Synthesis of Novel Saccharide Hydrazones

Article abstract of DOI:10.1080/00397911.2015.1021425

Synthesis of important heterocyclic hydrazine derivatives N-aminopyrrolidine, N-aminopiperidine, and N-aminoazepane from hydrazine hydrate and dihalogenides were examined and optimized. These heterocyclic hydrazine derivatives were used in condensation reactions with six different monosaccharides to form corresponding hydrazones. Biological evaluations of these novel compounds, which are simple acyclic nucleoside analogs, were done. L-Arabinose N-aminoazepane hydrazone showed minor anti-HIV activity, giving a starting point for further structural modifications. (Chemical Equation Presented).

Full text of DOI:10.1080/00397911.2015.1021425

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Synthesis of Novel Saccharide Hydrazones
Mihkel Ilissona, Kristjan Tomsona, Anastasia Selyutinab, Silver Türkc & Uno Mäeorga
a Institute of Chemistry, University of Tartu, Tartu, Estonia
b Institute of Technology, Univesrity of Tartu, Tartu, Estonia
c Faculty of Medicine, University of Tartu, Tartu, Estonia
Accepted author version posted online: 23 Feb 2015.
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To cite this article: Mihkel Ilisson, Kristjan Tomson, Anastasia Selyutina, Silver Türk & Uno Mäeorg (2015): Synthesis of Novel
Saccharide Hydrazones, Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic
Chemistry, DOI: 10.1080/00397911.2015.1021425
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Synthesis of novel saccharide hydrazones
Mihkel Ilisson1, Kristjan Tomson1, Anastasia Selyutina2, Silver Türk3, Uno
Mäeorg1
1Institute of Chemistry, University of Tartu, Tartu, Estonia 2Institute of Technology,
Univesrity of Tartu, Tartu, Estonia 3Faculty of Medicine, University of Tartu, Tartu,
Estonia
E-mail: uno.maeorg@ut.ee
Abstract
Synthesis of important heterocyclic hydrazine derivatives N-aminopyrrolidine, N-
aminopiperidine and N-aminoazepane from hydrazine hydrate and dihalogenides were
examined and optimized. These heterocyclic hydrazine derivatives were used in
condensation reactions with six different monosaccharides to form corresponding
hydrazones. Biological evaluations of these novel compounds, which are simple acyclic
nucleoside analogues, were done. L-arabinose N-aminoazepane hydrazone showed minor
anti-HIV activity, giving a starting point for further structural modifications.
KEYWORDS: Heterocycle, hydrazine, saccharide, hydrazone, HIV
INTRODUCTION
1

Hydrazones is a class of chemical compounds analogous with imines. They can be
synthesized with a condensation reaction from a hydrazine derivative and a carbonyl
compound. Saccharide hydrazones, which contain heterocycles can be observed as
acyclic nucleoside mimetics. These compounds have shown biological activity against
bacteria[1-3], viruses[4,5], fungi[1,2] and cancer cells[6].
Structurally simple heterocyclic hydrazine derivatives, such as N-aminopyrrolidine, N-
aminopiperidine and N-aminoazepane are important building blocks in organic synthesis.
There are several bioactive molecules known, which contain aforementioned
fragments[7,8]. However, there are only few methods available for synthesis of these
heterocyclic hydrazine derivatives. Most commonly secondary amines are used as a
starting material and electrophilic amination[9] or nitrosoylation[10] with subsequent
reduction[11] is carried out. These methods have some setbacks – electrophilic amination
includes using unstable reagents such as chloramines and hydroxylamines while
nitrosoylation route provides highly toxic and carcinogenic nitroso-intermediates.
Synthesis of N-aminopyrrolidine, N-aminopiperidine and N-aminoazepane can be also
carried out via polyanion strategy[12-15], developed out in our workgroup. However, this
route requires using protection-deprotection steps and column chromatography as a
purification method, which makes this method inconvenient to use in a larger scale.
In current work, efforts were concentrated on synthesis of heterocyclic hydrazine
derivatives using hydrazine hydrate and suitable dihalogenides as starting materials.
2

Previously, some procedures for the synthesis of N-aminopyrrolidine and N-
aminopiperidine have been described[16,17], but they were found out to be either
unoptimized or non-reproducible. To the best of our knowledge, N-aminoazepane have
not been synthesized from hydrazine hydrate and 1,6-dihalohexane before.
Synthesized hydrazine derivatives were used in condensation reactions with
monosaccharides to form corresponding hydrazones. These novel compounds were used
in experiments against human immunodeficiency virus (HIV)-based virus-like particles,
bacteria and fungi in order to characterize their antiretroviral, antibacterial and antifungal
activities.
RESULTS AND DISCUSSION
Synthesis Of Heterocyclic Hydrazine Derivatives
Initial experiments were started using just a slight excess (1.2 eq) of hydrazine hydrate
compared to the dihalogenides. Also, sodium hydroxide solution was used in order to
keep the reaction medium basic. This procedure gave desired products in very low yields
(1.6-7.2%), probably due to formation of N,N’-bisubstituted hydrazine derivatives and
elimination and substitution reactions caused by the use of sodium hydroxide (Scheme 1).
Therefore we decided to significantly increase the excess of hydrazine hydrate, which
decreases the amount of disubstitution and also binds the hydrogen halide formed during
the reaction, making the use of inorganic base unnecessary.
In the synthesis of N-aminoazepane, a polymerization occurred in our initial experiments.
We speculate that the polymerization was favored against the cyclization because of the
3

conformational hindrances and high concentration of reagents (see Scheme 2). Formation
of seven-membred rings has high energy barrier and the polymerization is preferred at
higher concentrations. Therefore, the next experiments for the synthesis of N-
aminoazepane were carried out in significantly diluted solutions and without the presence
of sodium hydroxide to avoid the polymerization.
N-aminopyrrolidine was purified by vacuum distillation. For N-aminopiperidine and N-
aminoazepane the distillation was not applied, because of the decomposition, which
occurred during the distillation process. On the other hand the corresponding high purity
products were already obtained by extraction of the reaction mixture and evaporation of
the solvent.
During the optimization of the reaction conditions we reached to good yields (45-74%) of
hydrazine derivatives (see Scheme 3) considering that the synthesis is a simple one-step
process and inexpensive and easily accessible reagents were used. Yields can be probably
improved somewhat further, when to use even larger excess of hydrazine hydrate in the
reactions.
Synthesis And Biological Evaluation Of Saccharide Hydrazones
For the synthesis of saccharide hydrazones, previously synthesized heterocyclic
hydrazine derivatives 1-3 and six different aldoses – L-arabinose, D-galactose, D-
mannose, D-ribose, L-rhamnose and 2-deoxy-D-ribose – were used (see Table 1).
4

Procedure used for these condensation reactions were analogous to the one used in
German scientists H. Stroh and H. Scharnow’s work[18]
.
All the reactions were clean according to the TLC and NMR and were completed within
several hours. After the completion, solvents and other volatiles were removed in vacuum
and residue was recrystallized in a suitable solvent. In some cases, the product was a
liquid and therefore column chromatography was used for purification. Even though
triethylamine doped eluent was used, we could see some decomposition by TLC.
However, this decomposition turned out to be insignificant, as we got excellent yields
and did not detect any impurities with NMR.
18 synthesized novel hydrazones 4-21 were subjected to biological evaluations. Their
activity was examined against E. coli, S. aureus and C. albicans. In these experiments it
was confirmed that selected compounds did not have any effect on these bacteria and
fungi.
Toxicology tests revealed that all synthesized hydrazones are non-toxic to mammalian
cells at the concentration of 1 mM. Saccharide hydrazones 4-21 were also tested for anti-
HIV properties. It was found out, that compound 6 have minor effect (IC50 ≈ 400 μM)
against HIV. Also, compound 21 showed some activity (IC50 1 mM) against HIV.
Other 16 hydrazones turned out to be ineffective towards inhibiting HIV.
EXPERIMENTAL
5

Synthesis
All the used reagents and solvents were obtained commercially from Sigma-Aldrich
company and used without further purification.
Thin layer chromatography was performed on Macherey-Nagel Alugram® SIL G/UV
254 silica gel plates. For visualization, 1% phosphomolybdic acid solution in ethanol or
10% sulfuric acid solution in ethanol were used.
For column chromatography, Merck Kieselgel 70-230 mesh silica gel was used.
FTIR spectra were measured with Perkin-Elmer Spectrum BXII FTIR spectrometer
equipped with Interspectrum (Estonia) zinc selenide ATR crystal. Wavelenghts in spectra
are presented in cm-1.
NMR spectra were recorded with Bruker Avance II 200 and Bruker Avance III HD
spectrometers. 1H NMR spectra were measured at frequencies 200 MHz and 700 MHz.
13C NMR spectra were measured at 50 MHz and 176 MHz. TMS was used as an internal
standard. Chemical shifts are presented in ppm, decoupling constants in Hz.
HRMS spectra were measured on Thermo Electron LTQ Orbitrap spectrometer with ESI
ethod.
PROCEDURE FOR PREPARATION OF N-AMINOPYRROLIDINE (1)
6

In a 100 ml two-necked flask, hydrazine hydrate (24.25 ml, 0.5 mol) was dissolved in 40
ml of methanol. Mixture was heated to reflux and 1,4-dibromobutane (17.91 ml, 0.15
mol) was added dropwise within 1 h. Reaction mixture was left stirring at reflux for 24 h.
Methanol was removed by fractional distillation. Residue was basified with 75 g of 40%
NaOH and extracted 10 times with Et2O. Combined extracts were dried on anhydrous
Na2SO4 and filtrated. Solvent was removed under reduced pressure and the product was
purified by vacuum distillation (bp 46 ˚C/30 mbar). This process yielded 6.374 g (49%)
of N-aminopyrrolidine[17], a clear colorless liquid.
PROCEDURE FOR PREPARATION OF N-AMINOPIPERIDINE (2)
In a 100 ml two-necked flask, hydrazine hydrate (14.55 ml, 0.3 mol) was dissolved in 25
ml of methanol. Solution was heated to reflux and 1,5-dibromopentane (13.62 ml, 0.1
mol) was added dropwise within 1 h. Reaction mixture was left stirring at reflux for 24 h.
Methanol was removed under reduced pressure and residue was basified with 40 g of
40% NaOH solution. Mixture was extracted 8 times with Et2O and combined extracts
were dried on anhydrous Na2SO4 and filtrated. Solvent was removed under reduced
pressure (40 ˚C at 40 mbar). Process yielded 7.380 g (74%) of N-aminopiperidine[17], a
light yellow liquid, which was used in subsequent experiments without further
purification.
PROCEDURE FOR PREPARATION OF N-AMINOAZEPANE (3)
In a 500 ml two-necked flask, hydrazine hydrate (24.25 ml, 0.5 mol) was dissolved in
360 ml of methanol. Solution was heated to reflux and 1,6-dibromohexane (23.07 ml,
7

0.15 mol) was added dropwise within 1,5 h. After 24 h of stirring at reflux, the methanol
was evaporated under reduced pressure. Residue was basified with 55 g of 40% NaOH
solution and extracted 10 times with Et2O. Combined extracts were dried on anhydrous
Na2SO4 and filtrated. Solvent was removed under reduced pressure (40 ˚C at 25 mbar).
Process yielded 7.695 g (45%) of N-aminoazepane[16], a light yellow liquid, which was
used in subsequent experiments without further purification.
GENERAL PROCEDURE FOR PREPARATION OF SACCHARIDE
HYDRAZONES (4-21)
In a 100 ml flask, 8 mmol of hydrazine derivative (N-aminopyrrolidine, N-
aminopiperidine or N-aminoazepane) was dissolved in 15 ml of methanol. 5 mmol of
monosaccharide was added and mixture was stirred at the methanol reflux. After 0.75 –
4.5 h of stirring, the reaction was complete according to the TLC. Volatiles were
removed under reduced pressure and the residue was recrystallized in MTBE-ethanol
mixture or purified by column chromarography (eluent: ethanol-benzene-triethylamine
29:10:1). Additional details about the specific procedures can be found in Table 1 and in
the supplementary information.
Biological Evaluation
ANTI-RETROVIRAL EVALUATION
Materials Used
Dimethyl sulfoxide (DMSO) and polybrene were purchased from Sigma Aldrich (USA).
All reagents and media used for cell cultivation were purchased from Naxo OÜ (Estonia).
8

U2OS human osteosarcoma cells were obtained from ATCC and grown in Iscove's
Modified Dulbecco's Medium (IMDM) (supplemented with 10% fetal bovine serum
(FBS), 100 U Penicillin and 100 µg/ml Streptomycin (Pen/Strep)) at 37° C in the
presence of 5% CO2.
CYTOTOXICITY ASSAY
Cytotoxicity was measured using the xCELLigence RTCA DP Instrument (ACEA
Biosciences, Inc). U2OS cells were seeded on E-plate 16 (3,5*103 cells per well) and
incubated for 24 hours. Then compounds (4-21) dissolved in DMSO (or equivalent
amount of DMSO as a vehicle control) were added and incubation was continued for
another 24 hours. Impedance signals were recorded throughout the incubation.
ANALYSIS OF ANTIVIRAL ACTIVITY
Antiretroviral activity was analyzed using HIV-1 based virus like particles (VLPs),
obtained using ViraPower Lentiviral Expression System according to the manufacture
(Invitrogen) instructions. The RNA packed into VLPs was engineered to encode a
reporter gene (Gaussia luciferase (Gluc)) which is, however, expressed only when RNA
is reverse transcribed into DNA by HIV reverse transcriptase present in VLPs. The day
before the experiment U2OS cells were seeded on 24-well plate (5*104 cells per well).
Next day cells were infected with HIV-1 VLPs at a multiplicity of infection (MOI) = 0.01
infectious units/cell in the presence of polybrene 6 µg/ml and analyzed compounds (or
DMSO as a vehicle control). After 1 hour of incubation (37°C, 5%CO2) cells were
washed and full media with analyzed compounds (or DMSO) was added. Cells were
9

incubated for 24 hours with compounds and then in fresh media without compounds for
another 24 hours. After this cells were lysed and Gluc activity in cells was measured
using Renilla Luciferase Assay System and Glomax 20/20 Luminometer (Promega). Gluc
signal was normalized to the total protein concentration (measured with Bio-Rad protein
assay).
ANTI-MICROBIAL EVALUATION
Synthesized hydrazones (4-21) was tested for antimicrobial activity against Escherischia
coli ATCC 700336, Staphylococcus aureus ATCC 43300 and Candida albicans ATCC
MYA-2876 by disk-diffusion assay. Müller-Hinton plates (Oxoid, Basingstoke, UK)
were inoculated with bacteria suspended in distilled water (turbidity McFarland 0.5) by
using sterile cotton swabs. Sterilized filter paper disks were placed on the inoculated
plates. 50 μg hydrazones in 50 microliters of distilled water were pipetted onto the disks.
Then the plates were incubated for 24 hours at 37°. None of the tested hydrazones formed
zones of inhibition against any of the tested microorganisms.
SUPPLEMENTARY INFORMATION
Supplemental data for this article can be accessed on the publisher’s website.
ACKNOWLEDGEMENTS
This work was supported by Estonian Science Foundation (grant No. 8794) and Research
Targeted Financing projects SF0180032s12, institutional research funding projects
10

IUT20-17 of the Estonian Ministry of Education and Research and Competence center
Mesosystems - Theory and Applications project TK-114.
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12

Table 1. Procedure details for the synthesis of saccharide
hydrazones.
Hydrazone Hydrazine
derivative
Monosaccharide Reaction
time (h)
Purification
method*
50:50
50:50
50:50
25:75
50:50
67:33
50:50
67:33
67:33
CC
Isolated
yield (%)
93
L-arabinose
D-galactose
Dmannose
D-ribose
3
4
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
3.5
3
85
5
85
6
4
96
7
4.5
2
77
8
71
9
3.5
4
93
10
11
12
13
14
15
16
17
18
77
2
68
3.25
4
85
80:20
100:0
80:20
75:25
100:0
83
3
71
L-rhamnose
2.5
3.75
3.25
80
83
74
13

2-deoxy-D-
ribose
1.5
1
CC
88
98
73
19
20
21
1
2
3
CC
0.75
100:0
* For purification method MTBE:EtOH solvent ratio is written if the product was
purified by recrystallization. CC indicates purification by column chromatography with
standard eluent composition (ethanol-benzene-triethylamine 29:10:1).
14

Scheme 1. Possible reaction paths in the synthesis of heterocyclic hydrazine derivatives.
15

Scheme 2. Cyclization and polymerization route of (6-bromohexyl)hydrazine.
16

Scheme 3. Yields and optimized reaction conditions for the synthesis of heterocyclic
hydrazine derivatives.
17