Synthesis and antibacterial activity of novel neamine derivatives: Preponderant role of the substituent position on the neamine core

Article abstract of DOI:10.1039/c2ob07065k

A series of neamine derivatives were prepared from the cyclic carbonate and sulfate of 1,3,2′,6′-tetraazido-3′,4′,-di-O- acetylneamine. Ring opening reactions with diversely substituted amines result in the formation of the corresponding carbamates or sulfonic acids with good overall yields. The antibacterial activities of the synthesized products against E. coli (DH5α) and S. aureus (RN4220) were evaluated. With isolated single regioisomers, the preponderant effect of the 5-positions of the carbamate substituent on the neamine core was demonstrated.

Full text of DOI:10.1039/c2ob07065k

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PAPER
Synthesis and antibacterial activity of novel neamine derivatives:
preponderant role of the substituent position on the neamine core†
Nicolas Gernigon,a Valérie Bordeau,b Fabienne Berrée,a Brice Felden*b and Bertrand Carboni*a
Received 9th December 2011, Accepted 24th April 2012
DOI: 10.1039/c2ob07065k
A series of neamine derivatives were prepared from the cyclic carbonate and sulfate of 1,3,2′,6′-
tetraazido-3′,4′,-di-O-acetylneamine. Ring opening reactions with diversely substituted amines result in
the formation of the corresponding carbamates or sulfonic acids with good overall yields. The
antibacterial activities of the synthesized products against E. coli (DH5α) and S. aureus (RN4220) were
evaluated. With isolated single regioisomers, the preponderant effect of the 5-positions of the carbamate
substituent on the neamine core was demonstrated.
to the A-site of the 16S ribosomal RNA.1 Therefore, not surpris-
ingly, this pseudodisaccharide was used as a starting scaffold to
Introduction
Aminoglycosides have been used as antibacterials for decades,
but it is only recently that they have been recognized for their
antiviral properties and their importance in the treatment of
genetic diseases.1 Their use, however, has been slowed down by
problems associated with bacterial resistance, nephrotoxicity or
ototoxicity resulting from the non-specific binding of RNAs.2
Aminoglycosides exert their biological functions by binding to
the A-site and decoding the region of the prokaryotic ribosomal
RNA small subunit.3 They interact with unpaired adenine resi-
dues in the decoding loop, displacing the unpaired adenines and
locking them into a “flipped-out” orientation similar to that
which occurs during mRNA decoding.4 Consequently, they
interfere with the protein synthesis in bacteria, in turn inducing
cell death.
The key structural feature of this family of antibiotics is strep-
tamine, a 1,3-diaminocyclohexanetetraol unit or 2-deoxystrept-
amine in cases where the 2-hydroxy function is deoxygenated as
in most aminoglycosides (Scheme 1).5 Analysis of the structural
elements common to the natural or semisynthetic aminoglyco-
side family highlights that a number of them (e.g. neomycin,
ribostamycin, kanamycin B, arbekacin) contain a neamine core.2
While showing itself to have a modest antibiotic activity, it con-
stitutes however a minimal motif which is required for binding
generate new potential antibiotics with the aim of circumventing
microbial resistance while retaining activity and decreasing the
associated toxicity.6 However, even though major progresses
have been made in the understanding of RNA–ligand recognition
principles in recent years, the design of RNA-specific ligands
remains a difficult task. RNA is a dynamic target and mutual
interactions can greatly modify their conformations.7 The most
significant contributions related to association phenomena result
from electrostatic interactions between the protonated amino
groups and the negatively charged phosphates of the oligoribo-
nucleotide backbone.8 Keeping this in mind, we decided to
maintain these key functional groups and have developed a syn-
thetic strategy that allows the easy introduction of polar substitu-
ents on the 5- or 6-O-position of the neamine core. Two
compounds 1 and 2 possessing, respectively, a cyclic carbonate
or a sulfate moiety at the 5,6-positions were selected as key
building blocks (Scheme 1).9 These five-membered rings are
likely to undergo a variety of ring opening reactions in the pres-
ence of various nucleophiles.10
aInstitut des Sciences Chimiques de Rennes, UMR 6226
CNRS-Université de Rennes 1, Campus de Beaulieu, 35042 Rennes,
France. E-mail: bertrand.carboni@univ-rennes1.fr
bLaboratoire de Biochimie Pharmaceutique, INSERM U835-UPRES
EA2311, Université de Rennes 1, 2, avenue du Pr Léon Bernard, 35043
Rennes, France. E-mail: brice.felden@univ-rennes1.fr
†Electronic supplementary information (ESI) available: Synthesis of 3
and 4, biological assays for 8a and 8a + 8b and 1H and 13C NMR
spectra of neamine derivatives 1, 2 and 5–18. See DOI: 10.1039/
c2ob07065k
Scheme 1 Synthetic routes to neamine derivatives.
4720 | Org. Biomol. Chem., 2012, 10, 4720–4730
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In this report, we describe the synthesis of these two novel
neamine derivatives, 1 and 2, their reactivity towards amines and
a biological evaluation of the antibacterial activities of the result-
ing products on Gram positive and negative bacteria.
chloride. Examination of the 1H NMR spectrum of the crude
material revealed the presence of two regioisomers (1 : 1), as
indicated by two signals of anomeric protons at 5.54 and
5.30 ppm. By contrast, the ring opening of the cyclic sulphate 2
did not occur under these conditions. Thus, after an optimization
process, the reaction was carried out by using propylamine as
solvent. Here too, a 1 : 1 mixture of products 6a and 6b (two
H-1′ at 5.38 and 5.37 ppm and two double doublets at 4.64 and
4.34 ppm for HC(OSO3H), H-5 or H-6) was obtained and a
complete deacetylation occurring at the 3′ and 4′ positions was
observed (Scheme 3).
We then initiated the synthesis of other neamine derivatives
and hypothesized that introducing additional polar substituents
on the neamine core would result in higher interactions within
the 16S rRNA binding site.8 The previous transformations were
therefore applied similarly to other amines possessing sup-
plementary amino, hydroxy or imidazole groups. Once the car-
bamates were prepared, they were treated with sodium methylate
in methanol, followed by hydrogenation in the presence of plati-
num oxide and hydrochloric acid,19 inducing the formation of
fully deprotected neamine derivatives 7–11 (Scheme 4). In the
case of the sulfonic acids, the azido groups were directly reduced
Results and discussion
Neamine hydrochloride was prepared using a reported procedure
through methanolysis of the commercially available neomycin
sulfate.11 It was then protected as its tetraazido derivative in a
82% yield using freshly prepared triflyl azide in the presence of
copper sulfate.12,13 The choice of this amino protecting group,
first reported by Wong and co-workers,12 offers advantages com-
pared with carbamate or N-trityl groups.14 These take into
account a better stability, an easier chemoselective deprotection,
and a greater solubility and the absence of rotamers that compli-
cate the interpretation of the NMR spectra. The regioselective
triacetylation at positions 6, 3′ and 4′ was undertaken with
excess of acetic anhydride in a 1 : 1 CH2Cl2–pyridine mixture to
give compound 3.15 Side products, mainly the tetraacetylated
derivative, were easily separated by column chromatography and
converted after treatment with MeONa in methanol to the corre-
sponding tetrahydroxy derivative to be recycled.16 Treatment of
3 with 1.3 equivalents of NaOH 0.1 N in THF regioselectively
produced the free 5, 6 diol 4.17 This results in the disappearance
of the peaks at 4.94 ppm (H-6) and at 169.2 and 20.1 ppm
1
(COCH3), respectively in the H NMR and 13C NMR spectra.
Compound 4 can be converted to the corresponding carbonate 1
with triphosgene in CH2Cl2 in the presence of pyridine in a 94%
yield. In parallel, the sulfate 2 was synthesized by using thionyl
chloride and subsequent oxidation with ruthenium trichloride in
the presence of NaIO4 (yield, 79%). 1 and 2 were characterised
by spectroscopic data (1H NMR, 13C NMR and mass spectra) in
agreement with the proposed structures (Scheme 2).
Scheme 3 Ring opening of 1 and 2 with n-propylamine.
With these two building blocks in hand, we first studied their
reactivity towards propylamine as a model experiment. The
nucleophilic attack on the carbonate 1 was followed by ring
opening to give the carbamates 5a and 5b.18 A complete conver-
sion was observed after 2 h at room temperature in methylene
Scheme 2 Synthesis of the carbonate- and sulfate-containing neamine
derivatives 1 and 2.
Scheme 4 Synthesis of the neamine derivatives 7–14.
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Table 1 The MICs of the aminoglycoside derivatives 7–14 and 18
under identical conditions after the ring opening of the cyclic
sulfate moiety to produce compounds 12–14.
compared to neamine (μg mL−1
)
To compare the influence of the position of the carbamate sub-
stituent on the neamine core, we tried to separate regioisomers a
and b. Many attempts were realized at different steps of the syn-
thetic sequence. Finally, the separation of 8a and 8b was
achieved just before the last hydrogenation step by HPLC on a
C18 reversed-phase column (Scheme 4). Furthermore, to unmis-
takably establish the structure of each isomer, 8a was separately
synthesized from the triacetate derivative 3. After treatment with
triphosgene to obtain the chloroformate 15 in 84% yield, the
addition of 3-azidopropylamine, followed by deprotection of the
hydroxyl groups and hydrogenation with H2 in the presence of
PtO2 and hydrochloric acid, afforded compound 8a in 61%
overall yield (three steps, Scheme 5).
Entry Compound E. coli (strain DH5α) S. aureus (strain RN4220)
a
b
c
d
e
f
g
h
i
Neamine
7a + 7b
8a + 8b
8a
8b
9a + 9b
10a + 10b 250 50
11a + 11b 150 50
12a + 12b >400
30 10
>400
70 15
50 15
>100
22
>400
35
45
>100
50
250 50
200 50
>400
8
7
5
250 50
5
j
k
l
13a + 13b 50 10
14a + 14b 200 50
60 10
300 50
300 50
18
250 50
The comparative antibacterial activities of 7–14, 18 and
neamine were evaluated by determining the minimum inhibitory
concentrations (MICs) against E. coli (DH5α) and S. aureus
(RN4220) cells as described in the Experimental section
(Table 1).
The antibacterial activities of eleven new neamine derivatives
against gram positive and negative bacteria are presented in
Table 1. The introduction of an OCONH-nPr substituent instead
of an hydroxyl group at position 5 or 6 causes a loss of activity
(entries a and b). When the n-propyl extremity of the carbamate
moiety was replaced by a 3-aminopropyl, the activity was
restored close to that of neamine (entries a and c), whereas the
MICs are higher in the cases of OCONH–(CH2)3–OH or
OCONH–(CH2)2–imidazole (entries g and h). Furthermore,
introducing two aminopropyl chains abolished the antibacterial
activity against gram positive and negative bacteria (entry l). It is
worthy to note the negative influence of an increase in the
number of carbon atoms in the aminoalkyl chain observed for
E. coli., while this modification has little effect for S. aureus
(RN4220) (entries c and f). It suggests that despite strong
sequence conservation, the decoding sites of each bacterium
have a slightly different fold. Similar observations can also be
drawn with the sulfonic acid derivatives (entries i, j and k). The
most remarkable result of these antibacterial assays is the critical
influence of the position of the carbamate substituent on the
neamine core (entries d and e). The comparison of the two iso-
lated regioisomers 8a and 8b demonstrated that compound 8a is
the most active (see the ESI† for details). It indicates that carba-
mate substitution at position 6 is detrimental to the antibacterial
activity whereas substitutions at position 5 are tolerated and have
only a minor negative functional influence compared to the
neamine (Table 1).
Scheme 5 Synthesis of neamine derivative 8a.
At last, to increase the panel of the neamine derivatives and to
increase the number of ammonium groups present on the amino-
glycoside core, the bis-carbamate 18 was prepared from 16, an
intermediate in the preparation of 8 (1 : 1 mixture of two regio-
isomers). Addition of 4-nitrophenyl chloroformate gave the car-
bonates 17a + 17b in 71% yield. A sequence similar to that
described above afforded product 18 (53% yield, three steps,
Scheme 6).
Conclusions
In this study, we developed efficient methods for synthesizing
cyclic carbonates and sulfates of 1,3,2′,6′-tetraazido-3′,4′,-di-O-
acetylneamine which are key precursors for the preparation of
novel neamine derivatives. The reported ring opening reactions
are applicable to different nucleophiles, allowing various chemi-
cal modifications of other aminoglycosides used in clinics.
Although the final products showed antibacterial activities
Scheme 6 Synthesis of the bis-carbamate 18.
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weaker than that of neamine, biological tests revealed the deter-
minant influence of the position of the carbamate substituent on
the antibacterial activity. This report is expected to serve as a
basis to generate compounds of higher activities against human
and animal bacterial pathogens.
(CH3), 20.6 (CH3); HRMS (electrospray) Calcd. for
C17H20N12O9Na [M + Na]+: 559, 1374, found: 559, 1371. [α]
+148.1 (c 0.45, CH2Cl2).
3′,4′-Di-O-acetyl-5,6-[sulfonylbis(oxy)]-1,3,2′,6′-tetraazidone-
amine 2
To a solution of 4 (2 g, 3.9 mmol) in anhydrous CH2Cl2
(40 mL) with NEt3 (1.9 mL, 13.7 mmol) at 0 °C was added
SOCl2 (425 μL, 5.9 mmol). The reaction was refluxed for 2 h.
The solution was cooled to room temperature, neutralized by
saturated NH4Cl aqueous solution (10 mL). The layers were sep-
arated and the organic layer was concentrated under reduced
pressure. The residue was taken up with EtOAc (50 mL), washed
with H2O (2 × 5 mL), brine (1 × 5 mL), dried over MgSO4 and
concentrated to obtain the corresponding sulfite which was used
without further purification. It was dissolved in a mixture of
CCl4 (20 mL) and acetonitrile (20 mL) and treated with NaIO4
(1.26 g, 5.9 mmol), RuCl3 (40 mg, 5%mol) and water (20 mL).
After stirring for 2 h, the reaction mixture was diluted with
diethyl ether (50 mL) and the layers were separated. The organic
layer was washed with H2O (2 × 10 mL), saturated NaHCO3 (1
× 10 mL), dried over MgSO4, and concentrated in vacuo. The
residue was purified by column chromatography using 20% to
25% EtOAc in cyclohexane to obtain 2 (1.8 g, 3.1 mmol, 79%)
Experimental section
General procedures
Tetrahydrofuran was distilled from sodium/benzophenone,
methylene chloride from P2O5 and methanol from sodium.
Analytical thin layer chromatography was performed on Merck
Silica Gel 60 F254 plates. Silica gel 60H (200–300 mesh) was
used for column chromatography. The compounds were charac-
terized by 1H and 13C NMR techniques including COSY,
1
HMQC and HMBC experiments. H NMR spectra (300 MHz,
500 MHz) and 13C NMR (75 MHz, 125 MHz) were recorded on
a Bruker AC 300 and AC 500 spectrometer with TMS as the
internal reference. Chemical shifts are given in ppm and coup-
ling constants J in Hz. High-resolution mass spectra (HRMS)
were recorded on a Bruker MicrOTOF-Q II spectrometer (Centre
Regional de Mesures Physiques de l’Ouest). Optical rotations
were measured by a Rudolph Research Analytical Autopol III
Automatic Polarimeter. The HPLC separation of the neamine
derivative 8 was carried out on a C18 reversed-phase column
(Interchim, 10 × 250 mm) on an Agilent 1200 system with diode
array UV detector (220 nM). Samples were eluted at a flow rate
of 3 mL min−1, using the gradient: 0.1% TFA in acetonitrile/
0.1% TFA in water 40/60 v/v. 3-Azidopropylamine and 6-azido-
hexylamine were prepared according to reported procedures.20
1
(Rf 0.7 EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, CDCl3)
δ 5.46 (dd, J = 9.9, 9.9 Hz, 1H, H-3′), 5.32 (d, J = 3.7 Hz, 1H,
H-1′), 5.05 (dd, J = 9.8, 9.8 Hz, 1H, H-4′), 4.61 (dd, J = 10.2,
10.2 Hz, 1H, H-5), 4.48 (dd, J = 10.1, 10.1 Hz, 1H, H-6), 4.27
(ddd, J = 3.4, 4.9, 9.8 Hz, 1H, H-5′), 4.09–3.95 (m, 2H, H-1,
H-4), 3.71 (ddd, J = 5.2, 9.1, 14.2 Hz, 1H, H-3), 3.47 (dd, J =
3.6, 10.6 Hz, 1H, H2′), 3.39–3.33 (m, 2H, H-6′), 2.59 (ddd, J =
5.1, 5.1, 14.1 Hz, 1H, H-2eq), 2.11 (s, 3H, CH3), 2.08 (s, 3H,
CH3), 1.67 (ddd, J = 12.1, 12.1, 12.1 Hz, 1H, H-2ax); 13C NMR
(75 MHz, CDCl3) δ 170.1 (CH3–CvO), 169.7 (CH3–CvO),
96.9 (C-1′), 84.5 (C-5), 84.2 (C-6), 75.4 (C-4), 70.0 (C-5′), 69.6
(C-4′), 69.0 (C-3′), 60.6 (C-2′), 58.8 (C-3), 55.6 (C-1), 50.8
(C-6′), 32.9 (C-2), 20.7 (CH3), 20.6 (CH3); HRMS (electrospray)
Calcd. for C16H20N12O10NaS [M + Na]+: 595.1044, found:
595.1050. [α] +136.7 (c 0.64, CH2Cl2).
1. Synthesis of cyclic carbonate 1 and sulfate 2. 5,6-[Carbo-
nylbis(oxy)]-3′,4′-di-O-acetyl-1,3,2′,6′-tetraazidoneamine 1
To a solution of 4 (2 g, 3.9 mmol) in anhydrous CH2Cl2
(40 mL) and pyridine (3.15 mL, 39 mmol) at −78 °C under an
Ar atmosphere was added a solution of triphosgene (1.7 g,
5.85 mmol) in anhydrous CH2Cl2 (10 mL). The reaction was
stirred overnight and allowed to warm to room temperature. The
solution was diluted with CH2Cl2 (50 mL) and neutralized with
saturated NH4Cl aqueous solution (10 mL). The organic phase
was separated from the aqueous phase, washed with HCl 1N
(2 × 5 mL), saturated NaHCO3 (1 × 5 mL), dried over MgSO4
and filtered. The filtrate was evaporated under reduced pressure
and purified by column chromatography using 20% to 30%
EtOAc in cyclohexane to give 1 (1.99 g, 3.7 mmol, 94%) (Rf 0.7
2. Preparation of the neamine derivatives 7–11 via the cyclic
carbonate 1. General procedure A: addition of amines to car-
bonate 1: To a solution of 1 (200 mg, 0.37 mmol) in anhydrous
CH2Cl2 (5 mL) was added the amine (0.58 mmol). The reaction
was stirred at room temperature for 2 h. Quantitative conversion
of the starting material was observed by TLC. Solvent was elimi-
nated under reduced pressure and the residue was taken up with
EtOAc (10 mL). The organic layer was washed with HCl 1N (2
× 1 mL), H2O (2 × 1 mL), brine (1 mL), dried over MgSO4 and
concentrated in vacuo. The residue was purified through silica
gel chromatography.
General procedure B: cleavage of the acetates: Sodium meth-
oxide was first prepared by slow addition, under an Ar atmos-
phere, of sodium (56 mg, 2 mmol) to anhydrous methanol
(5 mL). 0.05 mL of this solution (0.02 mmol) was slowly added
to a solution of the acetate (0.2 mmol) in anhydrous methanol
(5 mL). The reaction mixture was stirred overnight at room temp-
erature. The solution was diluted with methanol (5 mL) before
the addition of a cation-exchange resin (Amberlyst® 15,
1
EtOAc–cyclohexane 2 : 3). H NMR (500 MHz, CDCl3) δ 5.49
(dd, J = 9.2, 9.2 Hz, 1H, H-3′), 5.42 (d, J = 3.6 Hz, 1H, H-1′),
5.05 (dd, J = 9.3, 9.3 Hz, 1H, H-4′), 4.25 (ddd, J = 4.3, 4.3,
10.1 Hz, 1H, H-5′), 4.20 (dd, J = 9.9, 9.9 Hz, 1H, H-5), 4.10
(dd, J = 10.2, 10.2 Hz, 1H, H-6), 4.02 (dd, J = 8.9, 8.9 Hz, 1H,
H-4), 3.99–3.94 (m, 1H, H-1), 3.73–3.68 (m, 1H, H-3), 3.46
(dd, J = 3.7, 10.7 Hz, 1H, H-2′), 3.38 (d, J = 4.4 Hz, 2H, H-6′),
2.58 (ddd, J = 5.2, 5.2, 14.0 Hz, 1H, H-2eq), 2.12 (s, 3H, CH3),
2.08 (s, 3H, CH3), 1.64 (ddd, J = 12.5, 12.5, 12.5 Hz, 1H,
H-2ax); 13C NMR (125 MHz, CDCl3) δ 170.0 (CH3–CvO),
169.7 (CH3–CvO), 152.5 (CvO), 96.4 (C-1′), 80.8 (C-5), 80.6
(C-6), 75.7 (C-4), 69.9 (C-5′), 69.7 (C-4′), 69.1 (C-3′), 60.6
(C-2′), 59.7 (C-3), 56.1 (C-1), 50.9 (C-6′), 33.6 (C-2), 20.7
This journal is © The Royal Society of Chemistry 2012
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20–50 mesh, H+ form). The mixture was stirred for 1 h. The
resin was filtered off and the filtrate was concentrated in vacuo.
General procedure C: reduction of the azide functions: Azide
(0.2 mmol) was dissolved in anhydrous methanol (5 mL). PtO2
(100% wt). HCl 12 N (370 μL) was added and the reaction was
stirred overnight under an atmospheric pressure of dihydrogen at
room temperature. The mixture was filtered through celite, sol-
vents were eliminated under reduced pressure and the residue
was lyophilised to give the final product which was used without
further purification.
5-[[(Propylamino)carbonyl]oxy]-neamine tetrahydrochloride
7a and 6-[[(propylamino)carbonyl]oxy]-neamine tetrahydro-
chloride 7b
By following the general procedure C, a 1 : 1 mixture of
regioisomers 7a and 7b was obtained (90 mg, 0.16 mmol, 81%).
1H NMR (300 MHz, D2O) δ 5.81 (d, J = 3.2 Hz, 1H, H-1′), 5.45
(d, J = 3.4 Hz, 1H, H-1′), 4.80 (dd, J = 9.0, 9.0 Hz, 1H), 4.20
(dd, J = 9.8, 9.8 Hz, 1H), 4.00–2.90 (m, 24H), 2.50–2.34 (m,
2H, H-2eq), 1.96–1.76 (m, 2H, H-2ax), 1.44–1.26 (quint., J = 7.1
Hz, 4H, H-2′′), 0.75 (t, J = 7.3 Hz, 6H, H-3′′); 13C NMR
(75 MHz, D2O) δ 157.3 (CvO), 157.1 (CvO), 95.7 (C-1′),
93.5 (C-1′), 77.2, 76.8, 74.0, 73.4, 73.4, 71.2, 70.6, 70.2, 69.7,
69.2, 68.0, 68.0, 53.4, 53.1, 49.4, 48.8, 48.2, 48.0, 42.5 (C-6′),
42.5 (C-6′), 40.1 (C-1′′), 39.9 (C-1′′), 28.2 (C-2), 27.9 (C-2),
22.1 (C-2′′), 22.1 (C-2′′), 10.5 (C-3′′), 10.5 (C-3′′); HRMS (elec-
trospray) Calcd. for C16H33N5O7Na [M + Na]+: 430.2278,
found: 430.2277.
2.1 Preparation of 7a + 7b. 3′,4′-Di-O-acetyl-5-[[(propyl-
amino)carbonyl]oxy]-1,3,2′,6′-tetraazidoneamine 5a and 3′,4′-
di-O-acetyl-6-[[(propylamino)carbonyl]oxy]-1,3,2′,6′-tetraazi-
doneamine 5b
By following the general procedure A, a 1 : 1 mixture of regioi-
somers 5a + 5b was obtained (187 mg, 0.31 mmol, 85%)
(chromatography conditions: 20% to 30% EtOAc in cyclo-
1
hexane, Rf 0.4 EtOAc–cyclohexane 2 : 3). H NMR (300 MHz,
2.2 Preparation of 8a + 8b. 5-[[(3-Azidopropylamino)carbo-
nyl]oxy]-3′,4′-di-O-acetyl-1,3,2′,6′-tetraazidoneamine and 6-
[[(3-azidopropylamino)carbonyl]oxy]-3′,4′-di-O-acetyl-1,3,2′,6′-
tetraazidoneamine
CDCl3) δ 5.54 (d, J = 3.3 Hz, 1H, H-1′), 5.48 (dd, J = 10.5, 10.5
Hz, 1H, H-3′), 5.45 (dd, J = 10.6, 10.6 Hz, 1H, H-3′), 5.30 (d,
J = 3.2 Hz, 1H, H-1′), 5.21 (t, J = 5.6 Hz, 1H, NH), 5.05 (dd,
J = 9.7, 9.7 Hz, 2H, H-4′), 4.97 (t, J = 5.6 Hz, 1H, NH), 4.86
(dd, J = 8.9, 8.9 Hz, 1H, CH–OCvO–NHR), 4.75 (dd, J = 9.8,
9.8 Hz, 1H, CH–OCvO–NHR), 4.52–4.44 (m, 1H, H-5′),
4.42–4.34 (m, 1H, H-5′), 4.11 (d, J = 6.2 Hz, 1H, OH), 3.98 (d,
J = 3.3 Hz, 1H, OH), 3.69 (dd, J = 8.8 Hz, 1H), 3.62–3.10 (m,
19H), 2.42–2.30 (m, 2H, H-2eq), 2.10 (s, 6H, CH3–CvO), 2.06
(s, 6H, CH3–CvO), 1.70–1.45 (m, 4H, H-2ax, H-2′′), 0.94 (t,
J = 7.2 Hz, 6H, H-3′′); 13C NMR (75 MHz, CDCl3) δ 170.2
(CH3–CvO), 169.9 (CH3–CvO), 169.7 (CH3–CvO), 169.7
(CH3–CvO), 156.8 (CvO), 156.1 (CvO), 98.5 (C-1′), 98.5,
(C-1′), 82.5, 78.6, 78.1, 77.0, 76.3, 75.0, 70.8, 70.8, 70.0, 69.4,
69.3, 69.3, 61.4 (C–N3), 60.9 (C–N3), 60.3 (C–N3), 58.7
(C–N3), 58.3 (C–N3), 58.1 (C–N3), 50.8 (C-6′), 50.7 (C-6′), 43.2
(C-1′′), 43.1 (C-1′′), 31.8 (C-2), 31.6 (C-2), 23.0 (C-2′′), 22.9
(C-2′′), 20.7 (CH3–CvO), 20.7 (CH3–CvO), 20.6 (CH3–
CvO), 20.6 (CH3–CvO), 11.1 (C-3′′), 11.1 (C-3′′); HRMS
(electrospray) Calcd. for C20H29N13O9Na [M + Na]+: 618.2109,
found: 618.2090.
By following the general procedure A, a 1 : 1 mixture of regioi-
somers was obtained (200 mg, 0.31 mmol, 84%). (Chromato-
graphy conditions: 20% to 30% EtOAc in cyclohexane) (Rf 0.4
1
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, CDCl3) δ 5.55
(t, J = 5.2 Hz, 1H, NH), 5.48 (d, J = 3.7 Hz, 1H, H-1′), 5.42 (dd,
J = 9.6, 9.6 Hz, 2H, H-3′), 5.28 (d, J = 4.2 Hz, 1H, H-1′), 5.25
(t, J = 6.0 Hz, 1H, NH), 5.03 (dd, J = 9.7, 9.7 Hz, 2H, H-4′),
4.87 (dd, J = 9.2, 9.2 Hz, 1H, CH–OCvO–NHR), 4.76 (dd, J =
9.8, 9.8 Hz, 1H, CH–OCvO–NHR), 4.46 (ddd, J = 3.9, 6.7,
9.8 Hz, 1H, H-5′), 4.35 (ddd, J = 2.9, 4.7, 9.9 Hz, 1H, H-5′),
4.04 (d, J = 7.2 Hz, 1H, OH), 3.94 (d, J = 4.0 Hz, 1H, OH),
3.70–3.20 (m, 22H), 2.40–2.30 (m, 2H, H-2eq), 2.09 (s, 6H,
CH3), 2.04 (s, 6H, CH3), 1.86–1.74 (m, 4H, H-2′′), 1.62 (ddd,
J = 12.6, 12.6, 12.6 Hz, 1H, H-2ax), 1.47 (ddd, J = 12.2, 12.2,
12.2 Hz, 1H, H-2ax); 13C NMR (75 MHz, CDCl3) δ 170.2
(CH3–CvO), 170.0 (CH3–CvO), 169.8 (CH3–CvO), 169.7
(CH3–CvO), 156.7 (CvO), 156.1 (CvO), 98.6 (C-1′), 98.5,
(C-1′), 82.7, 78.6, 77.9, 76.7, 76.1, 74.8, 70.8, 69.9, 69.4, 69.3,
69.3, 69.2, 61.4 (C–N3), 60.8 (C–N3), 60.1 (C–N3), 58.6 (C–
N3), 58.3 (C–N3), 58.1 (C–N3), 50.8 (C-6′), 50.6 (C-6′), 49.0
(C-3′′), 49.0 (C-3′′), 39.0 (C-1′′), 38.8 (C-1′′), 31.7 (C-2), 31.7
(C-2), 28.8 (C-2′′), 28.7 (C-2′′), 20.7 (CH3), 20.6 (CH3), 20.6
(CH3), 20.6 (CH3); HRMS (electrospray) Calcd. for
C20H28N16O9Na [M + Na]+: 659.2123, found: 659.2134.
5-[[(Propylamino)carbonyl]oxy]-1,3,2′,6′-tetraazidoneamine
and 6-[[(propylamino)carbonyl]oxy]-1,3,2′,6′-tetraazidoneamine
By following the general procedure B, a 1 : 1 mixture of
regioisomers was obtained (71 mg, 0.14 mmol, 69%). (Rf 0.2
1
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, CDCl3) δ 5.54
(d, J = 3.3 Hz, 1H, H-1′), 5.30 (d, J = 3.2 Hz, 1H, H-1′), 5.21 (t,
J = 5.6 Hz, 1H, NH), 4.97 (t, J = 5.6 Hz, 1H, NH), 4.86 (dd, J =
8.9, 8.9 Hz, 1H, CH–OCvO–NHR), 4.75 (dd, J = 9.8, 9.8 Hz,
1H, CH–OCvO–NHR), 4.52–4.44 (m, 1H, H-5′), 4.42–4.34
(m, 1H, H-5′), 4.11 (d, J = 6.2 Hz, 1H, OH), 3.98 (d, J = 3.3 Hz,
1H, OH), 3.69 (dd, J = 8.8 Hz, 1H), 3.62–3.10 (m, 27H),
2.42–2.30 (m, 2H, H-2eq), 1.70–1.45 (m, 4H, H-2ax, H-2′′), 0.94
(t, J = 7.2 Hz, 6H, H-3′′); 13C NMR (75 MHz, CDCl3) δ 156.8
(CvO), 156.1 (CvO), 98.5 (C-1′), 98.5, (C-1′), 82.5, 78.6,
78.1, 77.0, 76.3, 75.0, 70.8, 70.8, 70.0, 69.4, 69.3, 69.3, 61.4
(C–N3), 60.9 (C–N3), 60.3 (C–N3), 58.7 (C–N3), 58.3 (C–N3),
58.1 (C–N3), 50.8 (C-6′), 50.7 (C-6′), 43.2 (C-1′′), 43.1 (C-1′′),
31.8 (C-2), 31.6 (C-2), 23.0 (C-2′′), 22.9 (C-2′′), 11.1 (C-3′′),
11.1 (C-3′′); HRMS (electrospray) Calcd. for C16H25N13O7Na
[M + Na]+: 534.1898, found: 534.1901.
5-[[(3-Azidopropylamino)carbonyl]oxy]-1,3,2′,6′-tetraazido-
neamine and 6-[[(3-azidopropylamino)carbonyl]oxy]-1,3,2′,6′-
tetraazidoneamine
By following the general procedure B, we obtained a mixture
(1 : 1) of white solids (100 mg, 0.18 mmol, 91%). (Rf 0.20
1
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, Acetone-d6) δ
6.66 (s, br, 1H), 6.52 (s, br, 1H), 5.64 (d, J = 3.7 Hz, 1H, H-1′),
5.30 (d, J = 3.4 Hz, 1H, H-1′), 4.92 (dd, J = 9.1, 9.1 Hz, 1H),
4.81 (dd, J = 9.8, 9.8 Hz, 1H), 4.26–4.20 (m, 1H, H-5′),
4.20–4.14 (m, 1H, H-5′), 3.94 (dd, J = 8.9, 10.2 Hz, 1H), 3.91
(dd, J = 8.8, 10.4 Hz, 1H), 3.80–3.62 (m, 8H), 3.62–3.40 (m,
14H), 3.40–3.20 (m, 7H), 3.12 (dd, J = 3.4, 10.4 Hz, 1H, H-2′),
2.45–2.30 (m, 2H, H-2éq), 1.90–1.75 (m, 4H, H-2′′), 1.68 (ddd,
J = 12.5, 12.5, 12.5 Hz, 1H, H-2ax), 1.53 (ddd, J = 12.2, 12.2,
4724 | Org. Biomol. Chem., 2012, 10, 4720–4730
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1
12.2 Hz, 1H, H-2ax); 13C NMR (75 MHz, Acetone-d6) δ 156.5
(CvO), 156.1 (CvO), 98.6 (C-1′), 98.5 (C-1′), 80.2, 77.7, 77.5,
76.7, 75.5, 75.0, 72.1, 71.9, 71.6, 71.5, 71.4, 70.8, 63.4 (C–N3),
62.9 (C–N3), 60.5 (C–N3), 59.4 (C–N3), 59.3 (C–N3), 58.6
(C–N3), 51.4 (C-6′), 51.4 (C-6′), 48.7 (C-3′′), 48.6 (C-3′′), 38.3
(C-1′′), 38.1 (C-1′′), 31.7 (C-2), 31.6 (C-2), 29.5 (C-2′′), 29.3
(C-2′′); HRMS (electrospray) Calcd. for C16H24N16O7Na
[M + Na]+: 575.1911, found: 575.1912.
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, Acetone-d6) δ
6.57 (t, J = 5.4 Hz, 1H, NH), 6.38 (t, J = 5.5 Hz, 1H, NH), 5.64
(d, J = 3.6 Hz, 1H, H-1′), 5.31 (d, J = 3.6 Hz, 1H, H-1′), 4.88
(dd, J = 9.3, 9.3 Hz, 1H, CH–OCvO–NHR), 4.78 (dd, J = 9.2,
9.2 Hz, 1H, CH–OCvO–NHR), 4.26–4.12 (m, 2H, H-5′), 3.93
(dd, J = 10.3, 10.3 Hz, 1H), 3.90 (dd, J = 9.1, 9.1 Hz, 1H),
3.80–3.39 (m, 14H), 3.38–3.25 (m, 11H), 3.25–3.05 (m, 5H),
2.44–2.28 (m, 2H, H-2eq), 1.67 (ddd, J = 12.4, 12.4, 12.4 Hz,
1H, H-2ax), 1.65–1.30 (m, 17H); 13C NMR (75 MHz, Acetone-
d6) δ 156.6 (CvO), 156.0 (CvO), 98.5 (C-1′), 98.5 (C-1′),
80.2, 77.7, 77.6, 76.6, 75.6, 75.1, 72.0, 71.9, 71.5, 71.5, 71.3,
70.9, 63.4 (C–N3), 63.1 (C–N3), 60.6 (C–N3), 59.4 (C–N3), 59.3
(C–N3), 58.6 (C–N3), 51.4 (C-6′′), 51.4 (C-6′′), 51.1 (C-6′), 51.0
(C-6′), 40.9 (C-1′′), 40.6 (C-1′′), 31.6 (C-2′′), 31.5 (C-2′′), 29.6
(C-2), 29.5 (C-2), 28.6 (C-5′′), 28.5 (C-5′′), 26.2, 26.1, 26.0,
26.0; HRMS (electrospray) Calcd. for C19H30N16O7Na
[M + Na]+: 617.2381, found: 617.2372.
5-[[(3-Azidopropylamino)carbonyl]oxy]-neamine pentahydro-
chloride 8a and 6-[[(3-azidopropylamino) carbonyl]oxy]-
neamine pentahydrochloride 8b
By following the general procedure C, we obtained 8a and 8b
(97 mg, 0.16 mmol, 80%) as a mixture (1 : 1) of white solids.
1H NMR (300 MHz, D2O) δ 5.86 (d, J = 3.9 Hz, 1H, H-1′), 5.51
(d, J = 3.8 Hz, 1H, H-1′), 4.88 (dd, J = 9.2, 9.2 Hz, 1H), 4.80
(dd, J = 9.7, 9.7 Hz, 1H), 4.30 (dd, J = 10.1, 10.1 Hz, 1H),
4.10–3.10 (m, 23H), 3.00–2.92 (m, 4H), 2.50 (ddd, J = 4.3, 4.3,
12.9 Hz, 1H, H-2eq), 2.44 (ddd, J = 3.3, 3.3, 11.7 Hz, 1H,
H-2eq), 1.95 (ddd, J = 12.6, 12.6, 12.6 Hz, 1H, H-2ax), 1.91
(ddd, J = 12.6, 12.6, 12.6 Hz, 1H, H-2ax), 1.86–1.76 (m, 4H,
H-2′′); 13C NMR (75 MHz, D2O) δ 157.5 (CvO), 157.2
(CvO), 96.0 (C-1′), 93.6 (C-1′), 77.5, 77.0, 74.3, 73.5, 73.3,
71.4, 70.7, 70.3, 69.8, 69.3, 68.1, 68.1, 53.5, 53.1, 49.4, 48.8,
48.2, 48.0, 40.1 (C-6′), 40.0 (C-6′), 37.7, 37.7, 37.1, 37.1, 28.3,
27.9, 27.0, 27.0; HRMS (electrospray) Calcd. for C16H35N6O7
[M + H]+: 423.2567, found: 423.2571.
5-[[(3-Azidohexylamino)carbonyl]oxy]-neamine pentahydro-
chloride 9a and 6-[[(3-azidohexylamino) carbonyl]oxy]-
neamine pentahydrochloride 9b
By following the general procedure C, a 1 : 1 mixture of
regioisomers 9a and 9b was obtained (105 mg, 0.16 mmol,
1
82%). H NMR (300 MHz, D2O) δ 5.80 (d, J = 3.6 Hz, 1H,
H-1′), 5.43 (d, J = 3.8 Hz, 1H, H-1′), 5.00–4.70 (m, 3H), 4.24
(dd, J = 9.9, 9.9 Hz, 1H), 4.05–3.75 (m, 5H), 3.70 (dd, J = 10.6,
10.6 Hz, 1H), 3.65–2.90 (m, 16H), 2.90–2.80 (m, 4H, H-1′′),
2.50–2.30 (m, 2H, H-2eq), 2.00–1.75 (m, 2H, H-2ax), 1.60–1.30
(m, 8H, H-2′′, H-5′′), 1.30–1.10 (m, 8H, H-3′′, H-4′′); 13C NMR
(75 MHz, D2O) δ 157.3 (CvO), 157.1 (CvO), 95.8 (C-1′),
93.6 (C-1′), 77.3, 73.6, 73.5, 73.4, 71.3, 70.7, 70.3, 69.8, 69.3,
69.2, 68.1, 68.0, 53.5, 53.1, 49.4, 48.8, 48.2, 48.1, 40.6, 40.2,
40.2, 40.0, 39.5, 39.5, 28.5, 28.3, 28.0, 27.9, 26.6, 26.6, 25.4,
25.4, 25.3, 25.2; HRMS (electrospray) Calcd. for C19H41N6O7
[M + H]+: 465.3037, found: 465.3040.
2.3 Preparation of 9a + 9b. 5-[[(3-Azidohexylamino)carbo-
nyl]oxy]-3′,4′-di-O-acetyl-1,3,2′,6′-tetraazidoneamine and 6-
[[(3-azidohexylamino) carbonyl]oxy]-3′,4′-di-O-acetyl-1,3,2′,6′-
tetraazidoneamine
By following the general procedure A, a (1 : 1) mixture of regioi-
somers was obtained (195 mg, 0.29 mmol, 78%). (Chromato-
graphy conditions: 20% to 30% EtOAc in cyclohexane) (Rf 0.40
1
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, CDCl3) δ 5.52
(d, J = 3.4 Hz, 1H, H-1′), 5.45 (dd, J = 9.4, 9.4 Hz, 2H, H-3′),
5.28 (d, J = 3.7 Hz, 1H, H-1′), 5.14 (t, J = 5.8 Hz, 1H, NH),
5.05 (dd, J = 10.1, 10.1 Hz, 2H, H-4′), 4.96 (t, J = 5.8 Hz, 1H,
NH), 4.86 (dd, J = 9.1, 9.1 Hz, 1H, CH–OCvO–NHR), 4.74
(dd, J = 9.8, 9.8 Hz, 1H, CH–OCvO–NHR), 4.48 (ddd, J = 2.8,
4.5, 10.1 Hz, 1H, H-5′), 4.38 (ddd, J = 2.7, 4.7, 10.0 Hz, 1H,
H-5′), 4.05 (d, J = 6.3 Hz, 1H, OH), 3.95 (d, J = 3.6 Hz, 1H,
OH), 3.74–3.62 (m, 1H), 3.62–3.15 (m, 21H), 2.45–2.30 (m,
2H, H-2eq), 2.10 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.07 (s, 3H,
CH3), 2.06 (s, 3H, CH3), 1.70–1.30 (m, 18H); 13C NMR
(75 MHz, CDCl3) δ 170.2 (CH3–CvO), 170.0 (CH3–CvO),
169.7 (CH3–CvO), 169.7 (CH3–CvO), 156.8 (CvO), 156.1
(CvO), 98.5 (C-1′), 98.5 (C-1′), 82.7, 78.7, 78.0, 76.9, 76.3,
75.0, 70.8, 69.9, 69.4, 69.4, 69.3, 69.2, 61.4 (C–N3), 60.9 (C–
N3), 60.3 (C–N3), 58.7 (C–N3), 58.3 (C–N3), 58.1 (C–N3), 51.3
(C-6′′), 51.3 (C-6′′), 50.8 (C-6′), 50.7 (C-6′), 41.4 (C-1′′), 41.2
(C-1′′), 31.8 (C-2′′), 31.6 (C-2′′), 29.6 (C-2), 29.5 (C-2), 28.7
(C-5′′), 28.7 (C-5′′), 26.3, 26.3, 26.2, 26.1, 20.7 (CH3), 20.7
(CH3), 20.6 (CH3), 20.6 (CH3); HRMS (electrospray) Calcd. for
C23H33N16O9Na2 [M + 2Na]+: 723.2412, found: 723.2438.
5-[[(3-Azidohexylamino)carbonyl]oxy]-1,3,2′,6′-tetraazido-
neamine and 6-[[(3-azidohexylamino) carbonyl]oxy]-1,3,2′,6′-
tetraazidoneamine
2.4 Preparation of 10a + 10b. 3′,4′-Di-O-acetyl-5-[[[(2-(1H-
imidazol-3-yl)ethyl)amino]carbonyl]oxy]-1,3,2′,6′-tetraazidone-
amine and 3′,4′-di-O-acetyl-6-[[[(2-(1H-imidazol-3-yl)ethyl)
amino]carbonyl]oxy]-1,3,2′,6′-tetraazidoneamine
By following the general procedure A, a 1 : 1 mixture of regioi-
somers was obtained (175 mg, 0.27 mmol, 73%). (Chromato-
graphy conditions: 10% MeOH in CH2Cl2) (Rf 0.35 MeOH–
1
CH2Cl2 1 : 10). H NMR (300 MHz, Acetone-d6) δ 7.64 (s, 1H,
H-6′′), 7.63 (s, 1H, H-6′′), 6.91 (s, 2H, H-4′′), 6.71 (t, J = 5.4
Hz, 1H, NH), 6.49 (t, J = 5.5 Hz, 1H, NH), 5.91 (d, J = 3.4 Hz,
1H, H-1′), 5.47 (dd, J = 9.1, 9.1 Hz, 1H, H-3′), 5.47 (d, J = 3.9
Hz, 1H, H-1′), 5.41 (dd, J = 9.3, 10.7 Hz, 1H, H-3′), 5.04 (dd,
J = 10.0, 10.0 Hz, 1H, H-4′), 5.03 (dd, J = 10.0, 10.0 Hz, 1H,
H-4′), 4.97 (dd, J = 9.2, 9.2 Hz, 1H, CH–OCvO–NHR), 4.83
(dd, J = 9.8, 9.8 Hz, 1H, CH–OCvO–NHR), 4.51 (ddd, J = 3.4,
4.7, 9.6 Hz, 2H, H-5′), 3.90–3.55 (m, 9H), 3.55–3.25 (m, 11H),
2.90–2.70 (m, 4H, H-2′′), 2.50–2.30 (m, 2H, H-2eq), 2.10 (s, 3H,
CH3), 2.05 (s, 3H, CH3), 2.04 (s, 3H, CH3), 2.02 (s, 3H, CH3),
1.75 (ddd, J = 12.3, 12.3, 12.3 Hz, 1H, H-2ax), 1.58 (ddd,
J = 12.1, 12.1, 12.1 Hz, 1H, H-2ax); 13C NMR (75 MHz,
Acetone-d6) δ 169.5 (CH3–CvO), 169.4 (CH3–CvO), 169.3
(CH3–CvO), 169.2 (CH3–CvO), 156.2 (CvO), 156.0 (CvO),
135.0 (C-6′′), 135.0 (C-6′′), 135.0 (C-3′′), 135.0 (C-3′′), 116.0
(C-4′′), 113.2 (C-4′′), 98.1 (C-1′), 98.1 (C-1′), 80.0, 78.2, 77.5,
77.1, 75.2, 74.8, 69.8, 69.8, 69.3, 69.3, 69.2, 69.2, 60.6 (C–N3),
By following the general procedure B, a 1 : 1 mixture of
regioisomers was obtained (85 mg, 0.15 mmol, 72%). (Rf 0.20
This journal is © The Royal Society of Chemistry 2012
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60.6 (C–N3), 60.4 (C–N3), 59.0 (C–N3), 58.9 (C–N3), 58.4
(C–N3), 50.8 (C-6′), 50.7 (C-6′), 41.4 (C-1′′), 41.3 (C-1′′), 31.5
(C-2), 31.4 (C-2), 26.9 (C-2′′), 26.9 (C-2′′), 19.8 (CH3), 19.8
(CH3), 19.8 (CH3), 19.8 (CH3); HRMS (electrospray) Calcd. for
C22H30N15O9 [M + H]+: 648.2351, found: 648.2348.
5-[[[(2-(1H-Imidazol-3-yl)ethyl)amino]carbonyl]oxy]-1,3,2′,
6′-tetraazidoneamine and 6-[[[(2-(1H-imidazol-3-yl)ethyl)amino]-
carbonyl]oxy]-1,3,2′,6′-tetraazidoneamine
5.41 (dd, J = 9.4, 9.4 Hz, 1H, H-3′), 5.29 (d, J = 3.3 Hz, 1H,
H-1′), 5.03 (dd, J = 10.1, 10.1 Hz, 1H, H-4′), 5.02 (dd, J = 9.3,
9.3 Hz, 1H, H-4′), 4.87 (dd, J = 9.2, 9.2 Hz, 1H, CH–OCvO–
NHR), 4.73 (dd, J = 9.8, 9.8 Hz, 1H, CH–OCvO–NHR), 4.52
(s, br, 1H), 4.47 (ddd, J = 2.9, 4.3, 10.1 Hz, 1H, H-5′), 4.38
(ddd, J = 2.9, 4.4, 9.9 Hz, 1H, H-5′), 4.27 (d, J = 3.3 Hz, 1H,
OH), 3.75–3.60 (m, 5H), 3.60–3.45 (m, 7H), 3.45–3.20 (m,
12H), 2.35 (ddd, J = 3.4, 3.4, 13.2 Hz, 2H, H-2eq), 2.08 (s, 6H,
CH3), 2.05 (s, 6H, CH3), 1.72 (quint., J = 5.6 Hz, 4H, H-2′′),
1.62 (ddd, J = 12.6, 12.6, 12.6 Hz, 1H, H-2ax), 1.48 (ddd, J =
12.2, 12.2, 12.2 Hz, 1H, H-2ax); 13C NMR (75 MHz, Acetone-
d6) δ 169.5 (CH3–CvO), 169.4 (CH3–CvO), 169.2 (CH3–
CvO), 169.2 (CH3–CvO), 156.7 (CvO), 156.3 (CvO), 98.2
(C-1′), 98.2 (C-1′), 80.4, 78.2, 77.6, 77.6, 76.8, 75.6, 74.9, 69.9,
69.8, 69.4, 69.3, 69.2, 60.7 (C–N3), 60.6 (C–N3), 59.7 (C–N3),
59.2 (C–N3), 59.1 (C–N3), 58.9 (C–N3), 58.8 (C–N3), 58.5 (C–
N3), 50.7 (C-6′), 50.7 (C-6′), 38.3 (C-1′′), 38.1 (C-1′′), 32.7
(C-2′′), 32.6 (C-2′′), 31.4 (C-2), 31.3 (C-2), 19.8 (CH3), 19.8
(CH3), 19.8 (CH3), 19.8 (CH3).
By following the general procedure B, a 1 : 1 mixture of
regioisomers was obtained (67 mg, 0.12 mmol, 59%). (Rf
0.2 MeOH–CH2Cl2 1 : 10). 1H NMR (500 MHz, CD3OD) δ
8.12 (s, 1H, H-6′′), 8.10 (s, 1H, H-6′′), 7.09 (s, 1H, H-4′′), 7.08
(s, 1H, H-4′′), 5.66 (d, J = 3.6 Hz, 1H, H-1′), 5.25 (d, J = 3.7
Hz, 1H, H-1′), 4.87 (dd, J = 9.4, 9.4 Hz, 1H, CH–OCvO–
NHR), 4.65 (dd, J = 9.8, 9.8 Hz, 1H, CH–OCvO–NHR),
4.25–4.17 (m, 2H, H-5′), 3.88 (dd, J = 8.9, 10.5 Hz, 1H), 3.83
(dd, J = 8.9, 10.4 Hz, 1H), 3.69 (dd, J = 9.0, 9.0 Hz, 1H),
3.65–3.35 (m, 23H), 3.13 (dd, J = 3.8, 10.5 Hz, 1H, H-2′), 3.06
(dd, J = 3.8, 10.5 Hz, 1H, H-2′), 2.94–2.84 (quint., J = 7.0 Hz,
4H, H-2′′), 2.36–2.28 (m, 2H, H-2eq), 1.55 (ddd, J = 12.4, 12.4,
12.4 Hz, 1H, H-2ax), 1.47 (ddd, J = 12.1, 12.1, 12.1 Hz, 1H,
H-2ax); 13C NMR (125 MHz, CD3OD) δ 156.8 (CvO), 156.7
(CvO), 134.1 (C-6′′), 134.1 (C-6′′), 133.2 (C-3′′), 133.2 (C-3′′),
116.5 (C-4′′), 116.4 (C-4′′), 98.6 (C-1′), 98.4 (C-1′), 79.5, 77.7,
77.1, 77.0, 75.1, 74.6, 72.0, 71.9, 71.3, 71.2, 70.7, 70.4, 62.8
(C–N3), 60.2 (C–N3), 59.2 (C–N3), 59.2 (C–N3), 58.4 (C–N3),
58.4 (C–N3), 51.2 (C-6′), 51.1 (C-6′), 40.2 (C-1′′), 40.0 (C-1′′),
31.6 (C-2), 31.5 (C-2), 25.9 (C-2′′), 25.8 (C-2′′); HRMS (electro-
spray) Calcd. for C18H26N15O7 [M + H]+: 564.2140, found:
564.2148.
5-[[(3-Hydroxypropylamino)carbonyl]oxy]-1,3,2′,6′-tetraazi-
doneamine and 6-[[(3-hydroxypropylamino)carbonyl]oxy]-
1,3,2′,6′-tetraazidoneamine
By following the general procedure B, a 1 : 1 mixture of
regioisomers was obtained (77 mg, 0.15 mmol, 73%). (Rf 0.15
1
EtOAc–cyclohexane 2 : 3). H NMR (500 MHz, Acetone-d6) δ
5.65 (d, J = 3.7 Hz, 1H, H-1′), 5.30 (d, J = 3.8 Hz, 1H, H-1′),
4.91 (dd, J = 9.4, 9.4 Hz, 1H, CH–OCvO–NHR), 4.79 (dd,
J = 9.8, 9.8 Hz, 1H, CH–OCvO–NHR), 4.22 (ddd, J = 2.5, 5.2,
9.9 Hz, 1H, H-5′), 4.17 (ddd, J = 2.3, 5.2, 9.8 Hz, 1H, H-5′),
3.93 (dd, J = 8.7, 10.5 Hz, 1H), 3.89 (dd, J = 8.7, 10.5 Hz, 1H),
3.80–3.40 (m, 18H), 3.35–3.30 (m, 5H), 3.16 (dd, J = 3.7, 10.5
Hz, 1H, H-2′), 2.39 (ddd, J = 4.5, 4.5, 12.8 Hz, 1H, H-2eq), 2.35
(ddd, J = 4.1, 4.1, 12.7 Hz, 1H, H-2eq), 1.74 (m, 4H, H-2′′), 1.67
(ddd, J = 12.4, 12.4, 12.4 Hz, 1H, H-2ax), 1.53 (ddd, J = 12.4,
12.4, 12.4 Hz, 1H, H-2ax); 13C NMR (75 MHz, Acetone-d6) δ
156.8 (CvO), 156.4 (CvO), 98.6 (C-1′), 98.5 (C-1′), 80.1,
77.8, 77.5, 76.7, 75.5, 74.9, 72.1, 71.9, 71.5, 71.4, 71.3, 70.9,
63.4 (C–N3), 63.3 (C–N3), 60.5 (C–N3), 59.4 (C–N3), 59.3
(C–N3), 59.2 (C–N3), 59.1 (C–N3), 58.6 (C–N3), 51.4 (C-6′),
51.4 (C-6′), 38.2 (C-1′′), 38.2 (C-1′′), 32.6 (C-2′′), 32.4 (C-2′′),
31.7 (C-2), 31.6 (C-2); HRMS (electrospray) Calcd. for
C16H25N13O8Na [M + Na]+: 550.1847, found: 550.1852.
5-[[[(2-(1H-Imidazol-3-yl)ethyl)amino]carbonyl]oxy]-neamine
pentahydrochloride 10a and 6-[[[(2-(1H-imidazol-3-yl)ethyl)
amino]carbonyl]oxy]-neamine pentahydrochloride 10b
By following the general procedure C, a 1 : 1 mixture of
1
regioisomers was obtained (90 mg, 0.14 mmol, 70%). H NMR
(300 MHz, D2O) δ 8.49 (s, 2H, H-6′′), 7.18 (s, 2H, H-4′′), 5.84
(d, J = 3.4 Hz, 1H, H-1′), 5.42 (d, J = 3.6 Hz, 1H, H-1′), 4.80
(dd, J = 9.1, 9.1 Hz, 1H, CH–OCvO–NHR), 4.26 (dd, J = 9.6,
9.6 Hz, 1H, CH–OCvO–NHR), 4.05–3.75 (m, 6H), 3.71 (dd,
J = 10.2, 10.2 Hz, 1H), 3.65–3.15 (m, 17H), 2.90–2.80 (m, 4H),
2.50–2.35 (m, 2H, H-2eq), 2.00–1.80 (m, 2H, H-2ax); 13C NMR
(75 MHz, D2O) δ 157.2 (CvO), 157.0 (CvO), 133.1 (C-6′′),
133.1 (C-6′′), 130.6 (C-3′′), 130.5 (C-3′′), 116.3 (C-4′′), 116.2
(C-4′′), 95.7 (C-1′), 93.4 (C-1′), 77.3, 76.7, 74.2, 73.4, 73.2,
71.2, 70.7, 70.3, 69.8, 69.2, 68.1, 68.0, 53.5, 53.1, 49.4, 48.8,
48.2, 48.0, 40.2, 40.0, 39.5, 39.4, 28.2, 27.9, 24.4, 24.4; HRMS
(electrospray) Calcd. for C18H34N7O7 [M + H]+: 460.2520,
found: 460.2526, calcd. for C18H33N7O7Na [M + Na]+:
482.2339, found: 482.2332.
5-[[(3-Hydroxypropylamino)carbonyl]oxy]-neamine tetrahy-
drochloride 11a and 6-[[(3-hydroxypropylamino)carbonyl]oxy]-
neamine tetrahydrochloride 11b
By following the general procedure C, a 1 : 1 mixture of
regioisomers 11a and 11b was obtained (89 mg, 0.16 mmol,
1
78%). H NMR (300 MHz, D2O) δ 5.84 (d, J = 3.5 Hz, 1H,
H-1′), 5.45 (d, J = 3.7 Hz, 1H, H-1′), 4.82 (dd, J = 9.1, 9.1 Hz,
1H, CH–OCvO–NHR), 4.28 (dd, J = 9.6, 9.6 Hz, 1H, CH–
OCvO–NHR), 4.00–3.80 (m, 2H, H-5′), 3.97 (dd, J = 10.4,
10.4 Hz, 1H), 3.92 (dd, J = 10.1, 10.1 Hz, 1H), 3.83 (dd, J =
9.3, 9.3 Hz, 1H), 3.73 (dd, J = 9.8, 9.8 Hz, 1H), 3.65–3.00 (m,
22H), 2.50–2.35 (m, 2H, H-2eq), 2.00–1.75 (m, 2H, H-2ax), 1.63
(quint., J = 6.4 Hz, 4H, H-2′′); 13C NMR (75 MHz, D2O) δ
157.3 (CvO), 157.1 (CvO), 95.7 (C-1′), 93.5 (C-1′), 77.3,
76.7, 74.2, 73.5, 73.3, 71.3, 70.7, 70.3, 69.8, 69.2, 68.1, 68.1,
59.1, 59.1, 53.5, 53.1, 49.4, 48.8, 48.3, 48.1, 40.2 (C-6′), 40.0
(C-6′), 37.7 (C-1′′), 37.6 (C-1′′), 31.1 (C-2′′), 31.1 (C-2′′), 28.2
2.5 Preparation of 11a + 11b. 3′,4′-Di-O-acetyl-5-[[(3-hydro-
xypropylamino)carbonyl]oxy]-1,3,2′,6′-tetraazidoneamine and
3′,4′-di-O-acetyl-6-[[(3-hydroxypropylamino)carbonyl]oxy]-
1,3,2′,6′-tetraazidoneamine
By following the general procedure A, a 1 : 1 mixture of regioi-
somers was obtained (147 mg, 0.24 mmol, 64%). (Chromato-
graphy conditions: 20% to 30% EtOAc in cyclohexane) (Rf 0.35
1
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, Acetone-d6) δ
5.87 (t, J = 5.7 Hz, 1H, NH), 5.61 (t, J = 5.8 Hz, 1H, NH), 5.55
(d, J = 3.5 Hz, 1H, H-1′), 5.46 (dd, J = 9.4, 9.4 Hz, 1H, H-3′),
4726 | Org. Biomol. Chem., 2012, 10, 4720–4730
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(C-2), 27.9 (C-2); HRMS (electrospray) Calcd. for
C16H33N5O8Na [M + Na]+: 446.2227, found: 446.2236.
10.5 Hz, 1H, H-2′), 2.50 (ddd, J = 4.4, 4.4, 13.0 Hz, 1H, H-2eq),
2.41 (ddd, J = 4.1, 4.1, 13.3 Hz, 1H, H-2eq), 1.80 (quint., J = 6.4
Hz, 4H, H-2′′), 1.72 (ddd, J = 11.1, 11.1, 11.1 Hz, 1H, H-2ax),
1.60 (ddd, J = 12.2, 12.2, 12.2 Hz, 1H, H-2ax); 13C NMR
(75 MHz, Acetone-d6) δ 98.8 (C-1′), 97.6 (C-1′), 86.3, 84.3,
80.7, 76.0, 75.0, 74.7, 72.1, 72.0, 71.7, 71.5, 71.4, 70.8, 63.6,
63.2, 60.5 (C–N3), 59.5 (C–N3), 58.9 (C–N3), 58.5 (C–N3), 51.4
(C–N3), 48.8 (C–N3), 48.5 (C–N3), 48.5 (C–N3), 41.3 (C-6′),
41.1 (C-6′), 36.4, 36.4, 31.5 (C-2), 31.5 (C-2); HRMS (electro-
spray) Calcd. for C15H23N16O8S [M − H]−: 587.1606, found:
587.1610.
3. Preparation of the neamine derivatives 12–14 via the
cyclic sulfate 2. General procedure D: ring opening of the
sulfate 2: 2 (200 mg, 0.35 mmol) was dissolved in pure amine
(1 mL) and the mixture was stirred overnight at room tempera-
ture. The reaction was diluted with EtOAc (10 mL), washed with
HCl 1N (3 × 2 mL), H2O (2 × 1 mL), brine (1 mL), dried over
MgSO4 and concentrated under reduced pressure. The residue
was purified through silica gel chromatography.
3.1 Preparation of 12a + 12b. 5-(N-Propylamino)-6-sulfoxy-
1,3,2′,6′-tetraazidoneamine 6a and 6-(N-propylamino)-5-
sulfoxy-1,3,2′,6′-tetraazidoneamine 6b
5-(N-(3-Azidopropylamino))-6-sulfoxyneamine hexahydro-
chloride 13a and 6-(N-(3-azidopropylamino))-5-sulfoxyneamine
hexahydrochloride 13b
By following the general procedure D, a 1 : 1 mixture of regio-
isomers was obtained (123 mg, 0.22 mmol, 64%). (Chromato-
graphy conditions: 40% to 50% EtOAc in cyclohexane) (Rf 0.25
By following the general procedure C, a 1 : 1 mixture of
regioisomers 13a and 13b was obtained (88 mg, 0.13 mmol,
66%). H NMR (300 MHz, D2O) δ 5.88 (d, J = 3.7 Hz, 1H,
1
1
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, CDCl3) δ 5.71
H-1′), 5.65 (d, J = 3.4 Hz, 1H, H-1′), 4.85–4.60 (m, 2H, H-5′),
4.50 (dd, J = 9.7, 9.7 Hz, 1H), 4.34 (dd, J = 9.8, 9.8 Hz, 1H),
4.10–2.80 (m, 26H), 2.55–2.35 (m, 2H, H-2eq), 1.90 (ddd, J =
12.8, 12.8, 12.8 Hz, 2H, H-2ax), 1.75 (quint., J = 7.6 Hz, 4H,
H-2′′); 13C NMR (75 MHz, D2O) δ 95.8 (C-1′), 93.9 (C-1′),
83.4, 80.3, 76.5, 74.9, 72.9, 71.6, 70.6, 70.5, 69.4, 69.3, 68.0,
67.6, 53.5, 52.6, 49.7, 49.7, 49.0, 48.6, 48.2, 48.1, 40.8, 40.7,
40.1, 39.6, 28.0, 27.9, 27.0, 26.8; HRMS (electrospray) Calcd.
for C15H33N6O8S [M − H]−: 457.2081, found 457.2084.
(t, J = 6.0 Hz, 1H, NH), 5.38 (t, J = 4.0 Hz, 1H, H-1′), 5.37 (t,
J = 4.0 Hz, 1H, H-1′), 5.14 (t, J = 6.0 Hz, 1H, NH), 4.80–4.70
(m, 5H), 4.64 (dd, J = 8.5, 8.5 Hz, 1H), 4.34 (dd, J = 9.7, 9.7
Hz, 1H), 4.28–4.15 (m, 1H, H-5′), 4.15–4.05 (m, 1H, H-5′),
3.99 (dd, J = 9.7, 9.7 Hz, 2H), 3.85–3.70 (m, 1H), 3.70–3.10
(m, 20H), 2.50–2.30 (m, 2H, H-2eq), 1.70–1.50 (m, 6H, H-2′′,
H-2ax), 0.98 (t, J = 7.3 Hz, 3H, H-3′′), 0.97 (t, J = 7.3 Hz, 3H,
H-3′′); 13C NMR (75 MHz, CDCl3) δ 98.8 (C-1′), 97.6 (C-1′),
86.3, 84.3, 80.7, 76.0, 75.0, 74.7, 72.1, 72.0, 71.7, 71.5, 71.4,
70.8, 60.5 (C–N3), 59.5 (C–N3), 58.9 (C–N3), 58.5 (C–N3), 51.4
(C–N3), 48.8 (C–N3), 48.5 (C–N3), 48.5 (C–N3), 41.3 (C-6′),
41.1 (C-6′), 36.4, 36.4, 31.5 (C-2), 31.5 (C-2), 11.1 (C-3′′), 11.1
(C-3′′); HRMS (electrospray) Calcd. for C15H24N13O8S
[M − H]−: 546.1592, found: 546.1580.
3.3 Preparation of 14a
+
14b. 5-(N-(3-Hydroxypropyl-
amino))-6-sulfoxy-1,3,2′,6′-tetraazidoneamine and 6-(N-(3-
hydroxypropylamino))-5-sulfoxy-1,3,2′,6′-tetraazidoneamine
By following the general procedure D, a 1 : 1 mixture of regioi-
somers was obtained (138 mg, 0.25 mmol, 71%). (Chromato-
graphy conditions: 40% to 50% EtOAc in cyclohexane) (Rf 0.2
1
5-(N-Propylamino)-6-sulfoxyneamine pentahydrochloride 12a
and 6-(N-propylamino)-5-sulfoxyneamine pentahydrochloride
12b
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, Acetone-d6) δ
6.49 (t, J = 5.7 Hz, 1H, NH), 6.22 (t, J = 5.9 Hz, 1H, NH), 5.34
(d, J = 3.6 Hz, 1H, H-1′), 5.33 (d, J = 3.5 Hz, 1H, H-1′), 4.62
(dd, J = 8.9, 8.9 Hz, 1H), 4.40 (dd, J = 9.7, 9.7 Hz, 1H), 4.25
(ddd, J = 2.5, 5.3, 9.8 Hz, 1H, H-5′), 4.15 (ddd, J = 2.5, 5.1, 9.8
Hz, 1H, H-5′), 4.00–3.40 (m, 21H), 3.40–3.30 (m, 4H), 3.21
(dd, J = 3.8, 10.5 Hz, 1H, H-2′), 2.49 (ddd, J = 4.3, 4.3, 13.0
Hz, 1H, H-2eq), 2.40 (ddd, J = 4.2, 4.2, 13.0 Hz, 1H, H-2eq),
1.86–1.74 (quint., J = 3.6 Hz, 4H, H-2′′), 1.73 (ddd, J = 12.4,
12.4, 12.4 Hz, 1H, H-2ax), 1.59 (ddd, J = 12.1, 12.1, 12.1 Hz,
1H, H-2ax); 13C NMR (75 MHz, Acetone-d6) δ 98.8 (C-1′), 97.7
(C-1′), 86.2, 84.3, 80.6, 76.1, 75.0, 74.7, 72.1, 72.0, 71.7, 71.5,
71.4, 70.8, 63.6, 63.1, 59.8 (C–N3), 59.4 (C–N3), 59.3 (C–N3),
59.3 (C–N3), 58.9 (C–N3), 58.5 (C–N3), 51.4 (C-6′), 51.4 (C-6′),
41.7 (C-2′′), 41.4 (C-2′′), 32.0, 31.9, 31.5 (C-2), 31.5 (C-2);
HRMS (electrospray) Calcd. for C15H24N13O9S [M − H]−:
562.1541, found: 562.1549.
By following the general procedure C, a 1 : 1 mixture of
regioisomers 12a and 12b was obtained (96 mg, 0.15 mmol,
1
77%). H NMR (300 MHz, D2O) δ 5.88 (d, J = 3.7 Hz, 1H,
H-1′), 5.65 (d, J = 3.4 Hz, 1H, H-1′), 4.85–4.60 (m, 2H, H-5′),
4.50 (dd, J = 9.7, 9.7 Hz, 1H), 4.34 (dd, J = 9.8, 9.8 Hz, 1H),
4.10–2.80 (m, 22H), 2.55–2.35 (m, 2H, H-2eq), 1.90 (ddd, J =
12.8, 12.8, 12.8 Hz, 2H, H-2ax), 1.75 (quint., J = 7.6 Hz, 4H,
H-2′′), 0.98 (t, J = 7.3 Hz, 3H, H-3′′), 0.97 (t, J = 7.3 Hz, 3H,
H-3′′); 13C NMR (75 MHz, D2O) δ 95.8 (C-1′), 93.9 (C-1′),
83.4, 80.3, 76.5, 74.9, 72.9, 71.6, 70.6, 70.5, 69.4, 69.3, 68.0,
67.6, 49.7, 49.7, 49.0, 48.6, 48.2, 48.1, 40.8, 40.7, 40.1, 39.6,
28.0, 27.9, 27.0, 26.8, 11.1, 11.1; HRMS (electrospray) Calcd.
for C15H38N5O8S [M + H]+: 448.2441, found: 448.2428.
3.2 Preparation of 13a + 13b. 5-(N-(3-Azidopropylamino))-6-
sulfoxy-1,3,2′,6′-tetraazidoneamine and 6-(N-(3-azidopropyl-
amino))-5-sulfoxy-1,3,2′,6′-tetraazidoneamine
5-(N-(3-Hydroxypropylamino))-6-sulfoxyneamine pentahydro-
chloride 14a and 6-(N-(3-hydroxypropylamino))-5-sulfoxyne-
amine pentahydrochloride 14b
By following the general procedure D, a 1 : 1 mixture of regioi-
somers was obtained (154 mg, 0.26 mmol, 76%). (Chromato-
graphy conditions: 40% to 50% EtOAc in cyclohexane) (Rf 0.25
By following the general procedure C, a 1 : 1 mixture of
regioisomers 14a and 14b was obtained (75 mg, 0.12 mmol,
1
1
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, Acetone-d6) δ
58%). H NMR (300 MHz, D2O) δ 5.86 (d, J = 3.6 Hz, 1H,
5.55 (d, J = 3.8 Hz, 1H, H-1′), 5.53 (d, J = 3.8 Hz, 1H, H-1′),
4.63 (dd, J = 9.0, 9.0 Hz, 1H), 4.42 (dd, J = 9.7, 9.7 Hz, 1H),
4.23 (ddd, J = 2.4, 5.3, 9.8 Hz, 1H, H-5′), 4.15 (ddd, J = 2.5,
5.1, 9.8 Hz, 1H, H-5′), 4.00–3.23 (m, 25H), 3.20 (dd, J = 3.8,
H-1′), 5.66 (d, J = 3.4 Hz, 1H, H-1′), 4.80–4.60 (m, 2H, H-5′),
4.47 (dd, J = 9.8 Hz, 1H), 4.31 (dd, J = 9.8 Hz, 1H), 4.10–3.75
(m, 7H), 3.70–3.45 (m, 8H), 3.45–3.25 (m, 6H), 3.25–3.15 (m,
5H), 2.45 (m, 2H, H-2eq), 1.91 (ddd, J = 12.6, 12.6, 12.6 Hz,
This journal is © The Royal Society of Chemistry 2012
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2H, H-2ax), 1.76–1.64 (quint., J = 6.4 Hz, 4H, H-2′′); 13C NMR
(75 MHz, D2O) δ 95.9 (C-1′), 93.9 (C-1′), 83.2, 80.1, 76.7, 74.8,
72.9, 71.4, 70.6, 70.5, 69.5, 69.3, 68.0, 67.7, 58.6, 58.6, 53.4,
52.6, 49.6, 48.6, 48.2, 48.1, 40.7, 40.6, 40.1, 39.6, 31.2, 31.1,
28.0, 27.9; HRMS (electrospray) Calcd. for C15H32N5O9S
[M − H]−: 458.1921, found: 458.1918.
(CH3), 20.5 (CH3); HRMS (electrospray) Calcd. for
C22H30N16O10Na [M + Na]+: 701.2228, found: 701.2223.
5-[[(3-Azidopropylamino)carbonyl]oxy]-1,3,2′,6′-tetraazido-
neamine
By following the general procedure B, the corresponding O-
deprotected neamine derivative was obtained (83 mg,
0.15 mmol, 75%). (Rf 0.20 EtOAc–cyclohexane). 1H NMR
(300 MHz, Acetone-d6) δ 6.66 (s, br, 1H), 5.29 (d, J = 3.6 Hz,
1H, H-1′), 4.92 (dd, J = 9.2, 9.2 Hz, 1H, H-5), 4.22 (ddd,
J = 2.2, 4.9, 9.6 Hz, 1H, H-5′), 3.91 (dd, J = 8.7, 10.5 Hz, 1H),
3.80–3.60 (m, 3H), 3.60–3.40 (m, 8H), 3.40–3.20 (m, 3H), 3.14
(dd, J = 3.6, 10.4 Hz, 1H, H-2′), 2.35 (ddd, J = 3.7, 3.7, 12.8
Hz, 1H, H-2eq), 1.84 (quint., J = 6.7 Hz, 2H, H-2′′), 1.52 (ddd,
J = 12.2, 12.2, 12.2 Hz, 1H, H-2ax); 13C NMR (75 MHz,
Acetone-d6) δ 156.6 (CvO), 98.6 (C-1′), 77.7, 77.5, 75.5, 72.1,
71.5, 70.9, 63.0 (C–N3), 60.4 (C–N3), 59.3 (C–N3), 51.4 (C-6′),
48.6 (C-3′′), 38.3 (C-1′′), 31.7 (C-2), 29.4 (C-2′′); HRMS
(electrospray) Calcd. for C16H24N16O7Na [M + Na]+: 575.1911,
found: 575.1912.
4. Synthesis of 8a. 5-[(Chlorocarbonyl)oxy]-1,3,2′,6′-tetra-
azido-6,3′,4′-tri-O-acetylneamine 15
To a solution of 3 (2 g, 3.6 mmol) and pyridine (2.63 mL,
32.4 mmol) in anhydrous CH2Cl2 (40 mL) under an Ar atmos-
phere was added a solution of triphosgene (3.2 g, 10.8 mmol) in
anhydrous CH2Cl2 (10 mL). The reaction mixture was stirred
overnight. The solution was then diluted with CH2Cl2 (50 mL)
and neutralized with saturated NH4Cl aqueous solution (10 mL).
The organic phase was separated, washed with HCl 1N (2 ×
5 mL), saturated NaHCO3 (1 × 5 mL), dried over MgSO4 and
filtered. The filtrate was evaporated under reduced pressure to
give 15 (1.9 g, 3.1 mmol, 84%) which was used without any
other purification due to its poor stability (Rf 0.75 EtOAc–cyclo-
5-[[(3-Azidopropylamino)carbonyl]oxy]-neamine pentahydro-
chloride 8a
1
hexane 2 : 3). H NMR (300 MHz, CDCl3) δ 5.40 (dd, J = 9.4,
By following the general procedure C, 8a was obtained
(97 mg, 0.16 mmol, 87%). 1H NMR (300 MHz, D2O) δ 5.50 (d,
J = 3.8 Hz, 1H, H-1′), 4.88 (dd, J = 9.2, 9.2 Hz, 1H), 4.30 (dd,
J = 10.1, 10.1 Hz, 1H), 3.96 (dd, J = 10.1, 10.1 Hz, 1H),
4.90–3.10 (m, 10H), 3.00–2.92 (m, 2H), 2.45 (ddd, J = 3.3, 3.3,
11.7 Hz, 1H, H-2eq), 1.91 (ddd, J = 12.6, 12.6, 12.6 Hz, 1H,
H-2ax), 1.86–1.76 (m, 2H, H-2′′); 13C NMR (75 MHz, D2O) δ
157.4 (CvO), 93.6 (C-1′), 77.5, 73.2, 71.4, 70.3, 69.8, 68.1,
53.1, 49.4, 48.8, 40.0 (C-6′), 37.7, 37.0, 27.9, 27.0; HRMS
(electrospray) Calcd. for C16H35N6O7 [M + H]+: 423.2562,
found: 423.2565.
10.3 Hz, 1H, H-3′), 5.16 (d, J = 3.8 Hz, 1H, H-1′), 5.08–4.96
(m, 3H, H-4′, H-6, H-5), 4.40 (ddd, J = 3.0, 4.6, 10.1 Hz, 1H,
H-5′), 3.76–3.44 (m, 4H), 3.42–3.24 (m, 2H), 2.46 (ddd, J = 4.5,
4.5, 13.4 Hz, 1H, H-2eq), 2.13 (s, 3H, CH3), 2.08 (s, 3H, CH3),
2.04 (s, 3H, CH3), 1.67 (ddd, J = 12.8, 12.8, 12.8 Hz, 1H,
H-2ax); 13C NMR (75 MHz, CDCl3) δ 170.0 (CH3–CvO),
169.7 (CH3–CvO), 169.6 (CH3–CvO), 150.2 (CvO), 98.7
(C-1′), 80.7, 78.7, 73.1, 70.6, 69.7, 69.2, 61.0 (C–N3), 58.1 (C–
N3), 57.4 (C–N3), 50.7 (C-6′), 31.5 (C-2), 20.6 (CH3), 20.6
(CH3), 20.4 (CH3).
5-[[(3-Azidopropylamino)carbonyl]oxy]-1,3,2′,6′-tetraazido-
6,3′,4′-tri-O-acetylneamine
5. Synthesis of 8b. 6-[[(3-Azidopropylamino)carbonyl]oxy]-
1,3,2′,6′-tetraazidoneamine
To a solution of 15 (200 mg, 0.33 mmol) in anhydrous
CH2Cl2 (5 mL) was added 3-azidopropylamine (50 mg,
0.50 mmol). The reaction was stirred at room temperature for
2 h. Quantitative conversion of the starting material was
observed by TLC. The solvent was eliminated under reduced
pressure and the residue was taken up with EtOAc (10 mL). The
organic layer was washed with HCl 1N (2 × 1 mL), H2O (2 ×
1 mL), brine (1 × 1 mL), dried over MgSO4 and concentrated in
vacuo. The residue was purified through silica gel chromato-
graphy to give a white solid (209 mg, 0.31 mmol, 93%).
(Chromatography conditions: 20% to 30% of EtOAc in cyclo-
hexane) (Rf 0.50 EtOAc–cyclohexane 2 : 3). 1H NMR
(500 MHz, CDCl3) δ 5.41 (dd, J = 9.2, 10.8 Hz, 1H, H-3′), 5.26
(t, J = 5.9 Hz, 1H, NH), 5.22 (d, J = 3.8 Hz, 1H, H-1′), 5.03 (dd,
J = 9.7, 9.7 Hz, 1H, H-4′), 5.00 (dd, J = 9.4, 10.0 Hz, 1H, H-6),
4.90 (dd, J = 10.0, 10.0 Hz, 1H, H-5), 4.33 (ddd, J = 2.7, 4.8,
10.1 Hz, 1H, H-5′), 3.72–3.66 (m, 1H), 3.64 (dd, J = 9.8, 9.8
Hz, 1H), 3.60–3.52 (m, 1H), 3.40–3.20 (m, 7H), 2.42 (ddd, J =
4.5, 4.5, 13.3 Hz, 1H, H-2eq), 2.05 (s, 3H, CH3), 2.03 (s, 3H,
CH3), 2.01 (s, 3H, CH3), 1.80–1.72 (m, 2H, H-2′′), 1.61 (ddd,
J = 12.5, 12.5, 12.5 Hz, 1H, H-2ax); 13C NMR (125 MHz,
CDCl3) δ 170.1 (CH3–CvO), 169.9 (CH3–CvO), 169.7 (CH3–
CvO), 154.7 (CvO), 98.4 (C-1′), 78.1, 74.6, 74.3, 69.6, 69.4,
69.3, 60.6 (C–N3), 58.4 (C–N3), 57.4 (C–N3), 50.7 (C-6′), 48.8
(C-3′′), 38.7 (C-1′′), 31.6 (C-2′′), 28.8 (C-2), 20.6 (CH3), 20.6
8b was obtained from the 1 : 1 mixture previously prepared,
by separation with reversed-phase HPLC (C18 reversed-phase
column, Interchim, 10 × 250 mm). 1H NMR (300 MHz,
Acetone-d6) δ 5.76 (t, J = 5.4 Hz, 1H), 5.62 (d, J = 3.7 Hz, 1H,
H-1′), 4.81 (dd, J = 9.8, 9.8 Hz, 1H), 4.18 (ddd, J = 2.6, 5.2, 9.8
Hz, 1H, H-5′), 4.10 (br, 1H), 3.94 (dd, J = 8.9, 10.3 Hz, 2H),
3.71–3.45 (m, 10H), 3.40–3.32 (m, 3H), 2.45 (ddd, J = 4.4, 4.4,
12.9 Hz, 1H, H-2eq), 1.89 (quint., J = 6.6 Hz, 2H, H-2′′), 1.70
(ddd, J = 12.4, 12.4, 12.4 Hz, 1H, H-2ax); 13C NMR (75 MHz,
Acetone-d6) δ 156.1 (CvO), 98.5 (C-1′), 80.1, 76.75, 75.1,
72.0, 71.6, 71.4, 63.5 (C–N3), 59.4 (C–N3), 58.6 (C–N3), 51.5
(C-6′), 48.6 (C-3′′), 38.1 (C-1′′), 31.7 (C-2), 29.3 (C-2′′). [α]
+13.75 (c 0.8, H2O).
6-[[(3-Azidopropylamino)carbonyl]oxy]-neamine pentahydro-
chloride 8b
By following the general procedure C, 8b was obtained
1
(115 mg, 0.19 mmol, 94%). H NMR (500 MHz, D2O) δ 5.88
(d, J = 3.9 Hz, 1H, H-1′), 4.82 (dd, J = 9.6, 9.6 Hz, 1H), 4.04
(dd, J = 9.5, 9.5 Hz, 1H), 3.99–3.93 (m, 2H), 3.88 (dd, J = 9.4,
9.4 Hz, 1H), 3.61–3.52 (m, 2H), 3.48–3.40 (m, 3H), 3.29–3.15
(m, 3H), 2.99 (ddd, J = 7.5, 7.5, 7.5 Hz, 2H), 2.52 (ddd, J = 4.3,
4.3, 12.8 Hz, 1H, H-2eq), 1.96 (ddd, J = 12.7, 12.7, 12.7 Hz, 1H,
H-2ax), 1.84 (quint., J = 6.8 Hz, 2H, H-2′′); 13C NMR
(125 MHz, D2O) δ 157.2 (CvO), 96.0 (C-1′), 77.0, 74.3, 73.5,
4728 | Org. Biomol. Chem., 2012, 10, 4720–4730
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70.6, 69.3, 68.1, 53.5, 48.2, 48.0, 40.1 (C-6′), 37.7, 37.1, 28.2,
26.9. [α] +57.1 (c 0.35, H2O).
13.2 Hz, 1H, H-2eq), 2.08 (s, 3H, CH3), 2.05 (s, 3H, CH3), 1.78
(quint., J = 6.6 Hz, 4H, H-2′′), 1.63 (ddd, J = 12.6, 12.6, 12.6
Hz, 1H, H-2ax); 13C NMR (75 MHz, CDCl3) δ 169.9 (CH3–
CvO), 169.7 (CH3–CvO), 155.1 (CvO), 154.9 (CvO),
98.4 (C-1′), 78.1, 75.3, 74.7, 69.7, 69.4, 69.3, 60.7 (C–N3), 58.5
(C–N3), 57.9 (C–N3), 50.7 (C-6), 48.9 (C-3′′), 48.8 (C-3′′), 38.8
(C-1′′), 38.5 (C-1′′), 31.6 (C-2), 29.0 (C-2′′), 28.9 (C-2′′), 26.9
(CH3), 26.9 (CH3); HRMS (electrospray) Calcd. for
C24H34N20O10Na [M + Na]+: 785.2665, found: 785.2659.
5,6-Di-[[(3-azidopropylamino)carbonyl]oxy]-1,3,2′,6′-
tetraazidoneamine
6. Synthesis of 18. 5-[[(3-Azidopropylamino)carbonyl]oxy]-
3′,4′-di-O-acetyl-6-(4-nitrophenylcarbonate)-1,3,2′,6′-tetraazido-
neamine 17a and 6-[[(3-azidopropylamino)carbonyl]oxy]-3′,4′-
di-O-acetyl-5-(4-nitrophenylcarbonate)-1,3,2′,6′-tetraazidone-
amine 17b
To a solution of 16a and 16b (150 mg, 0.24 mmol), pre-
viously obtained in the preparation of 8a and 8b, in anhydrous
CH2Cl2 (5 mL) was added 4-nitrophenyl chloroformate (476 mg,
2.36 mmol). The reaction was stirred at room temperature over-
night. Quantitative conversion of the starting material was
observed by TLC. The solvent was eliminated under reduced
pressure and the residue was taken up with EtOAc (20 mL). The
organic layer was washed with NaOH 1N (5 × 1 mL), H2O (2 ×
1 mL), HCl 1N (2 × 1 mL), H2O (2 × 1 mL), brine (1 × 1 mL)
and dried over MgSO4. The residue was purified through silica
gel chromatography using 30% of EtOAc in cyclohexane to
afford a 1 : 1 mixture of 17a and 17b (135 mg, 0.17 mmol, 71%)
(Rf 0.50 EtOAc–cyclohexane 2 : 3). 1H NMR (300 MHz,
CDCl3) δ 8.29 (d, J = 9.1 Hz, 4H, H-6′′), 7.40 (d, J = 9.1 Hz,
2H, H-5′′), 7.38 (d, J = 9.1 Hz, 2H, H-5′′), 5.46 (dd, J = 9.3,
10.4 Hz, 1H, H-3′), 5.44 (dd, J = 9.2, 10.6 Hz, 1H, H-3′), 5.36
(t, J = 6.0 Hz, 1H, NH), 5.30–5.15 (m, 4H), 5.10–4.90 (m, 4H),
4.72 (dd, J = 10.0, 10.0 Hz, 1H), 4.50–4.40 (m, 2H, H-5′),
3.90–3.50 (m, 6H), 3.50–3.20 (m, 14H), 2.55–2.40 (m, 2H,
H-2eq), 2.08 (s, 3H, CH3), 2.08 (s, 3H, CH3), 2.05 (s, 3H, CH3),
2.04 (s, 3H, CH3), 1.90–1.60 (m, 6H, H-2′′, H-2ax); 13C NMR
(75 MHz, CDCl3) δ 170.0 (CH3–CvO), 169.9 (CH3–CvO),
169.7 (CH3–CvO), 169.6 (CH3–CvO), 155.3, 155., 154.9,
154.7, 151.9, 151.8, 145.7, 145.6, 126.2, 126.2, 125.5, 125.4,
121.8, 121.8, 121.6, 121.6, 99.0 (C-1′), 98.4 (C-1′), 80.0, 79.5,
78.8, 78.1, 74.9, 74.2, 69.9, 69.7, 69.6, 69.6, 69.3, 69.3, 60.8
(C–N3), 60.8 (C–N3), 58.3 (C–N3), 58.2 (C–N3), 57.9 (C–N3),
57.2 (C–N3), 50.7 (C-6′), 50.7 (C-6′), 49.0 (C-3′′), 48.8 (C-3′′),
39.0 (C-1′′), 38.7 (C-1′′), 31.6 (C-2′′), 31.4 (C-2′′), 28.9 (C-2),
28.9 (C-2), 26.9 (CH3), 26.9 (CH3), 26.9 (CH3), 26.9 (CH3);
HRMS (electrospray) Calcd. for C27H31N17O13Na [M + Na]+:
824.2185, found: 824.2169.
By following the general procedure B, the deacylated product
was obtained (102 mg, 0.15 mmol, 75%). (Rf 0.25 EtOAc–
1
cyclohexane 2 : 3). H NMR (300 MHz, Acetone-d6) δ 6.50 (d,
J = 5.9 Hz, 2H, OH), 5.25 (d, J = 3.5 Hz, 1H, H-1′), 5.03 (dd,
J = 9.6, 9.6 Hz, 1H, CH–OCvO–NHR), 4.88 (dd, J = 10.0,
10.0 Hz, 1H, CH–OCvO–NHR), 4.28 (s, 2H, NH), 4.26–4.16
(m, 1H, H-5′), 3.90 (dd, J = 8.9, 10.3 Hz, 1H), 3.83–3.65 (m,
3H), 3.65–3.50 (m, 2H), 3.50–3.35 (m, 5H), 3.35–3.20 (m, 4H),
3.13 (dd, J = 3.7, 10.5 Hz, 1H, H-2′), 2.45 (ddd, J = 4.1, 4.1,
12.8 Hz, 1H, H-2eq), 1.82 (quint., J = 7.0 Hz, 4H, H-2′′), 1.72
(ddd, J = 12.1, 12.1, 12.1 Hz, 1H, H-2ax); 13C NMR (75 MHz,
Acetone-d6) δ 155.3 (CvO), 155.2 (CvO), 98.6 (C-1′), 77.4,
74.7, 74.4, 72.1, 71.5, 70.7, 62.9 (C–N3), 59.2 (C–N3), 58.3
(C–N3), 51.3 (C-6), 48.7 (C-3′′), 48.6 (C-3′′), 38.2 (C-1′′), 38.0
(C-1′′), 31.5 (C-2), 29.1 (C-2′′), 29.0 (C-2′′); HRMS (electro-
spray) Calcd. for C20H30N20O8Na [M + Na]+: 701.2453, found:
701.2446.
5,6-Di-[[(3-azidopropylamino)carbonyl]oxy]-neamine hexa-
hydrochloride 18
By following the general procedure C, 18 was obtained
1
(119 mg, 0.16 mmol, 81%). H NMR (300 MHz, D2O) δ 5.45
(d, J = 3.8 Hz, 1H, H-1′), 5.06 (dd, J = 7.5, 7.5 Hz, 1H, CH–
OCvO–NHR), 4.40 (dd, J = 8.8, 10.1 Hz, 1H, CH–OCvO–
NHR), 3.98 (dd, J = 8.8, 10.3 Hz, 1H), 3.92–3.84 (m, 1H, H-5′),
3.72–3.58 (m, 2H), 3.45 (dd, J = 9.0, 9.0 Hz, 1H), 3.50–3.34
(m, 4H), 3.34–3.20 (m, 2H), 3.20–3.10 (m, 2H), 3.00–2.90 (m,
4H, H-1′′), 2.50 (ddd, J = 4.1, 4.1, 12.6 Hz, 1H, H-2eq), 2.00
(ddd, J = 12.7, 12.7, 12.7 Hz, 1H, H-2ax), 1.78 (quint., J = 7.5
Hz, 4H, H-2′′); 13C NMR (75 MHz, D2O) δ 156.6 (CvO),
156.5 (CvO), 93.5 (C-1′), 75.7, 72.9, 72.8, 70.2, 69.9, 68.0,
53.1, 48.5, 47.7, 39.9 (C-6′), 37.9 (C-3′′), 37.8 (C-3′′), 37.0
(C-1′′), 37.0 (C-1′′), 27.8 (C-2), 26.9 (C-2′′), 26.9 (C-2′′);
HRMS (electrospray) Calcd. for C20H43N8O8 [M + H]+:
523.3204, found: 523.3201.
5,6-Di-[[(3-azidopropylamino)carbonyl]oxy]-3′,4′-di-O-acetyl-
1,3,2′,6′-tetraazidoneamine
To a solution of 17a + 17b (135 mg, 0.17 mmol) in anhydrous
CH2Cl2 (5 mL) was added 3-azidopropylamine (26 mg,
0.26 mmol). The reaction was stirred at room temperature for
2 h. Quantitative conversion of the starting material was
observed by TLC. The solvent was eliminated under reduced
pressure and the residue was taken up with EtOAc (10 mL). The
organic layer was washed with NaOH 1N (5 × 1 mL), H2O (2 ×
1 mL), HCl 1N (2 × 1 mL), H2O (2 × 1 mL), brine (1 × 1 mL)
and dried over MgSO4. The residue was purified through silica
gel chromatography using 25% of EtOAc in cyclohexane to
afford the desired product (113 mg, 0.14 mmol, 87%). (Rf 0.40
7. Biological evaluation. MICs (minimum inhibitory con-
centrations) were determined in a Mueller–Hinton (MH) broth
according to the following procedure: E. coli (DH5α) and
S. aureus strains (RN4220) cells were grown in 10 mL of a
Mueller–Hinton broth overnight at 37 °C. Subsequently 100 μL
of this suspension was mixed with 9.9 mL of fresh Mueller–
Hinton and incubated at 37 °C for 3–5 h. 5 mL of this inoculum
was added to 45 mL of fresh Mueller–Hinton broth containing
phenol red at 0.02 g L−1 and 1% glucose. 90 μL of the resulting
mixture was introduced to a sterile 96-wells microplate. Stock
solutions ranging from 50 μM to 10 mM in HEPES (10 mM,
pH = 7.4) were prepared for the chemicals described in
Scheme 3, 4 and 5. The various concentrations of the
1
EtOAc–cyclohexane 2 : 3). H NMR (300 MHz, CDCl3) δ 5.43
(dd, J = 9.5, 10.4 Hz, 1H, H-3′), 5.33 (t, J = 5.7 Hz, 1H, NH),
5.24 (d, J = 3.7 Hz, 1H, H-1′), 5.23 (m, 1H, NH), 5.03 (dd, J =
10.0, 10.0 Hz, 2H, H-4′, CH–OCvO–NHR), 4.81 (dd, J = 10.0,
10.0 Hz, 1H, CH–OCvO–NHR), 4.50–4.42 (m, 1H, H-5′),
3.75–3.50 (m, 3H), 3.45–3.15 (m, 11H), 2.41 (ddd, J = 4.3, 4.3,
This journal is © The Royal Society of Chemistry 2012
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compounds were added from the freshly prepared stock solutions
to the microplate. Negative (MH broth without bacteria) and
positive (MH with bacteria without the chemical that was
replaced by HEPES) controls were included and the microplate
was then incubated overnight at 37 °C. The MICs were deter-
mined by visual examination of the microplates, by observing
the change of colour of the growth media to a bright yellow
resulting from the acid production of the growing bacteria in the
course of the assay (test). No colour change reflected the
absence of growth, because there was no acid production. For
each chemical, MIC determinations were performed indepen-
dently at least four times, in duplicate for each set of exper-
iments, the MICs collected being reproducible.
13 Recently, imidazole-1-sulfonyl azide hydrochloride was described as an
inexpensive, shelf-stable diazotransfer reagent for the generationof azides
from primary amines. (a) N. M. Smith, M. J. Greaves, R. Jewell,
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17 Compound 4 was already obtained from 3 via selective protection of the
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tylation of the 3′ and 4′ hydroxy groups and deprotection of the cyclohex-
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19 Hydrogenation reactions were not reproducible using other catalysts (Pd–
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This journal is © The Royal Society of Chemistry 2012