI-FT: m/z: calcd for
Chem. Eur. J. 2008, 14, 2465 – 2481
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2475

E. M. Carreira et al.
CH2Cl2 (12 mL). After 3.5 h at room temperature solvents were evapo-
rated and purification over a short silica plug (hexane/EtOAc 1:1) afford-
5H), 5.90–5.84 (m, 1H), 5.81–5.75 (m, 1H), 5.21 (dd, J=11, 9 Hz, 1H),
5.11 (d, J=12 Hz, 1H), 5.04–4.99 (m, 2H), 4.89 (t, J=10 Hz, 1H), 4.77
(d, J=3 Hz, 1H), 4.54 (dd, J=7, 6 Hz, 1H), 4.32 (ddd, J=10, 10, 3 Hz,
1H), 4.22 (s, 4H), 4.00 (dt, J=10, 3 Hz, 1H), 3.49 (s, 3H), 3.23 (dd, J=
15, 10 Hz, 1H), 3.10 (dd, J=15, 2 Hz, 1H), 3.17–3.07 (m, 2H), 2.32–2.22
(m, 2H), 2.04 (s, 3H), 1.89 (s, 3H), 1.68 (tt, J=15, 8 Hz, 2H), 1.59 (tt,
J=15, 8 Hz, 2H), 1.49–1.33 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d
= 170.8, 170.5, 169.5, 155.5, 135.9, 128.3, 128.0, 127.8, 126.2, 124.6, 98.4,
70.7, 70.6, 66.9, 65.5, 56.0, 53.6, 53.3, 52.8, 52.0, 43.1, 33.9, 29.7, 26.1, 23.7,
20.7, 20.5 ppm; HRMS MALDI: m/z: calcd for C29H41N3O11S: 662.2354;
found: 662.2343 [M+Na]+.
ed the sulfonyl chloride (175 mg, 43%) as a white solid. Rf
= 0.54
(hexane/EtOAc 1:1); 1H NMR (300 MHz, CDCl3): d = 5.51 (dd, J=10,
9 Hz, 1H), 4.98 (d, J=4 Hz, 1H), 4.88 (dd, J=10, 10 Hz, 1H), 4.85 (dd,
J=10, 5 Hz, 1H), 4.54 (ddd, J=10, 9, 2, 1H), 3.96 (dd, J=15, 10 Hz,
1H), 3.79 (dd, J=15, 2 Hz, 1H), 3.49 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H),
2.01 ppm (s, 3H); 13C NMR (75 MHz, CDCl3): d = 170.1, 170.0, 169.7,
96.8, 70.7, 70.3, 69.4, 66.3, 64.7, 56.2, 20.6 ppm; ESI-FT: m/z: calcd for
C26H28O6: 425.0280; found: 425.0273 [M+Na]+.
Methyl
methyl-2,3,4-tri-O-acetyl-a-d-glucopyranoside: Triethylamine (200 mL,
1.43 mmol) and [6-(2,5-dihydro-1H-pyrrol-1-yl)-6-oxohexyl]amine
6-((N-(6-(2,5-dihydro-1H-pyrrol-1-yl)-6-oxohexyl)sulfamoyl)-
Methyl
6-((N-(6-(3,4-dihydroxypyrrolidin-1-yl)-6-oxohexyl)sulfamoyl)-
methyl-3,4-di-O-acetyl-2-deoxy-2-[(benzyloxycarbonyl)amino]-a-d-gluco-
pyranoside (20): The isolated oil (1.32 g, 2.06 mmol) was dissolved in ace-
tone/tert-butanol/water 15:6:2 (20 mL) was added N-methylmorpoline
oxide (560 mg, 4.12 mmol) and potassium osmate (76.0 mg, 0.210 mmol).
After 18 h at room temperature the reaction was quenched by adding
sodium thiosulfate (360 mg, 2.10 mmol) and Florisil (360 mg). This was
stirred for an additional hour. Filtration followed by flash chromatogra-
phy (CH2Cl2/MeOH 20:1) afforded the desired product 20 (1.27 g, 91%)
as a colorless oil. Rf = 0.38 (CH2Cl2/MeOH 10:1); 1H NMR (300 MHz,
CDCl3): d = 7.38–7.28 (m, 5H), 5.20 (dd, J=10, 10 Hz, 1H), 5.15–5.08
(m, 2H), 5.03 (d, J=12 Hz, 1H), 4.94 (dd, J=15, 10 Hz, 1H), 4.89–4.81
(m, 1H), 4.77 (dd, J=8, 4 Hz, 1H), 4.34 (dd, J=10, 2 Hz, 1H), 4.30–4.18
(m, 2H), 3.99 (ddt, J=11, 3, 4 Hz, 1H), 3.65 (dd, J=11, 6 Hz, 1H), 3.61–
3.53 (m, 1H), 3.48 (s, 3H), 3.44 (dd, J=11, 6 Hz, 1H), 3.27 (ddd, J=15,
10, 10 Hz, 1H), 3.18–3.07 (m, 3H), 2.30–2.20 (m, 2H), 2.04 (s, 3H), 1.89
(s, 3H), 1.86–1.71 (m, 2H), 1.70–1.52 (2H), 1.47–1.34 ppm (m, 2H);
13C NMR (CDCl3, 75 MHz): d = 172.0, 170.6, 169.7, 155.8, 135.9, 128.4,
128.2, 127.9, 98.4, 71.4, 70.8, 69.9, 67.1, 65.6, 56.2, 53.7, 52.3, 51.1, 50.4,
43.0, 33.7, 33.7, 29.4, 26.0, 23.8, 23.7, 20.8, 20.6 ppm; IR (neat): n˜ = 3316,
(63.0 mg, 344 mmol) were added to a solution of the sulfonyl chloride
(115 mg, 287 mmol) in CH2Cl2 (3.0 mL).[31] After stirring for 2.5 h at room
temperature the solvent was evaporated and the crude was purified im-
mediately by flash chromatography (hexane/EtOAc 1:1
!
hexane/
EtOAc 1:10), which afforded the desired compound (95.0 mg, 61%) as a
colorless oil. Rf = 0.40 (CH2Cl2/MeOH 10:1); [a]2D4 = +73.08 (c = 1.05,
CHCl3); 1H NMR (300 MHz, CDCl3): d = 5.85–5.72 (m, 2H), 5.43 (dd,
J=10, 9 Hz, 1H), 4.94 (d, J=4 Hz, 1H), 4.91 (d, J=3 Hz, 1H), 4.82 (dd,
J=10, 10 Hz, 1H), 4.79 (dd, J=10, 3 Hz, 1H), 4.33 (ddd, J=10, 10, 3 Hz,
1H), 4.18 (s, 4H), 3.46 (s, 3H), 3.21 (dd, J=15, 10 Hz, 2H), 3.14–3.03 (m,
2H), 2.24 (t, J=7 Hz, 2H), 2.03 (s, 3H), 2.01 (s, 3H), 1.96 (s, 3H), 1.70–
1.51 (m, 4H), 1.45–1.33 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d =
171.1, 170.0, 169.80, 169.7, 126.2, 124.7, 96.6, 71.0, 70.5, 69.4, 65.0, 56.0,
53.2, 52.7, 52.0, 43.0, 33.8, 29.6, 26.0, 23.6, 20.5 ppm; IR (film): n˜ =2938,
2860, 1750, 1639, 1617, 1433, 1369, 1326, 1221, 1149, 1044, 771 cmꢀ1
;
MALDI-FT: m/z: calcd for C23H36N2O11S: 571.1932; found: 571.1923
[M+Na]+.
Methyl 6-((N-(6-(3,4-dihydroxypyrrolidine-1-yl)-6-oxohexyl)sulfamoyl)-
methyl-2,3,4-tri-O-acetyl-a-d-glucopyranoside (19): The above isolated
oil (95.0 mg, 173 mmol) was dissolved in acetone/water/tert-butanol 15:6:2
(3.5 mL) and sequentially N-methyl morpholine oxide (47.0 mg,
346 mmol) and potassium osmate dihydrate (6.5 mg, 17 mmol) were
added. After stirring at room temperature for 16 h the reaction mixture
was quenched with sodium dithionite (30.0 mg, 173 mmol) and Florisil
(30.0 mg) by stirring for an additional 30 min. The reaction mixture was
filtrated and after concentration of the filtrate, the compound was puri-
fied by flash chromatography (CH2Cl2/MeOH 15:1). The desired product
19 (93.0 mg, 92%) was obtained as a colorless oil. Rf = 0.28 (CH2Cl2/
MeOH 10:1); [a]2D5 = +56.18 (c = 1.01, CHCl3); 1H NMR (300 MHz,
CDCl3): d = 5.43 (dd, J=10, 9 Hz, 1H), 5.18–5.10 (m, 1H), 4.93 (d, J=
4 Hz, 1H), 4.85 (dd, J=10, 10 Hz, 1H), 4.81 (dd, J=10, 3 Hz, 1H), 4.35
(ddd, J=10, 10, 3 Hz, 1H), 4.31–4.20 (m, 2H), 3.73–3.66 (m, NH), 3.65–
3.55 (m, 2H), 3.48 (s, 3H), 3.24 (dd, J=15, 10 Hz, 1H), 3.20–3.02 (m,
3H), 2.41 (t, J=4 Hz, 1H), 2.27–2.20 (m, 2H), 2.07 (s, 3H), 2.04 (s, 3H),
1.99 (s, 3H), 1.68–1.50 (m, 4H), 1.44–1.32 ppm (m, 2H); 13C NMR
(75 MHz, CDCl3): d = 172.3, 170.3, 169.9, 169.8, 96.7, 71.3, 71.0, 70.6,
69.8, 69.4, 65.0, 56.0, 55.2, 52.1, 51.0, 50.3, 46.2, 42.9, 33.6, 29.5, 25.9, 23.7,
20.6, 20.5 ppm; IR (film): n˜ =3313, 2939, 1749, 1620, 1452, 1369, 1326,
1220, 1148, 1043, 772 cmꢀ1; MALDI-FT: m/z: calcd for C23H38N2O13S:
605.1987; found: 605.1978 [M+Na]+.
2940, 1741, 1617, 1525, 1451, 1371, 1323, 1222, 1145, 1039, 917, 737 cmꢀ1
;
HRMS MALDI: m/z: calcd for C29H43N3O13S: 674.2589; found: 674.2636
[M+H]+.
Methyl
6-(N-(6-(3,4-dihydroxypyrrolidine-1-yl)-6-oxohexyl)sulfamoyl)-
methyl-3,4-di-O-acetyl-2-deoxy-2-{[(9H-fluoren-9-ylmethyl)carbonyl]ami-
no}-a-d-glucopyranoside (21): 10% Pd/C (19.3 mg, 0.180 mmol) was
added to a solution of 20 (1.20 g, 1.80 mmol) under inert atmosphere in
methanol (18.0 mL). Then a hydrogen-balloon (1.0 atm) was applied to
the reaction, evacuation of the inert atmosphere, which was replaced by
hydrogen, was performed three times. The reaction mixture was stirred
vigorously for 18 h. Filtration over celite and concentration afforded the
crude product and was used without further purification in the next step.
The isolated product was dissolved in water/dioxane 1:1 (18.0 mL) and
sodium bicarbonate (0.580 g, 5.40 mmol) and N-(9H-fluoren-2-ylmethox-
yoxycarbonyl)succinimide (670 mg, 2.00 mmol) were added. After 30 min
a white solid crashed out, this was filtered and washed with CH2Cl2
(60 mL) and water (60 mL). The organic phase was separated from the
water layer, and the water layer was extracted with EtOAc (3”100 mL).
The combined organic layers were washed with saturated NaCl (50 mL)
and dried over MgSO4. The organic phase was concentrated and flash
chromatography (CH2Cl2/MeOH 10:1) afforded the desired product 21
(14.5 g, 26% over 2 steps) as a white foam. M.p. 92–958C; Rf = 0.20
(CH2Cl2/MeOH 10:1); [a]D24
= +58.38 (c =
1.00, CHCl3); 1H NMR
Methyl
6-((N-(6-(2,5-dihydro-1H-pyrrol-1-yl)-6-oxohexyl)sulfamoyl)-
(300 MHz, CDCl3): d = 7.77 (d, J=7 Hz, 2H), 7.55 (dd, J=7, 6 Hz, 2H),
7.41 (dd, J=7, 7 Hz, 2H), 7.32 (dd, J=7, 7 Hz, 2H), 5.25 (dd, J=10,
2 Hz, 1H), 5.18 (d, J=10 Hz, 1H), 4.94 (dt, J=14, 10 Hz, 1H), 4.78 (dd,
J=9, 3 Hz, 1H), 4.70 (dd, J=13, 7 Hz, 1H), 4.42 (dd, J=10, 7 Hz, 1H),
4.36–4.14 (m, 5H), 4.01 (tt, J=10, 3 Hz, 1H), 3.68–3.62 (m, 2H), 3.61
(dd, J=10, 6 Hz, 1H), 3.52 (d, J=2 Hz, 4H), 3.45 (d, J=5 Hz, 1H), 3.41
(d, J=6 Hz, 1H), 3.32–3.18 (m, 2H), 3.16–3.08 (m, 3H), 2.25 (dt, J=7,
2 Hz, 2H), 2.06 (s, 3H), 1.96 (s, 3H), 1.70–1.53 (m, 4H), 1.42 ppm (dd,
J=7, 7 Hz, 2H); 13C NMR (75 MHz, CDCl3): d = 172.1, 170.7, 169.7,
155.8, 143.4, 141.1, 127.7, 127.0, 124.9, 119.9, 98.4, 71.4, 70.8, 70.6, 69.9,
67.2, 65.6, 56.2, 53.8, 52.3, 51.1, 50.4, 47.0, 43.0, 33.7, 29.5, 26.0, 23.8,
20.8 ppm; IR (film): n˜ =3330, 2942, 1747, 1619, 1523, 1450, 1367, 1325,
1240, 1148, 1044, 758 cmꢀ1; MALDI-FT: m/z: calcd for C36H47N3O13S:
784.2722; found: 784.2735 [M+Na]+.
methyl-3,4-di-O-acetyl-2-deoxy-2-[(benzyloxycarbonyl)amino]-a-d-gluco-
pyranoside:[48] Triphenylphosphine (14.3 g, 55.0 mmol) was added por-
tionwise at 08C to solution of 18 (5.60 g, 10.9 mmol) in CH2Cl2. Then sul-
furyl chloride (4.40 mL, 55.0 mmol) was added dropwise and the resulting
mixture was stirred for 20 h at room temperature. The mixture was con-
centrated and purified over a short silica plug with hexane/EtOAc 1:1.
From the resulting unstable sulfonyl chloride (4.28 g, 80%) a portion
(1.90 g, 3.85 mmol) was immediately dissolved again in CH2Cl2 (35 mL).
Sequentially triethylamine (2.44 mL, 17.5 mmol) and [6-(2,5-dihydro-1H-
pyrrol-1-yl)-6-oxohexyl]amine[31] (650 mg, 3.50 mmol) were added. After
stirring for 10 min at room temperature concentration of the solvent fol-
lowed by flash chromatography (CH2Cl2/MeOH 40:1) afforded the de-
sired product (1.40 g, 50% over 2 steps) as a colorless oil. Rf = 0.28
(CH2Cl2/MeOH 20:1); 1H NMR (300 MHz, CDCl3): d = 7.38–7.28 (m,
2476
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 2465 – 2481

Natural Products
FULL PAPER
Methyl N-[(6-piperazinyl-6-oxohexylsulfamoyl)-methyl-2,3,4-tri-O-acetyl-
a-d-glucopyranosyl]-AmB: Silica gel (312 mg) was suspended in CH2Cl2
(2.0 mL) and water (312 mL), then sodium periodate (44.0 mg, 203 mmol)
was added to give a flocky suspension. To the reaction mixture 19
(59.0 mg, 101 mmol) was added and stirred at room temperature for 1 h.
The suspension was filtered and the filter cake washed with CH2Cl2 (2”
5.0 mL) and EtOAc (2”5.0 mL). The solvents were removed under re-
duced pressure. To the resulting white solid, AmB (78.0 mg, 84.0 mmol)
was added, followed by DMF (1.7 mL), conc. HCl (1 drop) and
NaBH3CN (7.0 mg, 0.11 mmol) and the suspension that quickly cleared
was stirred for 16 h. Amberlite IRA-743 (70.0 mg) was added and the
suspension was stirred for an additional 1 h. After filtration the solvent
was removed and the solid purified by flash chromatography (CHCl3/
MeOH/H2O 20:6:1). The resulting yellow solid (91 mg, 73%) was dried
washed with CH2Cl2 (2”5.0 mL) and EtOAc (2”5.0 mL). The filtrate
was concentrated and the dialdehyde used without further purification.
To sodium cyanoborohydride (4.4 mg, 71 mmol) in DMF (1.1 mL) was
added the dialdehyde and AmB (51 mg, 55 mmol), followed by one drop
of conc. HCl. This mixture was stirred for 18 h. The solvent was evapo-
rated and flash chromatography (CHCl3/MeOH/H2O 40:8:1) afforded the
protected AmB derivative (54 mg, 59%) as a yellow solid. The isolated
yellow solid (25 mg, 15 mmol) was dissolved in MeOH/water 3:2
(0.30 mL) and potassium carbonate was added at room temperature.
After stirring for 20 min at room temperature piperidine (45 mL,
0.45 mmol) was added and an additional 1 h was stirred. Evaporation of
the solvent and flash chromatography (CHCl3/MeOH/H2O 10:6:1) af-
forded 23 (13 mg, 66%) as
a
yellow solid. 1H NMR (500 MHz,
[D6]DMSO): d = 6.87 (t, J=6 Hz, 1H), 6.49–6.20 (m, 8H), 6.17 (t, J=
4 Hz, 2H), 6.12–6.02 (m, 2H), 5.95 (dd, J=15, 9 Hz, 1H), 5.82 (s, 1H),
5.43 (dd, J=15, 10 Hz, 1H), 5.36 (s, 1H), 5.21 (d, J=5 Hz, 1H), 4.79 (s,
2H), 4.75 (s, 1H), 4.65 (s, 1H), 4.59 (s, 1H), 4.50–4.33 (m, 4H), 4.32 (s,
1H), 4.27–4.14 (m, 3H), 4.10–4.03 (m, 1H), 4.02–3.94 (m, 1H), 3.86–3.77
(m, 3H), 3.58–3.36 (m, 14H), 3.15–3.05 (m, 4H), 2.99–2.80 (m, 5H),
2.76–2.68 (m, 2H), 2.67–2.61 (m, 1H), 2.38 (d, J=10 Hz, 1H), 2.33–2.21
(m, 3H), 2.17 (d, J=6 Hz, 1H), 2.10–1.99 (m, 1H), 1.94–1.82 (m, 2H),
1.72 (d, J=7.0 Hz, 1H), 1.62–1.51 (m, 4H), 1.51–1.43 (m, 5H), 1.42–1.36
(m, 2H), 1.35–1.22 (m, 6H), 1.18 (d, J=6 Hz, 3H), 1.11 (d, J=6 Hz,
3H), 1.04 (d, J=6 Hz, 3H), 0.98 (t, J=7 Hz, 1H), 0.91 ppm (d, J=7 Hz,
3H); 13C NMR (125 MHz, [D6]DMSO): d = 174.9, 170.4, 170.2, 136.7,
136.6, 133.8, 133.6, 133.4, 133.1, 132.3, 132.1, 131.8, 131.7, 131.1, 128.5,
97.0, 96.9, 77.1, 77.0, 74.2, 73.7, 73.5, 73.2, 73.1, 72.6, 69.6, 69.1, 68.8, 67.6,
67.3, 66.1, 65.3, 65.1, 57.2, 55.4, 54.8, 53.1, 50.6, 49.5, 46.1, 46.0, 44.7, 44.3,
44.2, 42.3, 42.2, 42.1, 42.0, 41.9, 36.7, 35.0, 32.1, 29.3, 29.0, 28.9, 25.8, 25.6,
24.4, 18.4, 18.1, 16.9, 12.0, 10.5 ppm; MALDI-FT: m/z: calcd for
C64H104N4O24S: 1367.6653; found: 1367.663 [M+Na]+.
by lyophilisation. Rf
=
0.67 (CHCl3/MeOH/H2O 10:6:1); 1H NMR
(500 MHz, [D6]DMSO): d = 6.98 (dd, J=6, 6 Hz, 1H), 6.48–6.20 (m,
8H), 6.16 (dd, J=4, 4 Hz, 2H), 6.12–6.03 (m, 2H), 5.98–5.91 (m, 1H),
5.80 (s, 1H), 5.44 (dd, J=15, 10 Hz, 1H), 5.32 (s, 1H), 5.27 (dd, J=10,
10 Hz, 1H), 5.23–5.18 (m, 1H), 4.93–4.84 (m, 3H), 4.81–4.72 (m, 3H),
4.65–4.58 (m, 1H), 4.40–4.3 (m, 3H), 4.27–4.15 (m, 3H), 4.14–4.09 (m,
1H), 4.08–4.03 (m, 1H), 4.01–3.93 (m, 1H), 3.81 (s, 1H), 3.57–3.49 (m,
1H), 3.49–3.42 (m, 2H), 3.38–3.28 (m, 4H), 3.24 (dd, J=15, 8 Hz, 2H),
3.21 (d, J=3 Hz, 1H), 3.14–3.06 (m, 3H), 2.97–2.80 (m, 4H), 2.37 (d, J=
9 Hz, 1H), 2.30–2.23 (m, 3H), 2.17 (d, J=6 Hz, 2H), 2.01 (s, 3H), 2.00 (s,
3H), 1.96 (s, 3H), 1.93–1.82 (m, 3H), 1.78–1.70 (m, 1H), 1.63–1.36 (m,
14H), 1.35–1.21 (m, 8H), 1.18 (d, J=6 Hz, 3H), 1.11 (d, J=6 Hz, 3H),
1.04 (d, J=6 Hz, 3H), 0.91 (d, J=7 Hz, 3H); 13C NMR (125 MHz,
[D6]DMSO): d = 170.4, 170.1, 169.5, 169.4, 169.3, 162.2, 136.6, 133.8,
133.6, 133.4, 133.1, 132.3, 132.0, 131.8, 131.1, 97.0, 95.9, 79.0, 77.0, 73.6,
73.5, 70.3, 69.6, 69.5, 69.2, 69.1, 68.7, 67.6, 67.5, 67.4, 66.1, 65.3, 64.6, 55.1,
51.9, 51.8, 50.1, 49.7, 46.1, 45.9, 44.6, 44.1, 42.2, 42.0, 41.9, 35.6, 35.0, 32.1,
30.7, 30.3, 29.3, 28.9, 25.8, 24.3, 20.4, 20.3, 20.2, 18.3, 18.0, 16.9, 11.9 ppm;
IR (KBr): n˜ = 3386, 2933, 1752, 1663, 1437, 1371, 1326, 1224, 1042, 1011,
752 cmꢀ1; UV (H2O): 416, 391, 371, 352 cmꢀ1; MALDI-FT: m/z: calcd for
C70H109N3O28S: 1472.6991; found: 1472.701 [M+H]+.
N,N-Di-(3-hydroxypropyl)-AmB (25): To a solution of 3-(tert-butyldime-
thylsiloxy)propanal[77] (300 mg, 1.60 mmol) and AmB (1) (365 mg,
0.400 mmol) in DMF (3.00 mL) was added NaBH3CN (100 mg,
1.60 mmol) followed by a drop of conc. HCl. After 16 h at room tempera-
ture, Amberlite IRA-743 (400 mg) was added and the mixture was stirred
for an hour. After filtration, the solution was concentrated down and
added dropwise to diethyl ether (250 mL). The yellow precipitate was fil-
tered and purified by flash chromatography (CHCl3/MeOH/H2O 40:8:1).
The isolated yellow solid was dissolved in MeOH (5.00 mL) in a plastic
bottle. At 08C, diluted HF·pyridine solution (1.00 mL, prepared from
0.500 mL of the 70% HF·pyridine commercial solution and 0.500 mL of
pyridine) was added. After 2 h at room temperature, the solution was
concentrated and added dropwise to diethyl ether (250 mL). The yellow
precipitate was filtered and washed with diethyl ether (2”100 mL) pro-
Methyl N-[(6-piperazinyl-6-oxohexylsulfamoyl)-methyl-a-d-glucopyrano-
syl]-AmB (22): The yellow solid (50 mg, 34 mmol) was dissolved in
MeOH/H2O 3:2 (1.7 mL) and powdered potassium carbonate (21 mg,
153 mmol) was added at room temperature. After 40 min the solvents
were concentrated and flash chromatography (CHCl3/MeOH/H2O
10:6:1) afforded 22 as a yellow powder (30 mg, 65%). Rf = 0.42 (CHCl3/
MeOH/H2O 10:6:1); 1H NMR (500 MHz, [D6]DMSO): d
= 6.85–6.80
(m, 1H), 6.46–6.20 (m, 8H), 6.19–6.13 (m, 2H), 6.09 (d, J=11 Hz, 1H),
6.06 (d, J=10 Hz, 1H), 5.96 (dd, J=15, 9 Hz, 1H), 5.74 (s, 1H), 5.69–
5.63 (m, 1H), 5.55–5.48 (m, 1H), 5.44 (dd, J=15, 10 Hz, 1H), 5.32 (s,
1H), 5.21 (d, J=5 Hz, 1H), 4.81–4.78 (m, 2H), 4.76 (d, J=4 Hz, 1H),
4.64 (d, J=5 Hz, 1H), 4.51 (d, J=4 Hz, 1H), 4.45–4.30 (m, 3H), 4.22 (s,
2H), 4.16 (t, J=9, 1H), 4.10–4.02 (m, 1H), 4.00–3.90 (m, 1H), 3.80 (dt,
J=10, 1 Hz, 2H), 3.56–3.34 (m, 4H), 3.22 (dd, J=10, 4 Hz, 2H), 3.16–
3.10 (m, 2H), 3.08 (d, J=10 Hz, 1H), 3.05 (d, J=9 Hz, 1H), 2.96–2.83
(m, 5H), 2.76–2.69 (m, 1H), 2.68–2.62 (m, 1H), 2.40 (d, J=10.1, 2H),
2.30 (dd, J=15, 7 Hz, 2H), 2.27–2.19 (m, 3H), 2.16 (d, J=6 Hz, 2H),
1.91–1.77 (m, 3H), 1.72 (d, J=7 Hz, 2H), 1.62–1.36 (m, 14H), 1.32–1.21
(m, 8H), 1.17 (d, J=6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz,
3H), 0.98 (d, J=7 Hz, 1H), 0.91 (d, J=7 Hz, 3H), 0.80 ppm (d, J=7 Hz,
1H); 13C NMR (125 MHz, [D6]DMSO): d = 170.5, 170.3, 133.9, 133.7,
133.2, 132.3, 132.1, 132.0, 131.8, 131.1, 99.7, 97.1, 96.1, 77.0, 73.5, 72.8,
72.2, 71.7, 69.7, 69.1, 68.8, 67.7, 67.1, 66.1, 65.3, 60.2, 56.9, 54.7, 53.2, 50.8,
49.5, 46.3, 46.1, 44.7, 44.3, 44.2, 42.3, 42.2, 42.1, 42.0, 41.9, 39.9, 39.7, 39.5,
35.0, 32.2, 29.3, 29.0, 28.9, 25.8, 25.7, 24.4, 18.4, 18.1, 16.9, 16.4, 12.2, 12.0,
10.6, 8.6 ppm; IR (neat): n˜ = 3332, 2929, 1715, 1623, 1586, 1415, 1316,
viding the desired compound 25 as
a yellow solid (104 mg, 28%).
1H NMR (500 MHz, [D6]DMSO): d = 6.47–6.06 (m, 13H), 5.94 (dd, J=
15, 9 Hz, 1H), 5.90 (s, 1H), 5.45 (dd, J=15, 10 Hz, 1H), 5.36 (s, 1H),
5.22–5.20 (m, 1H), 4.79–4.76 (m, 2H), 4.72 (s, 1H), 4.61 (s, 1H), 4.45–
4.38 (m, 1H), 4.24–4.21 (m, 1H), 4.06–3.92 (m, 2H), 3.60–3.02 (m, OH),
2.31–2.26 (m, 1H), 2.18 (d, J=6 Hz, 1H), 2.00–1.97 (m, 1H), 1.90–1.87
(m, 1H), 1.76–1.72 (m, 2H), 1.60–1.52 (m, 2H), 1.42–1.21 (m, 18H), 1.11
(d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92 ppm (d, J=7 Hz, 3H);
13C NMR (125 MHz, [D6]DMSO): d = 174.2, 170.5, 136.7, 133.7, 133.5,
133.2, 133.1, 132.5, 132.3, 132.2, 132.0, 131.8, 131.1, 129.0, 97.2, 77.1, 74.6,
73.7, 73.5, 73.1, 69.1, 68.8, 67.6, 66.8, 66.2, 65.3, 65.0, 64.4, 58.7, 56.8, 46.0,
44.7, 44.2, 42.3, 42.0, 36.7, 35.0, 28.9, 18.4, 17.8, 16.9, 12.0 ppm; MALDI-
FT: m/z: calcd for C53H85NO19: 1040.5894; found: 1040.5789 [M+H]+.
N,N-Di-(methyl-4-butanoate)-AmB (26): To a solution of methyl-4-oxo-
butanoate (520 mg, 4.50 mmol) and AmB (1) (820 mg, 0.890 mmol) in
DMF (5.00 mL) was added NaBH3CN (280 mg, 4.50 mmol) followed by a
drop of conc. HCl. After 18 h at room temperature, Amberlite IRA-743
(800 mg) was added and the mixture was stirred for an hour. After filtra-
tion, the solution was concentrated down and added dropwise to diethyl
ether (250 mL). The yellow precipitate was filtered and purified by flash
chromatography (CHCl3/MeOH/H2O 40:8:1) providing the desired com-
pound 26 as a yellow solid (450 mg, 45%). Rf = 0.30 (CHCl3/MeOH/
H2O 40:8:1); 1H NMR (500 MHz, [D6]DMSO): d = 6.46–6.06 (m, 13H),
5.95 (dd, J=15, 9 Hz, 1H), 5.82 (s, 1H), 5.45 (dd, J=15, 10 Hz, 1H), 5.32
1144, 996, 823, 760 cmꢀ1
; UV (H2O): l = 416, 391, 372, 352 nm;
MALDI-FT: m/z: calcd for C64H103N3O25S: 1390.6313; found: 1390.630
[MꢀH+Na2]+.
Methyl
N-[(6-piperazinyl-6-oxohexylsulfamoyl)-methyl]-2-deoxy-2-
amino-a-d-glucopyranosyl]-AmB (23): Water (0.18 mL) and sodium peri-
odate (27 mg, 0.13 mmol) were added under vigorous stirring to silica
(0.18 g) in chloroform (1.5 mL); then 21 (50.0 mg, 65.6 mmol) was added
and stirred at room temperature for 1 h. The suspension was filtered,
Chem. Eur. J. 2008, 14, 2465 – 2481
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2477

E. M. Carreira et al.
(s, 1H), 5.21–5.19 (m, 1H), 4.80–4.74 (m, 2H), 4.62 (s, 1H), 4.43–4.36 (m,
1H), 4.32 (s, 1H), 4.25–4.17 (m, 2H), 4.06–3.96 (m, 2H), 3.75 (s, 1H),
3.58 (s, 6H), 3.55–3.35 (m, OH), 3.11–3.08 (m, 1H), 2.78–2.72 (m, 1H),
2.64–2.59 (m, 1H), 2.31 (t, J=7 Hz, 4H), 2.17 (d, J=6 Hz, 1H), 1.98–1.23
(m, 21H), 1.18 (d, J=6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz,
3H), 0.92 ppm (d, J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d =
173.6, 173.2, 170.5, 136.9, 136.7, 133.8, 133.6, 133.5, 133.1, 132.4, 132.3,
132.1, 132.0, 131.8, 131.6, 131.1, 128.7, 97.4, 97.1, 80.3, 77.1, 76.3, 75.7,
74.4, 73.8, 73.7, 73.5, 69.4, 69.2, 68.9, 67.9, 67.6, 66.2, 65.4, 63.9, 56.9, 51.0,
50.2, 46.2, 44.6, 44.2, 42.3, 42.0, 35.0, 31.0, 29.0, 24.0, 18.4, 18.2, 16.9,
12.0 ppm; MALDI-FT: m/z: calcd for C55H85NO21: 1124.6005; found:
1124.5600 [M+H]+.
compound 29 as a yellow solid (44.0 mg, 35%). 1H NMR (500 MHz,
[D6]DMSO): d = 6.48–6.06 (m, 13H), 5.96–5.91 (m, 1H), 5.43 (dd, J=
15, 10 Hz, 1H), 5.22 (brs, 1H), 4.89–4.65 (m, 3H), 4.46–4.40 (m, 1H),
4.30–4.18 (m, 2H), 4.09–4.00 (m, 1H), 3.94–3.88 (m, 1H), 3.88–3.44 (brs,
OH), 3.14–3.00 (m, 7H), 2.88–2.64 (m, 3H), 2.31–2.26 (m, 1H), 2.17 (d,
J=6 Hz, 1H), 1.62–1.23 (m, 18H), 1.19 (d, J=6 Hz, 3H), 1.11 (d, J=
6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92 ppm (d, J=7 Hz, 3H); 13C NMR
(125 MHz, [D6]DMSO): d
= 171.8, 170.5, 136.7, 133.8, 133.6, 133.3,
133.1, 132.4, 132.1, 131.8, 131.2, 128.5, 97.0, 77.1, 74.1, 73.8, 73.4, 69.0,
68.7, 67.8, 67.7, 67.6, 67.3, 66.1, 65.5, 64.8, 64.4, 54.2, 46.4, 45.8, 45.7, 44.7,
44.0, 42.4, 41.9, 38.0, 35.0, 29.0, 25.8, 18.4, 18.2, 16.8, 12.0 ppm; MALDI-
FT: m/z: calcd for C55H93N5O16: 1080.6696; found: 1080.61690 [M+H]+.
N,N-Di-(4-butanoic acid)-AmB (27): To a solution of N,N-di-(methyl-4-
butanoate)-AmB (26) (100 mg, 0.089 mmol) in THF (5.00 mL) and H2O
(3.00 mL) was added aqueous LiOH solution (2.00 mL, 1.00m) at 08C.
After 1 h, the reaction mixture was acidified to pH 5 with dilute aqueous
hydrochloric acid solution. The solution was concentrated and added
dropwise to diethyl ether (250 mL). The yellow-brown precipitate was fil-
tered providing the desired compound 27 (42.0 mg, 43%). 1H NMR
(500 MHz, [D6]DMSO): d = 12.22 (brs, 2H), 6.60–5.85 (m, 15H), 5.55–
5.34 (s, 2H), 5.25–5.04 (m, 2H), 5.02–4.99 (m, 1H), 4.95–4.90 (m, 1H),
4.85–4.82 (m, 2H), 4.64–4.57 (m, 1H), 4.39 (s, 1H), 4.15–4.05 (m, 1H),
3.68 (m, OH), 3.33–3.12 (m, 1H), 2.37–2.30 (m, 2H), 2.17 (d, J=6 Hz,
1H), 2.00–1.90 (m, 1H), 1.71–1.28 (m, 21H), 1.18 (dd, J=10, 6 Hz, 3H),
1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.91 ppm (d, J=7 Hz, 3H);
13C NMR (125 MHz, [D6]DMSO): d = 173.7, 173.5, 170.4, 136.9, 136.7,
133.8, 133.6, 133.5, 133.1, 132.4, 132.3, 132.1, 132.0, 131.8, 131.6, 131.1,
128.7, 97.8, 97.1, 93.9, 93.3, 80.7, 77.1, 76.4, 75.9, 75.2, 73.9, 73.5, 73.3,
72.4, 68.9, 68.5, 67.7, 66.4, 66.0, 64.8, 61.6, 51.0, 50.2, 46.2, 44.6, 42.3, 42.0,
35.0, 31.2, 18.5, 18.4, 16.9, 12.0 ppm; MALDI-FT: m/z: calcd for
C55H85NO21: 1094.5536; found: 1094.5541 [MꢀH]ꢀ.
16-(N’-(1-Methylpiperazinyl)-carboxamide)-N,N-di-(3-aminopropyl)-
AmB (30): To a solution of 28 (200 mg, 0.135 mmol) in DMF (2.00 mL)
was added 1-methylpiperazine (0.100 mL, 1.00 mmol), 1-hydroxybenzo-
triazole (36.0 mg, 0.270 mmol), (benzotriazol-1-yloxy)-tripyrrolidinophos-
phonium hexafluorophosphate (100 mg, 0.190 mmol). After 18 h at room
temperature, the solution was concentrated down and the residue was
added dropwise to diethyl ether (250 mL). The yellow precipitate was di-
luted in DMF (2.00 mL) and triethylamine (0.100 mL, 1.00 mmol) and
Fmoc-Cl (100 mg, 0.360 mmol) was added. After 2 h, the mixture added
dropwise to diethyl ether (250 mL). The yellow precipitate was filtered
and purified by flash chromatography (CHCl3/MeOH/H2O 10:6:1). To
the isolated solid in DMSO (3.00 mL) was added piperidine (0.200 mL,
2.10 mmol). After 2 h at room temperature, the solution was added drop-
wise to diethyl ether (250 mL). The yellow precipitate was filtered and
washed with diethyl ether (2”100 mL) providing the desired compound
30 as a yellow solid (10.0 mg, 7%). 1H NMR (500 MHz, [D6]DMSO):
d=6.48–6.06 (m, 12H), 5.97–5.92 (m, 1H), 5.43 (dd, J=15, 10 Hz, 1H),
5.23 (brs, 1H), 4.89–4.52 (m, 4H), 4.33–4.17 (m, 6H), 4.08–3.99 (m, 4H),
3.90 (s, 1H), 3.50 (brs, OH), 3.12–3.06 (m, 4H), 2.72–2.64 (m, 4H), 2.41–
2.26 (m, 1H), 2.17 (d, J=6 Hz, 1H), 1.82–1.15 (m, 22H), 1.19 (d, J=
6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92 (d, J=7 Hz,
3H); 13C NMR (125 MHz, [D6]DMSO): d = 171.8, 170.5, 136.6, 134.0,
133.8, 133.4, 133.3, 132.7, 132.5, 132.4, 132.0, 131.4, 128.7, 96.9, 77.4, 74.3,
74.0, 73.7, 69.2, 69.0, 68.0, 67.9, 66.3, 65.9, 55.3, 54.9, 51.2, 47.7, 46.4, 45.7,
45.4, 44.9, 42.6, 42.1, 41.3, 35.2, 31.4, 30.8, 29.1, 26.2, 18.6, 18.5, 18.3, 17.1,
12.2 ppm; MALDI-FT: m/z: calcd for C58H97N5O16: 1120.4147; found:
1120.7003 [M+H]+.
N,N-Di-{N-(9-fluorenylmethoxycarbonyl)-3-aminopropyl}-AmB (28): To
a
solution of N-(9-fluorenylmethoxycarbonyl)-3-aminopropanal[72]
(150 mg, 0.510 mmol) and AmB (1) (157 mg, 0.170 mmol) in DMF
(3.00 mL) was added NaBH3CN (32.0 mg, 0.510 mmol) followed by a
drop of conc. HCl. After 16 h at room temperature, Amberlite IRA-743
(300 mg) was added and the mixture was stirred for an hour. After filtra-
tion, the solution was concentrated down and added dropwise to diethyl
ether (250 mL). The yellow precipitate was filtered and purified by flash
chromatography (CHCl3/MeOH/H2O 10:6:1) providing the desired com-
pound 28 as a yellow solid (200 mg, 80%). Rf = 0.70 (CHCl3/MeOH/
H2O 10:6:1); 1H NMR (500 MHz, [D6]DMSO): d = 7.87 (d, J=8 Hz,
4H), 7.68 (d, J=8 Hz, 4H), 7.40 (t, J=8 Hz, 4H), 7.31 (t, J=8 Hz, 4H),
6.46–6.06 (m, 12H), 5.96 (dd, J=14, 8 Hz, 1H), 5.84 (s, 1H), 5.44 (dd, J=
15, 10 Hz, 1H), 5.33 (s, 1H), 5.21 (brs, 1H), 4.79–4.60 (m, 4H), 4.43–4.19
(m, 10H), 4.07–3.97 (m, 2H), 3.81 (s, 1H), 3.55–3.41 (m, 3H), 3.31 (brs,
OH), 3.13–3.02 (m, 6H), 2.77 (s, 1H), 2.64 (s, 1H), 2.28 (s, 1H), 2.17 (d,
J=6 Hz, 1H), 1.93–1.24 (m, 18H), 1.19 (d, J=6 Hz, 3H), 1.11 (d, J=
7 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92 ppm (d, J=7 Hz, 3H); 13C NMR
16-(N’-(3-Dimethylaminopropyl)-carboxamide)-N,N-di-(3-aminopropyl)-
AmB (31): To a solution of 28 (100 mg, 0.068 mmol) in DMF (2.00 mL)
was added 3-dimethylaminopropylamine (17.0 mg, 0.136 mmol), 1-hy-
droxybenzotriazole (11.0 mg, 0.082 mmol), (benzotriazol-1-yloxy)-tripyr-
rolidinophosphonium hexafluorophosphate (35.0 mg, 0.068 mmol) and
diisopropylethylamine (20.0 mL, 0.102 mmol). After 24 h at room temper-
ature, the solution was concentrated down and the residue was added
dropwise to diethyl ether (250 mL). The yellow precipitate was filtered
and washed with diethyl ether (2”100 mL). To the yellow solid in
DMSO (3.00 mL) was added piperidine (0.200 mL, 2.10 mmol). After 2 h
at room temperature, the solution was added dropwise to diethyl ether
(250 mL). The yellow precipitate was filtered and washed with diethyl
ether (2”100 mL) providing the desired compound 31 as a yellow solid
(125 MHz, [D6]DMSO): d
= 177.6, 170.5, 162.2, 156.0, 143.9, 142.5,
140.6, 139.3, 137.3, 136.7, 133.8, 133.6, 133.4, 133.1, 132.4, 132.3, 132.1,
131.9, 131.8, 131.7, 131.2, 128.8, 127.5, 127.2, 127.0, 125.1, 121.3, 119.9,
101.2, 97.1, 77.1, 77.0, 73.9, 73.5, 69.5, 69.1, 68.8, 67.8, 67.5, 66.1, 65.2,
64.8, 58.2, 48.3, 46.7, 44.8, 44.6, 44.2, 42.0, 41.9, 38.4, 35.7, 35.0, 30.7, 28.9,
28.5, 18.4, 18.2, 16.9, 15.1, 12.0 ppm; MALDI-FT: m/z: calcd for
C87H107N3O21: 1504.7295; found: 1504.7289 [M+Na]+.
(36.0 mg, 47%). 1H NMR (500 MHz, [D6]DMSO): d
= 6.48–6.07 (m,
13H), 5.97–5.93 (m, 1H), 5.43 (dd, J=15, 10 Hz, 1H), 5.23 (brs, 1H),
4.86–4.83 (m, 4H), 4.35–4.31 (m, 1H), 4.26–4.24 (m, 1H), 4.08–3.97 (m,
2H), 3.92–3.80 (m, 1H), 3.65–3.44 (m, 5H), 3.17–3.08 (m, 2H), 2.31–2.24
(m, 2H), 2.17 (d, J=6 Hz, 1H), 2.12 (s, 3H), 2.08 (s, 3H), 1.90–1.20 (m,
23H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92 (d, J=7 Hz,
3H); 13C NMR (125 MHz, [D6]DMSO): d = 170.5, 162.2, 136.7, 136.3,
133.8, 133.6, 133.4, 133.1, 132.4, 132.2, 131.9, 131.8, 131.2, 128.5, 97.1,
77.0, 74.1, 73.8, 73.5, 69.1, 68.8, 67.9, 67.6, 66.1, 65.7, 62.7, 54.5, 47.9, 46.2,
45.8, 45.5, 44.9, 44.7, 44.0, 42.4, 42.0, 35.7, 35.0, 30.7, 30.6, 18.4, 18.1, 16.9,
12.0 ppm; MALDI-FT: m/z: calcd for C58H99N5O16: 1122.4306; found:
1122.7160 [M+H]+.
16-(N’-(2-Aminoethyl)-carboxamide)-N,N-di-(3-aminopropyl)-AmB (29):
To a solution of 28 (170 mg, 0.115 mmol) in DMF (3.00 mL) was added
N-Fmoc-ethylenediamine (49.0 mg, 0.172 mmol), 1-hydroxybenzotriazole
(19.0 mg, 0.138 mmol), (benzotriazol-1-yloxy)-tripyrrolidinophosphonium
hexafluorophosphate (60.0 mg, 0.115 mmol) and diisopropylethylamine
(40.0 mL, 0.230 mmol). After 36 h at room temperature, the solution was
concentrated down and
a flash chromatography (CHCl3/MeOH/H2O
40:8:1) provided the protected derivative of compound 29. The yellow
solid was dissolved in DMSO (3.00 mL) and was added piperidine
(0.200 mL, 2.10 mmol). After 2 h at room temperature, the solution was
added dropwise to diethyl ether (250 mL). The yellow precipitate was fil-
tered and washed with diethyl ether (2”100 mL) providing the desired
16-(N’-(4-(3-Aminopropyl)morpholine)carboxamide)-N,N-di-(3-amino-
propyl)-AmB (32): To a solution of 28 (100 mg, 0.068 mmol) in DMF
(2.00 mL)
was
added
4-(3-aminopropyl)-morpholine
(20.0 mg,
0.136 mmol), 1-hydroxybenzotriazole (11.0 mg, 0.082 mmol), (benzotria-
2478
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 2465 – 2481

Natural Products
FULL PAPER
zol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (35.0 mg,
0.068 mmol) and diisopropylethylamine (20.0 mL, 0.102 mmol). After 18 h
at room temperature, the solution was concentrated down and the resi-
due was added dropwise to diethyl ether (250 mL). The yellow precipi-
tate was filtered and washed with diethyl ether (2”100 mL). To the
yellow solid in DMSO (3.00 mL) was added piperidine (0.200 mL,
2.10 mmol). After 2 h at room temperature, the solution was added drop-
wise to diethyl ether (250 mL). The yellow precipitate was filtered and
washed with diethyl ether (2”100 mL) providing the desired compound
32 as a yellow solid (39.0 mg, 49%). 1H NMR (500 MHz, [D6]DMSO):
d=6.48–6.06 (m, 12H), 5.97–5.93 (m, 1H), 5.43 (dd, J=15, 10 Hz, 1H),
5.22 (brs, 1H), 4.90–4.35 (m, 7H), 4.26–4.20 (m, 4H), 4.05–3.99 (m, 4H),
3.90–3.77 (m, 4H), 3.56–3.46 (m, 7H), 3.16–3.01 (m, 4H), 2.90–2.80 (m,
4H), 2.33–2.28 (m, 4H), 2.17 (d, J=6 Hz, 1H), 1.98–1.24 (m, 18H), 1.19
(d, J=6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.91 (d,
J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d = 171.9, 170.5, 136.7,
136.5, 133.7, 133.6, 133.3, 133.1, 132.4, 132.1, 131.9, 131.8, 131.2, 128.5,
97.0, 77.1, 74.2, 74.0, 73.8, 73.7, 73.4, 69.0, 68.7, 67.8, 67.7, 66.1, 64.8, 64.5,
55.9, 53.3, 47.8, 47.6, 47.4, 47.3, 46.1, 44.9, 44.7, 44.6, 42.4, 41.9, 38.2, 35.0,
29.0, 26.1, 18.4, 18.1, 16.9, 12.0 ppm; MALDI-FT: m/z: calcd for
C60H101N5O17: 1164.4672; found: 1164.7265 [M+H]+.
1H), 4.26–4.23 (m, 2H), 4.09–3.90 (m, 2H), 3.70–3.40 (m, 7H), 3.15 (brs,
12H), 2.30–2.27 (m, 1H), 2.17 (d, J=6 Hz, 1H), 2.02–1.28 (m, 20H), 1.17
(dd, J=16, 6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.92
(d, J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d = 172.7, 170.5,
136.7, 133.8, 133.7, 133.4, 133.1, 132.3, 132.1, 131.8, 131.1, 128.6, 97.1,
77.1, 74.6, 73.9, 73.7, 73.5, 69.1, 68.8, 68.5, 68.0, 67.6, 66.1, 64.9, 64.0, 61.8,
52.8, 52.7, 47.9, 46.2, 44.6, 42.4, 42.0, 37.7, 35.0, 28.9, 25.0, 18.4, 18.1, 16.9,
12.0 ppm; MS MALDI-FT: m/z: calcd for C58H100N5O16
found: 1122.7106 [M]+.
: 1122.7160;
16-(N’-(Hexylcarboxamide)-N,N-di-(3-aminopropyl)-AmB (35): To a so-
lution of 28 (150 mg, 0.100 mmol) in DMF (2.00 mL) was added 1-hexan-
amine (66.0 mL, 0.500 mmol), 1-hydroxybenzotriazole (27.0 mg,
0.200 mmol), (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexa-
fluorophosphate (52.0 mg, 0.100 mmol). After 18 h at room temperature,
the solution was concentrated down and the residue was added dropwise
to diethyl ether (250 mL). The yellow precipitate was filtered and washed
with diethyl ether (2”100 mL). To the yellow solid in DMSO (3.00 mL)
was added piperidine (0.200 mL, 2.10 mmol). After 2 h at room tempera-
ture, the solution was added dropwise to diethyl ether (250 mL). The
yellow precipitate was filtered and washed with diethyl ether (2”
100 mL) providing the desired compound 35 as a yellow solid (15.0 mg,
13%). 1H NMR (500 MHz, [D6]DMSO): d = 6.48–5.94 (m, 12H), 5.88–
5.81 (m, 1H), 5.46–5.33 (m, 1H), 5.23 (brs, 1H), 4.78–4.58 (m, 4H), 4.37–
4.21 (m, 6H), 4.06–3.96 (m, 2H), 3.33 (brs, OH), 3.16–3.05 (m, 5H),
2.96–2.92 (m, 2H), 2.32–2.26 (m, 2H), 2.17 (d, J=6 Hz, 1H), 1.75–1.15
(m, 23H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.91 (d, J=7 Hz,
16-(N’-6-Aminohexanylcarboxamide)-N,N-di(3-aminopropyl)-AmB (33):
To a solution of 28 (300 mg, 0.202 mmol) in DMF (2.0 mL) was added
diisopropylamine (87 mL, 0.49 mmol), 1-hydroxybenzotriazole (38 mg,
0.28 mmol), (benzotriazol-1-yloxy)-tripyrrolidino-phosphonium hexa-
fluorophosphate (126 mg, 0.242 mmol) and 6-(9-fluorenylmethoxycarbon-
yl)-1-diaminohexane monohydrochloride (114 mg, 0.303 mmol). After
18 h at room temperature, the solution was added dropwise to diethyl
ether (200 mL). The yellow precipitate was filtered and washed with di-
ethyl ether (2”10 mL). The yellow solid was purified by flash chromatog-
raphy (CHCl3/MeOH/H2O 60:9:1). The isolated yellow solid was dis-
solved in DMSO (2 mL) and piperidine (0.100 mL) was added. After
10 min at room temperature, the solution was added dropwise to diethyl
ether (200 mL). The yellow precipitate was filtered and washed with di-
ethyl ether (2”10 mL) providing the desired compound 33 as a yellow
3H), 0.87 (d, J=5 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d
=
171.7, 170.5, 136.7, 135.4, 133.8, 133.6, 133.1, 132.5, 132.4, 132.1, 131.8,
131.4, 131.3, 128.4, 97.0, 77.1, 74.1, 73.8, 73.4, 73.0, 69.0, 68.7, 67.7, 66.3,
66.1, 65.5, 64.5, 55.7, 45.8, 45.7, 44.7, 44.6, 44.3, 44.1, 42.4, 42.0, 41.9, 38.4,
35.0, 31.0, 29.2, 26.1, 25.8, 22.0, 18.4, 18.0, 16.9, 13.8, 12.0 ppm; MS
MALDI-FT: m/z: calcd for C59H100N4O16: 1121.4425; found: 1121.7207
[M+H]+.
16-(N’-(1,2,4-Triazole-1-ethyl-carboxamide)-N,N-di-(3-aminopropyl)-
AmB (36): To a solution of 28 (330 mg, 0.223 mmol) in DMF (2.00 mL)
was added 1H-1,2,4-triazole-1-ethanamide monohydrochloride (50.0 mg,
0.450 mmol), 1-hydroxybenzotriazole (60.0 mg, 0.450 mmol), (benzotria-
zol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (60.0 mg,
0.223 mmol) and diisopropylethylamine (0.100 mL, 0.500 mmol). After
18 h at room temperature, the solution was concentrated down and the
residue was added dropwise to diethyl ether (250 mL). The yellow pre-
cipitate was filtered and washed with diethyl ether (2”100 mL). The
yellow precipitate was filtered and purified by flash chromatography
(CHCl3/MeOH/H2O 40:8:1). The isolated yellow solid was dissolved in
DMSO (5.00 mL) and piperidine (0.200 mL, 2.10 mmol) was added.
After 2 h at room temperature, the solution was added dropwise to dieth-
yl ether (250 mL). The yellow precipitate was filtered and washed with
diethyl ether (2”100 mL) providing the desired compound 36 as a yellow
1
solid (119 mg, 51%). H NMR (500 MHz, [D6]DMSO): d = 7.89 (s, 1H),
6.45 (dd, J=14, 11 Hz, 1H), 6.39–6.19 (m, 8H), 6.16 (s, 1H), 6.15 (d, J=
4 Hz, 1H), 6.08 (dd, J=15, 11 Hz, 2H), 5.94 (dd, J=15, 9 Hz, 1H), 5.42
(dd, J=15, 10 Hz, 1H), 5.28–5.16 (m, 1H), 4.41–4.30 (m, 2H), 4.29–4.20
(m, 3H), 4.18 (s, 1H), 4.11–3.97 (m, 4H), 3.93–3.84 (m, 3H), 3.57–3.39
(14H), 3.20–3.12 (m, 2H), 3.08 (t, J=11 Hz, 3H), 2.98–2.89 (m, 2H),
2.88–2.74 (m, 3H), 2.46–2.37 (m, 1H), 2.33–2.22 (m, 2H), 2.16 (d, J=
6 Hz, 2H), 2.00–1.92 (m, 1H), 1.92–1.80 (m, 2H), 1.76–1.67 (m, 1H),
1.63–1.44 (m, 9H), 1.44–1.36 (m, 6H), 1.35–1.29 (m, 5H), 1.29–1.21 (m,
6H), 1.17 (d, J=6 Hz, 3H), 1.10 (d, J=7, 3H), 1.03 (d, J=6 Hz, 3H),
0.90 ppm (d, J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d = 171.5,
170.4, 136.7, 136.6, 133.8, 133.6, 133.3, 133.1, 132.3, 132.3, 132.1, 131.8,
131.7, 131.1, 128.3, 97.0, 77.1, 74.0, 73.8, 73.5, 69.0, 68.7, 67.7, 67.5, 66.1,
64.9, 64.4, 63.2, 57.0, 47.1, 46.2, 44.7, 44.6, 42.3, 41.9, 41.4, 38.4, 35.9, 35.0,
33.1, 30.3, 29.3, 29.0, 26.3, 26.1, 26.0, 18.4, 18.3, 16.8, 12.0 ppm; MALDI-
FT: m/z: calcd for C59H101N5O16: 1136.7316; found 1136.730 [M+H]+.
solid (51.0 mg, 20%). 1H NMR (500 MHz, [D6]DMSO): d
= 8.48 (s,
1H), 8.20 (brs, 1H), 7.96 (s, 1H), 6.48–6.06 (m, 12H), 5.96–5.91 (m, 1H),
5.43 (dd, J=15, 10 Hz, 1H), 5.22 (brs, 1H), 4.53–3.90 (m, 15H), 3.52–
3.41 (m, 5H), 3.11–3.07 (m, 2H), 2.92–2.73 (m, 4H), 2.29–2.73 (m, 2H),
2.17 (d, J=6 Hz, 1H), 1.89–1.26 (m, 21H), 1.17 (dd, J=16, 6 Hz, 3H),
1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz, 3H), 0.91 ppm (d, J=7 Hz, 3H);
13C NMR (125 MHz, [D6]DMSO): d = 172.5, 172.4, 170.5, 151.2, 144.3,
136.7, 136.6, 133.8, 133.6, 133.3, 133.1, 132.4, 132.2, 132.1, 131.8, 131.2,
128.4, 97.0, 77.1, 74.2, 73.8, 73.5, 73.2, 70.6, 69.0, 68.7, 67.7, 67.6, 66.1,
65.0, 64.8, 62.7, 62.5, 57.0, 48.0, 47.6, 46.1, 44.7, 44.4, 42.4, 41.9, 38.7, 35.0,
29.0, 18.4, 18.3, 18.1, 16.9, 12.0 ppm; MALDI-FT: m/z: calcd for
C57H93N7O16: 1132.3856; found: 1132.6752 [M+H]+.
16-(N’-(2-Amino-N,N,N-trimethylethanaminium chloride)-carboxamide)-
N,N-di-(3-aminopropyl)-AmB (34): To
a solution of 28 (100 mg,
0.067 mmol) in DMF (2.00 mL) was added 2-amino-N,N,N-trimethyletha-
naminium chloride (19.0 mg, 0.134 mmol) in H2O (0.250 mL), 1-hydroxy-
benzotriazole (18.0 mg, 0.134 mmol), (benzotriazol-1-yloxy)-tripyrrolidi-
nophosphonium hexafluorophosphate (35.0 mg, 0.067 mmol) and diiso-
propylethylamine (30.0 mL, 0.134 mmol). After 18 h at room temperature,
the solution was concentrated down and the residue was added dropwise
to diethyl ether (250 mL). The yellow precipitate was filtered and washed
with diethyl ether (2”100 mL). The yellow precipitate was filtered and
purified by flash chromatography (CHCl3/MeOH/H2O 40:8:1). The iso-
lated yellow solid was dissolved in DMSO (5.00 mL) and piperidine
(0.200 mL, 2.10 mmol) was added. After 2 h at room temperature, the so-
lution was added dropwise to diethyl ether (250 mL). The yellow precipi-
tate was filtered and washed with diethyl ether (2”100 mL) providing
the desired compound 34 as a yellow solid (25.0 mg, 34%). 1H NMR
(500 MHz, [D6]DMSO): d = 6.48–6.06 (m, 12H), 6.00–5.90 (m, 1H), 5.43
(dd, J=15, 10 Hz, 1H), 5.21 (brs, 1H), 4.98–4.69 (m, 3H), 4.43–4.35 (m,
N,N-Di-(3-aminopropyl)-AmB methyl ester (37): To a solution of N,N-di-
(3-aminopropyl)-AmB (3) (150 mg, 0.140 mmol) in MeOH (10.0 mL) was
added
a 2m solution of trimethylsilyldiazomethane in diethyl ether
(0.700 mL, 14.0 mmol). After 3 h at room temperature, the solution was
concentrated down to a volume of 1.00 mL and then added dropwise to
diethyl ether (250 mL). The yellow precipitate was filtered and washed
with diethyl ether (2”100 mL) providing the desired compound 37 as a
yellow solid (90.0 mg, 59%). 1H NMR (500 MHz, [D6]DMSO): d
=
Chem. Eur. J. 2008, 14, 2465 – 2481
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2479

E. M. Carreira et al.
6.45–5.93 (m, 13H), 5.44 (dd, J=15, 10 Hz, 1H), 5.34 (brs, 1H), 5.22
(brs, 1H), 4.92–4.69 (m, 3H), 4.35–4.18 (m, 3H), 4.06–4.02 (m, 1H),
3.81–3.76 (m, 1H), 3.63 (s, 3H), 3.34 (brs, OH), 3.10–3.04 (m, 3H), 2.82–
2.63 (m, 3H), 2.39–2.24 (m, 1H), 2.17 (d, J=6 Hz, 1H), 1.89–1.28 (m,
18H), 1.18 (d, J=6 Hz, 3H), 1.11 (d, J=6 Hz, 3H), 1.04 (d, J=6 Hz,
3H), 0.91 ppm (d, J=7 Hz, 3H); 13C NMR (125 MHz, [D6]DMSO): d =
173.0, 170.4, 136.7, 134.1, 133.8, 133.1, 132.5, 132.1, 131.7, 131.2, 129.2,
97.2, 96.8, 80.2, 76.2, 75.6, 74.0, 73.1, 69.0, 68.1, 66.2, 65.3, 64.8, 64.0, 52.1,
51.1, 45.6, 44.8, 44.0, 42.1, 41.9, 28.8, 18.4, 18.2, 17.1, 16.9, 12.0 ppm;
MALDI-FT: m/z: calcd for C54H89N3O17: 1052.6270; found: 1052.6265
[M+H]+.
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Received: August 9, 2007
Published online: January 14, 2008
Chem. Eur. J. 2008, 14, 2465 – 2481
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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