Synthesis of unprotected CH2-skipped piperazine-pyridine alternating cycles with azide end-group

Article abstract of DOI:10.3987/COM-14-S(K)12

This work describes synthesis of CH2-skipped alternating piperazine-pyridine cycles with azide end-group starting from piperazine, 1-methylpiperazine and pyridine-2,6-dicarboxylic acid. This compound can be used to enhance binding efficiency by shielding repulsion between negatively charged phosphate groups in DNA-oligonucleotide hybridization techniques.

Full text of DOI:10.3987/COM-14-S(K)12

HETEROCYCLES, Vol. 90, No. 1, 2015
625
HETEROCYCLES, Vol. 90, No. 1, 2015, pp. 625 - 630. © 2015 The Japan Institute of Heterocyclic Chemistry
Received, 27th February, 2014, Accepted, 26th March, 2014, Published online, 2nd April, 2014
DOI: 10.3987/COM-14-S(K)12
SYNTHESIS
OF
UNPROTECTED
CH2-SKIPPED
PIPERAZINE-PYRIDINE ALTERNATING CYCLES WITH AZIDE
END-GROUP
Andi Kipper,1 Indrek Kalvet,1 Kaido Tämm,1 Lauri Sikk,1 Peeter Burk,1
Kuldar Kõiv,2 and Uno Mäeorg1*
1Institute of Chemistry, University of Tartu, Ravila 14 a, Tartu 50411, Estonia;
2Institute of Biomedicine and Translational Medicine, University of Tartu, Ravila
19, Tartu 50411, Estonia
Abstract – This work describes synthesis of CH2-skipped alternating
piperazine-pyridine cycles with azide end-group starting from piperazine,
1-methylpiperazine and pyridine-2,6-dicarboxylic acid. This compound can be
used to enhance binding efficiency by shielding repulsion between negatively
charged phosphate groups in DNA-oligonucleotide hybridization techniques.
Piperazine derivatives are useful in the treatment or prevention of many diseases like inflammation,1 bone
degradation,2 thrombosis3 and tumor metastasis.4 On the other hand oligomeric piperazine compounds
could be source of potential positive charge which is important factor in interactions of small molecules
with biological systems. Subsequent functionality and reactivity offer a good possibility to regulate
hydrophilicity/hydrophobicity, geometry, π-interaction and as a result design the appropriate molecule for
certain purposes.
The main factors affecting efficiency of techniques using hybridization between single-stranded
DNA-helix and oligonucleotide (i.e. PCR, NASBA, LAMP, DNA-chips, etc.) are binding strength and
-specificity of those two counterparts. It has been previously shown that functionalization of
oligonucleotide with side chains containing amino groups is one way to improve hybridization efficiency.5
In the same article authors have shown that oligonucleotide-DNA helix hybridization temperature depends
on the number of amino groups in the side-chain. According to our preliminary unpublished work, the most
effective motifs bearing amino-groups would be piperazine rings.6
The aim of current work was to develop simple and cost-effective synthetic route to CH2 skipped

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piperazine-pyridine alternating structures with useful end-group “handle” for further functionalization.
Similar unfunctionalized symmetrical structures have previously been synthesized by Rissanen et al. to
analyze its complexation with Cd metal.7 According to our knowledge, synthesis of such asymmetrical
molecules has not been previously described.
Synthesis of target molecule (Scheme 1) was performed starting only from pyridine-2,6-dicarboxylic acid,
piperazine and N-methylpiperazine using simple reagents available in every organic chemistry laboratory.
Pyridine-2,6-dicarboxylic acid was converted into dimethyl ester 1 by chlorinating with thionyl chloride
followed by refluxing with absolute methanol. Diester 2 was reduced with NaBH4 using slow methanol
addition procedure described by Soai et al.8 Methanol reacts with NaBH4 and there are constantly small
amounts of methoxyborohydrides in solution which have stronger reducing power than regular borohydride
and able to reduce methyl esters. This also allows us to avoid more expensive and dangerous LiAlH4 which
is commonly used for reduction of esters.
The key intermediate 3 was prepared by mono-bromination of 2 in concentrated aqueous HBr at reflux
temperature. Care must be taken to optimize reaction time – reaction is difficult to monitor by TLC as diol
2 remains in water-phase during extraction therefore giving false impression of high conversion while in
fact, most of the starting material is still unreacted. On the other hand too long reaction times increase the
amount of dibrominated product and reduce the yield of 3.
Compound 3 was coupled with 1-methylpiperazine under mild conditions using potassium carbonate as a
base giving 4 in excellent yield. The poor solubility of K2CO3 in reaction media (acetonitrile) did not
prevent reaction from taking place but simplified following work-up (only filtration and removal of
solvent). Compound 10 was obtained by treating 4 with thionyl chloride. The product of this reaction was
not stable enough to store overnight and was therefore prepared just before final coupling reaction.
Azide functionality was introduced into target structure by treating 3 with NaN3 yielding compound 5,
which in turn was converted into an electrophilic compound upon treatment with MsCl. Thus obtained
compound 6 was coupled with N-Boc-piperazine under previously described N-alkylation conditions
giving 8. Instead of Boc-protection, the use of excess of unprotected piperazine could be considered but
subsequently one has to be aware of the tedious removal of the leftover piperazine. 8 was then deprotected
with trifluoroacetic acid (TFA) and coupled with freshly prepared 10 under aforementioned N-alkylation
conditions to give target compound 11 in acceptable yield.
The main reason for the low yield of 10 and 11 is probably the instability of tertiary amines bearing chloride
group. Relative stability of compound 10 in solution of DCM and its decomposition upon concentration
suggests that the main reason of instability is intermolecular quaternary ammonium salt formation.

HETEROCYCLES, Vol. 90, No. 1, 2015
627
O
O
O
O
1. SOCl2
OH
NaBH4
MeOH (over 1.5 h)
75 °C, 12 h
N
N
N
HO
OH
O
O
OH
2. MeOH
t-BuOH
30 min
65 °C, 30 min
93%
92%
1
2
HBr
120 °C, 18 h
N
N
NaN3
NH
N3
OH
Br
N
OH
HN
DMF, 60 oC, 8 h
33%
89%
3
5
K2CO3
ACN, N2
MsCl, Et3N
DCM
20 °C, 30 min
Boc2O
DCM
0 to 20 °C, 30 min
NH
20 °C, 1 h
N
N
NBoc
OMs
N3
N
OH
HN
N
87%
87%
K2CO3
ACN, N2
97%
7
6
4
SOCl2
N2
20 °C, 14 h
-15 °C, 45 min
N
N3
N
N
NBoc
N
Cl
87%
N
8
TFA
49% (crude)
DCM
20 oC, 6 h
10
N
N3
N
NH
K2CO3
~100%
ACN, N2
9
-15 to 50 °C, 1 h
N
N
N
N
N
N
44% (separated)
11
N3
Scheme 1. Synthesis of 1-{6-[(4-{[6-{azidomethyl}pyridin-2-yl]methyl}piperazin-1-yl)methyl]pyridin-2-
yl}methyl- 4-methylpiperazine (11)
Synthesis of target molecule was performed in 11 steps (Scheme 1) from easily available, low-cost starting
materials and scalable synthetic methods in 6% yield from pyridine-2,6-dicarboxylic acid.

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EXPERIMENTAL
Starting materials, reagents and analytical-grade solvents were purchased from commercial sources.
Preparative chromatographic separations were performed on silica gel (70-230 mesh, Machery-Nagel
Kieselgel 60) and reaction progress was monitored by analytical thin-layer chromatography (TLC) on
pre-coated silica gel (Machery-Nagel ALUGRAM(R)) plates, spots were detected by UV light (λ 254 nm)
or KMnO4 staining. IR spectra were measured on a Perkin-Elmer Spectrum BXII FTIR spectrometer using
1
ATR technique with ZnSe. H and 13C NMR spectra were recorded on a Bruker Avance-II or Bruker
Avance-III at field strength 200/400 and 50/100 MHz for 1H and 13C respectively and samples measured in
CDCl3 (unless otherwise indicated) using TMS (δ 0.00) or residual CHCl3 (1H NMR δ 7.26 and 13C NMR δ
77.16) as an internal standard. Chemical shifts (δ) are expressed in ppm and coupling constants (J) in Hz,
multiplicity (br=broad, s=singlet, d=doublet, t=triplet, m=multiplet) and integrals are indicated. Moisture
and air sensitive reactions were carried out in vacuum-dried, septum capped flasks under nitrogen
atmosphere. Solvents were dried over 3Å molecular sieves.
Synthesis of dimethyl pyridine-2,6-dicarboxylate (1): Compound was synthesized according to
previously published procedure in 93% yield; NMR data matched literature values.9
Synthesis of 2,6-di(hydroxymethyl)pyridine (2): Compound was synthesized according to previously
published procedure in 92% yield;8 NMR data matched literature values.10
Synthesis of 2-(bromomethyl)-6-(hydroxymethyl)pyridine (3): Compound was synthesized according
to previously published procedure in 33% yield; NMR data matched literature values.11
Synthesis of 2-(hydroxymethyl)-6-[(4-methylpiperazine-1-yl)methyl]pyridine (4): Compound 3 (200
mg, 0.99 mmol) and N-methylpiperazine (109 μL, 1.2 mmol) were dissolved in anhydrous acetonitrile
(ACN) (8 mL) under nitrogen atmosphere. Anhydrous K2CO3 was added and reaction mixture stirred at
room temperature for 1 h. K2CO3 was filtered off and solvent evaporated under reduced pressure. Product
(197 mg, 0.89 mmol) was obtained as yellow oil in 90% yield. IR 3214 cm-1. 1H NMR (200 MHz, CDCl3) δ
= 7.65 (t, J = 7.7, 1H, Py), 7.30 (d, J = 7.6, 1H, Py), 7.20 (d, J = 7.6, 1H, Py), 4.77 (s, 1H, OH), 4.74 (s, 2H,
CH2OH), 3.68 (s, 2H, PyCH2N), 2.56 (m, 8H, CH2), 2.32 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3) δ 159.2
(Py), 157.4 (Py), 137.1 (Py), 121.7 (Py), 118.9 (Py), 64.3 (NCH2Py), 64.1 (PyCH2OH), 55.0 (NCH2CH2N),
52.9 (NCH2CH2N), 45.8 (CH3).
Synthesis of 2-(azidomethyl)-6-(hydroxymethyl)pyridine (5): Compound was synthesized according to
previously published procedure in 80% yield; NMR data matched literature values.12
Synthesis of 2-(azidomethyl)-6-(methanesulofonylmethyl)pyridine (6): Compound 5 (340 mg, 2.07
mmol) and Et3N (375 μL, 2.69 mmol) were dissolved in DCM (15 mL). MsCl (192 μL, 2.49 mmol) in
DCM (200 μL) was added dropwise over 15 min. After stirring for 15 min at room temperature, solvent was
removed under vacuum and residue filtered through plug of silica and silica washed with EtOAc. Filtrate

HETEROCYCLES, Vol. 90, No. 1, 2015
629
was concentrated and dried in vacuum. Title compound (435 mg, 1.80 mmol) was obtained as red oil in
87% yield. 1H NMR (200 MHz, CDCl3) δ = 7.81 (t, J = 7.8, 1H, Py), 7.44 (d, J = 7.6, 1H, Py), 7.35 (d, J =
7.9, 1H, Py), 5.34 (s, 2H, CH2OMs), 4.48 (s, 2H, CH2N3), 3.11 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3) δ
156.1 (Py), 154.0 (Py), 138.3 (Py), 122.0 (Py), 121.6 (Py), 71.4 (PyCH2OMs), 55.5 (CH2N3), 38.2 (CH3).
HRMS (ESI) calcd for C8H10N4O3S [M+H+] 243.05464, found 243.05576.
Synthesis of tert-butyl piperazine-1-carboxylate (7): Compound was synthesized according to
previously published procedure in 92% yield; NMR data matched literature values.13
Synthesis of 1-{[6-(azidomethyl)pyridin-2-yl]methyl}piperazine (9): Compound 6 (400 mg, 1.65 mmol),
compound 7 (333 mg, 1.79 mmol) and anhydrous K2CO3 (200 mg, 1.44 mmol) were suspended in
anhydrous ACN (10 mL) in nitrogen atmosphere. Reaction mixture was stirred for 10 h at room
temperature, after which it was filtered and filtrate concentrated in vacuum. Product was purified by
column chromatography (DCM:MeOH 20:1 → 10:1) which afforded compound 8 (477 mg, 1.43 mmol) as
a yellow oil in 87% yield. Compound 8 (477 mg, 1.43 mmol) was dissolved in DCM (37 mL) and
trifluoroacetic acid (2.4 mL, 31.5 mmol) was added at room temperature. After 6 h DCM was added (20
mL) and solution neutralized with 1M NaOH solution (40 mL), the organic phase was further washed with
1M NaOH solution (30 mL) and brine (15 mL). Combined water fractions were further extracted with
DCM (4 x 20 mL), organic fractions combined, dried over anhydrous Na2SO4, filtered and solute
evaporated under reduced pressure. After drying in vacuum, title compound (330 mg, 1.42 mmol) was
obtained as yellow oil in 99% yield. IR 3378, 2099 cm-1. 1H NMR (200 MHz, CDCl3) δ = 7.61 (t, J = 7.7,
1H, Py), 7.29 (d, J = 7.7, 1H, Py), 7.14 (d, J = 7.6, 1H, Py), 4.37 (s, 2H, CH2N3), 3.95 (s, 1H, NH), 3.58 (s,
13
2H, CH2NCH2Py), 3.08–2.76 (m, 4H, CH2NCH2Py), 2.62–2.21 (m, 4H, CH2NH). C NMR (50 MHz,
CDCl3) δ 158.5 (Py), 155.1 (Py), 137.3 (Py), 122.4 (Py), 120.3 (Py), 64.6 (PyCH2N), 55.5 (CH2N3), 53.5
(NCH2CH2N), 45.4 (NCH2CH2N). HRMS (ESI) calcd for C11H16N6 [M+H+] 233.15092, found 233.15009.
Synthesis of 1-{6-[(4-{[6-{azidomethyl}pyridin-2-yl]methyl}piperazin-1-yl)methyl]pyridin-2-yl}-
methyl-4-methylpiperazine (11): Compound 4 (47 mg, 0.21 mmol) was added to SOCl2 (2 mL) at -15 °C
under nitrogen atmosphere. After 45 min of stirring at -15 °C, H2O (2 mL) was added dropwise, followed
by neutralization with saturated aqueous solution of Na2CO3. Mixture was extracted with DCM (3 x 5 mL)
and combined organic fractions were dried over anhydrous Na2SO4 and filtered. Filtrate was concentrated
under reduced pressure. Obtained chloride was dissolved in anhydrous ACN (3 mL) to which compound 8
(48 mg, 0.21 mmol) and anhydrous K2CO3 (20 mg, 0.14 mmol) were added under nitrogen atmosphere at
-15 °C. Reaction mixture was heated to 50 °C and stirred for 1 h. Solid K2CO3 was filtered off and filtrate
concentrated under reduced pressure. Title compound (20 mg, 0.046 mmol) was obtained in 22% yield
from 8, after purification by column chromatography (CHCl3:MeOH:NH4OH, 100:35:3). IR 2100 cm-1. 1H
NMR (400 MHz, CDCl3) δ = 7.68 (t, J = 7.8 Hz, 1H, Py), 7.61 (t, J = 7.6 Hz, 1H, Py), 7.40 (d, J = 7.6 Hz,

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HETEROCYCLES, Vol. 90, No. 1, 2015
1H, Py), 7.3 (m, 2H, Py), 7.21 (d, J = 7.6 Hz, 1H, Py), 4.46 (s, 2H, CH2N3), δ = 3.68 (m, 6H,PyCH2N),
2.58–2.48 (m, 16H, NCH2CH2N), 2.30 (s, 3H, CH3). 13C NMR (50 MHz, CDCl3) δ = 159.0 (Py), 158.2 (Py),
158.0 (Py), 155.0 (Py), 137.3 (Py), 136.6 (Py), 122.3 (Py), 122.2 (Py), 121.2 (Py), 121.2 (Py), 120.2 (Py),
64.5 (PyCH2N), 64.4 (PyCH2N), 64.4 (PyCH2N), 55.7 (CH2N3), 55.1 (NCH2CH2N), 53.3 (NCH2CH2N),
53.3 (NCH2CH2N), 53.2 (NCH2CH2N), 46.0 (CH3) HRMS (ESI) calcd for C23H33N9 [M+H+] 436.29317,
found 436.29141.
ACKNOWLEDGEMENTS
This work was supported by Quattromed HTI Laborid OÜ, Estonian Science Targeted Projects No SF
0180032s12, Estonian Science Council (IUT20-17) and the Estonian Science Foundation Grants no. 8794.
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