A new process for deprotection of acetyl and benzoyl groups in synthesis of azacitidine

Article abstract of DOI:10.14233/ajchem.2018.21206

4-Amino-1-β-D-ribofuranosyl-s-triazin-2(1H)-one or azacitidine is a promising DNA demethylation inhibitor used for the treatment of myloneplastic, bone cancer and breast cancer. An efficient, cost-effective and convenient manufacturing process for the synthesis of azacitidine is described. The present research relates to the synthesis, deprotection, isolation and purification of azacitidine (1). In this process, more particularly 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is used as deprotection reagent for deprotection of O-acetyl, O-benzoyl to acquire azacitidine (1). The new process allows for the reliable and efficient production of drug substance similar overall yield. The new improved process has merits including enantiomeric purity, better crystallization and the product complies with the requirements of USP30.

Full text of DOI:10.14233/ajchem.2018.21206

Asian Journal of Chemistry; Vol. 30, No. 7 (2018), 1521-1524
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2018.21206
A New Process for Deprotection of Acetyl and Benzoyl Groups in Synthesis of Azacitidine
*
SRUJANA SUNEEL KUMAR and V. SETHURAMAN
Department of Chemistry, Periyar Maniammai Institute of Science & Technology , Periyar Nagar, Thanjavur-613 403, India
*Corresponding author: E-mail: sunilchemist4u@gmail.com
Received: 30 December 2017;
Accepted: 3 February 2018;
Published online: 31 May 2018;
AJC-18924
4-Amino-1-β-D-ribofuranosyl-s-triazin-2(1H)-one or azacitidine is a promising DNA demethylation inhibitor used for the treatment of
myloneplastic, bone cancer and breast cancer. An efficient, cost-effective and convenient manufacturing process for the synthesis of
azacitidine is described. The present research relates to the synthesis, deprotection, isolation and purification of azacitidine (1). In this
process, more particularly 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is used as deprotection reagent for deprotection of O-acetyl, O-
benzoyl to acquire azacitidine (1). The new process allows for the reliable and efficient production of drug substance similar overall yield.
The new improved process has merits including enantiomeric purity, better crystallization and the product complies with the requirements
of USP30.
Keywords: 5-Azacytosine, D-ribose, 5-Azacitidine, Vorbruggen condensation, Deprotection, DBU.
Winkly and Robins [8]reported a 5-azacitidine synthesis
process that relies on the coupling of the ‘bromo sugar’with a
silyl derivative of 5-azacytosine. Niedballa and Vorbrueggen
[9] reported the procedure that has been used historically for large
scale synthesis of 5-azacitidine for the treatment of myclody-
splastic syndromes.The industrial draw backs of the described
process are (a) silylation of 5-azacytosine with more molar
equivalents of hexamethyldisilazane (HMDS), more reaction
duration time for silylation (b) more consumption of expensive
stannic chloride (SnCl4) and variable amounts of tin from one
batch to another batch in lack of control of the tin level ensuing
the procedure is not suitable for producing API for human
use, (c) development of the emulsion in the work up procedure
of coupled mixture. Vorbrüggen and Ruh-Pohlenz [10] have
previously noted that silylated heterocycles and protected
1-O-acyl or 1-O-alkyl sugars in the presence of Fredal-Craft
catalyst like SnCl4 often form emulsion and colloids during
work up d) expensive and unstable methanolic ammonia usage
for deprotection of benzoylgroups e) very low yield 5 % from
the 1a and the purity of the final product (98 %) did not comply
with the requirement of USP 30 (> 99.8 %). Piskala et al. [11]
have also reported a procedure for the ribosylation of silver salts
of 5-azapyrimidine nucleobases. Specifically, the ribosylation
of the silver salt of 5-azacytosine with 2,4,5-tri-O-benzoyl-β-
D-ribosyl chloride gives a mixture of 5-azacitidine.
INTRODUCTION
4-Amino-1-β-D-ribofuranosyl-s-triazin-2(1H)-one or 5-
azacytidine, a cytidine nucleoside present in DNA and RNA.
Most of the cytidine nucleosides are used in the treatment of
myelodysplastic syndrome [1].Azacitidine is mainly employed
in in vitro methyl group interactions from DNA. This may
weaken the effects of gene silencing mechanisms that occurred
prior to the methylation. Methylation events are therefore,
believed to secure the DNA in a silenced state. Being a ribonu-
cleoside, it has been shown effective biological activity against
HIV and HTLV [2,3].
The synthesis of azacitidine was accomplished by Pískala
[4] by using methanolic ammonia as deprotecting reagent of
benzoyl groups at 0 °C. This route of synthesis was not suitable
for large-scale material generation. The concentration and
stability of methanolic ammonia leads to the failure of the
classical method of synthesis. Different routes for the synthesis
of compound 1 have been reported with low yield [4]. Azaci-
tidine is commercially available as Vidaza™ for intravenous
or subcutaneous treatment of acute forms of leukemia [5].
Vidaza is formulated at a pH of about 6.5 contains a small
quantity of sodium hydroxide and delivers l00 mg of azaci-
tidine in 10 mL of water when the lyophilized is reconstituted.
This concentrate is further diluted with normal saline or
Ringer’s lactate to yield a finally administered concentration
of 1 mg/mL [6]. The presence of sodium hydroxide in Vidaza
also results in limited stability [7].
Herein, we report a method for preparing azacitidine by
using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as depro-
tecting reagent for carbohydrates which is suitable for the large

1522 Kumar et al.
Asian J. Chem.
scale production of the material. This improved process has
some industrial advantages such as convenient operation,
economical material cost and moderate yield with the classical
method of synthesis.
2,3,5-Tri-O-acetyl-1-O-methyl-D-ribofuranose (2): To a
solution of 1-O-methyl-β-D-ribofuranose (1.0 Kg) in diisopropyl
ether (400 mL) was added and cooled to 4 °C. Then acetic
anhydride (80.0 g) and acetic acid (48.0 g) were added
followed by pyridine (25.0 g). The reaction mixture was cooled
to 0 °C and added concentrated sulphuric acid (88 g). The
reaction mixture was stirred for 24 h at 0 °C and added sodium
acetate (216.5g). Ethyl acetate (1.2 L) was added and stirred
for 5 min followed by the addition of saturated sodium bicar-
bonate to adjust the pH up to 7.3. Separated the layers and
aqueous layers is extracted with ethylaceate (1.0 L) and com-
bined organic layer dried with MgSO4 and concentrated to
obtain crude 2,3,5-tri-O-acetyl-1-O-methyl-L-ribofuranose
(1.5 Kg). Crystallization done with ethanol by heating to 45 °C
and ramp cooling to room temperature to obtain 950 g of 2,3,5-
tri-O-acetyl-1-O-methyl-D-ribofuranose. 1H NMR (400MHz,
CDCl3, TMS = 0 ppm): 2.08 (s, 3H), 2.09 (s 3H), 2.10 (s, 3H),
2.13 (s, 3H), 4.12-4.19 (m, 1H), 4.30-4.40 (m, 2H), 5.32-5.38
(m, 2H), 6.17 (s, 1H) ppm.
2,3,5-Tri-O-acetyl-β-D-ribofuranose-4-amino-1,3,5-
triazine (3): To a suspension of 5-azacytocin (276.0 g), hexa-
methyldisilazane (2.5 L) and trimethylsilylchloride (100 mL),
iodine (1.0 g) were added and heated to 125 °C to reflux for
2 h. 5-Azacytocin was dissolved and reflux is stopped after
the compete gas evaluation. The excess HMDS is evaporated
off in the vacuum to obtain off-white solid, which is silylated
azacytocin. The above solid was diluted with ethyl acetate (5.0
L) and 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (1.0 Kg) was
added. Cooled the reaction mixture to 10 °C for 30 min and
added SnCl4 (600 mL) over a period of 10 min. Temperature
raised for 24 °C. Reaction mixture became clear solution from
suspension. Stirred for 2 h, after the reaction monitoring by
HPLC added NaHCO3 (2.0 Kg) and Na2CO3 (2.0 Kg) and to
the reaction mixture. Added demineralized water (500 mL)
slowly to the reaction mixture over a period of 15 min and
filtered. Separated the organic layer and the aqueous layer is
extracted with ethyl acetate (1.0 L). The organic layer is washed
with cold 10 % sodium bicarbonate (2X1L). Organic layer is
separated and washed with 10 % NaCl solution. Organic layer
is separated and added charcoal (50 g) and heated to 50 °C for
over 2 h and filtered through the celite bed. Celite bed is washed
with ethyl acetate (500 mL). Organic layer is dried over magne-
sium sulphate. Silica gel (60-120 mesh) was added to the
organic layer, stirred for 1 h at room temperature and filtered
through the celite bed. Celite bed was washed with ethyl acetate
(500 mL). Organic layer is concentrated in vacuum to dryness
to obtain 1.25 Kg of compound 3 as a brown solid.Yield 85 %,
ee 90 % (chiral purity).
EXPERIMENTAL
Reactions involving oxygen and/or moisture sensitive
reagents were carried out under an atmosphere of nitrogen or
argon using anhydrous solvents. Anhydrous solvents were
obtained in the following manner: THF was dried over sodium/
benzophenone and distilled, methanol was dried over activated
molecular sieves (3 Å) and degassed. Acetonitrile was dried
over P2O5 and distilled, and DMF was dried over activated
molecular sieves (4 Å) and degassed. All the other solvents
were of technical quality and distilled prior to use and deionized
water was used throughout the process. Column chromato-
graphy was carried out on silica gel 60-120 mesh (0.040-0.063
mm) and 230-400 mesh under flash conditions. Thin layer
chromatography (TLC) was performed on aluminum plates
precoated with silica gel 60 F254 and Rf’s were determined
using the solvent system used to elute the column unless other-
wise specified. Solvent system are reported in vol:vol ratios.
Visualization of the spots was carried out using UV light (254
nm) and/or stanning under heating (H2SO4 stanning solution.
Preparation – 4 g of vanillin, 25 mL of conc. H2SO4, 80 mL of
acetic acid and 680 mL of MeOH. KMnO4 solution, prepa-
ration- 1 g of KMnO4, 6 g of K2CO3, 1.5 mL of 1.25 NaOH
solution and 100 mL of water.
Analytical HPLC was performed on a standard system with
a diode array UV detector and equipped with a Zorbax column
(5 µm) containing reversed-phase silica gel purospher RP 18
(5 µm).
Method-I: Eluent A water (0.1 % TFA), eluent B MeCN;
0-15 min gradient of B (3-30 %, 15-20 min 100 % B, 20-22
min gradient eluent A water.
1H NMR spectra were recorded at 400 MHz. 13C NMR
1
spectra were recorded at 100 MHz and were H-decoupled.
All spectra were measured at room temperature except some
samples in DMSO-d6 and D2O (standard 35 °C). All the NMR
spectra were referenced internally to solvent reference fre-
quencies wherever possible. All chemical shifts (δ) are quoted
in ppm and coupling constants (J) values are reported in Hz.
1
1
Assignment of signals was carried out using H, H-COSY,
NOESY spectra.
Infrared spectroscopy (IR) was either performed on a
spectrometer equipped with an ATR unit or on a machine
lacking the ATR unit, with solids being measured as KBr
Palates. Peaks are given as wave numbers (ν) in cm-1. UV/
visible spectroscopy: wavelengths of maximum absorption
(λmax) are reported in nm with the corresponding logarithmic
molar extinction coefficient (log (ε, dm3 mol-1 cm-1)) given in
parentheses. Melting points (m.p.) are determined by the
capillary method on a Buchi melter apparatus and are not
corrected. The specific optical rotations were measured with
Perkin Elmer 241 polarimeter using a sodium lamp (589 nm)
at room temperature. Mass spectra were recorded with an
Agilent system using electron spray-ionization (ESI) tech-
nique.
4-Amino-1-β-D-ribofuranosyl-s-triazin-2(1H)-one or
crude azacitidine (4): To a solution of 2,3,5-tri-O-acetyl-β-
D-ribofuranose-4-amino-1,3,5-triazine (1.0 Kg) which is dis-
solved in DBU (5.0 L) was added methanol (40 L) and stirred
for 6 h at 20-25 °C. Filtered the solid and washed with methanol
(500 mL) and suck dried for 30 min and subjected for drying
under vacuum tray drier to get crude 400 g of 4-amino-1-β-
D-ribofuranosyl-s-triazin-2(1H)-one.
Purification of crude 4-amino-1-β-D-ribofuranosyl-s-
triazin-2(1H)-one (1): To a solution of crude 4-amino-1-β-

Vol. 30, No. 7 (2018)
A New Process for Deprotection of Acetyl and Benzoyl Groups in Synthesis of Azacitidine 1523
D-ribofuranosyl-s-triazin-2(1H)-one (4) (400 g) in DBU (2.0
L) was heated to 90 °C and added methanol (10 L) and acetic
acid (400 mL) slowly over a period of 30 min. Cooled the
reaction mixture to room temperature, filtered the solid and
washed the product with methanol (500 mL) to get pure
4-amino-1-β-D-ribofuranosyl-s-triazin-2(1H)-one (I) (390 g),
m/z 244.4 (Scheme-I).
5.41 (t, 1H, J = 6.0 Hz), 4.30-4.41 (m, 3H), 2.11 (s, 3H), 2.10
(s, 3H) and 2.08 (s, 3H) 13C NMR (CDCl3, 100 MHz) 170.3,
169.6, 169.5, 166.0, 156.0, 153.1, 89.7, 79.9, 73.7, 69.9, 62.8,
20.7, 20.4 and 20.3 ppm.
RESULTS AND DISCUSSION
1,2,3,5-Tri-O-acetyl-β-D-ribofuranose was prepared by
the reported method [12] with acetyl chloride in the presence
of acetic acid and pyridine. Silylation of 5-azacytoise was
1H NMR (CDCl3, 500 MHz): d 8.18 (s, 1H), 7.69 (s, 1H),
6.33 (s, 1H), 5.82 (d, 1H, J = 3.0 Hz), 5.54 (t, 1H, J = 4.0 Hz),
Route of synthesis:
HO
HO
O
OH
O
O
H2SO4
Methanol
H
H
OH
OH
OH
OH
O
O
HO
O
O
O
O
(AcO)2O
H
O
H
Py,DIPEA
O
O
OH
OH
O
O
2
NH2
N
Si
N
HN
O
N
O
O
2
O
HMDS,TMSCl
Iodine
N
N
O
H
O
O
O
N
O
SnCl4,EtOAc
O
N
O
H
O
H
O
O
O
O
3
NH2
NH2
N
N
N
N
H
O
N
H
O
DBU,Methanol
O
O
O
HO
O
O
O
O
OH OH
4
O
3
NH2
N
N
DBU
N
O
Methanol
90 °C
HO
O
OH OH
Azacitidine
1
Scheme-I

1524 Kumar et al.
Asian J. Chem.
performed with N,N-bistrimethylsilylacetamide (BSA)
hexamethyldisilazane (HMDS) as reaction solvent. TMSCl,
TMSI, (NH4)2SO4 were tested as catalyst for the silylation reac-
tion initiation. The coupling reaction between 5-azacytocin
and protected carbohydrates carried out with different Lewis
acids in various solvents (Table-1). Work up of coupled reaction
furnished in the solution of ethyl acetate at ambient temperature
with the good purity. The coupling reaction phenomenon
indicates that the reaction proceeds through via SN1 like path
way, which is consistent with the hypothesis of Piskala. An
expensive SnCl4 molecular equivalent is studied here for the
coupling reaction. The usage of class-1 solvents like benzene,
carbon tetrachloride, carbon trichloride, ethylene dichloride
was avoided for the coupling reaction by the contemplation
of ICH guidelines. We have studied the number of solvents
for the successful coupling reaction such as tetrahydrofuran,
dichloromethane, acetonitrile, ethyl acetate. Finally, ethyl acetate
was provided the clear reaction mixture without any emulsion
at the time of work up. After the reaction completion, SnCl4
was removed by the NaHCO3 and Na2CO3mixture (1:1). Charcoal
treatment was done to improve the colour of the intermediate.
Neutral alumina and charcoal mixture was used to make slurry
of the coupled product and then filtered through the celite pad.
Conclusion
A new process for the preparation of azacitidine, including
crystallization has been provided with similar yield over 3
steps. Because of the effect of O-acetyl ribose and O-benzoyl
ribose, the resulting intermediates have some industrially
favourable physical properties. For example, compound 1 can
be simply separated from the crude and purified. We have
successfully executed the same process to prepare the 8-bromo-
3-methyl-7-(B-D-ribofuranosyl)purine-2,6-dione which is we
have reported earlier [13]. The advantages of the new process
ensure that the suggested approach is an efficient, cost effective
and industrially convenient process. Therefore, this metho-
dology could be employed for commercial production of
azacitidine in large scale.
ACKNOWLEDGEMENTS
The authors are grateful to Indian Institute of Technology
Madras, Chennai, India and Dr.Vivek Kumar, NIH University,
Maryland, USA) for their incredible support for my research
work.
REFERENCES
1. A. Cihák, Oncology, 30, 405 (1974);
https://doi.org/10.1159/000224981.
TABLE-1
2. P.T. Diamantopoulos, M. Michael, O. Benopoulou, E. Bazanis, G. Tzeletas,
J. Meletis, G. Vayopoulos and N.-A. Viniou Virol. J., 9, 1 (2012);
https://doi.org/10.1186/1743-422X-9-1.
3. R.C. Hartenstein and I. Fridovich, J. Org. Chem., 32, 1653 (1967);
https://doi.org/10.1021/jo01280a095.
4. A. Piskala, Collect. Czech. Chem. Commun., 32, 3966 (1967);
https://doi.org/10.1135/cccc19673966.
5. A.G. Dinmohamed, Y. van Norden, O. Visser, E F M. Posthuma, P.C.
Huijgens, P. Sonneveld,A.A. van de Loosdrecht and M. Jongen-Lavrencic,
Leukemia, 29, 2449 (2015);
DIFFERENT LEWIS ACIDS FOR COUPLING REACTION
Batch
SnCl4
Solvents for Purity by Tin levela
size (g) equiv. glycosylation HPLC, 4
Entries
in 7
10
19
30
60
15
15
17
1
2
3
4
5
6
7
5.0
5.0
5.0
1.0
1.5
2.0
2.5
1.5
1.5
1.5
DCE
DCE
DCE
DCE
DCE
DCE
DCE
75
98
98
98
97
98
98
5.0
10.0
100.0
500.0
https://doi.org/10.1038/leu.2015.220.
6. R. Garcaa-Delgado, D. de Miguel, A. Bailén, J.R. Gonzalez, J. Bargay,
J.F. Falantes, R. Andreu, F. Ramos, M. Tormo, S. Brunet, A. Figueredo,
J. Casaño, Á. Medina, L. Badiella, A.F. Jurado and G. Sanz, Leukemia
Res., 38, 744 (2014);
https://doi.org/10.1016/j.leukres.2014.03.004.
7. N.R. Palepu, P.C. Buxton and B.B. Teja, Stable Formulations of
Azacitidine, WO 2011014541 A1 (2011).
8. M.W. Winkley and R.K. Robins, J. Org. Chem., 35, 491 (1970);
https://doi.org/10.1021/jo00827a045.
9. U. Niedballa and H. Vorbrueggen, J. Org. Chem., 39, 3672 (1974);
https://doi.org/10.1021/jo00939a012.
10. H. Vorbrüggen and C. Ruh-Pohlenz, Org. React., 55, 100 (2000);
https://doi.org/10.1002/0471264180.or055.01.
11. D. Ionescu and P. Blumbergs, Synthesis of 5-Azacytidine, US Patent
8058424B2 (2011).
DCE = Dichloroethane
The deprotection of compound 4 performed with DBU,
methanol was used as solvent with DBU to furnish crude
azacitidine. The crude azacitidine was crystallized with DBU,
methanol and acetic acid (5:10:1) to get pure azacitidine.
Comparing the previously reported result of Piskala pro-
cedure, it was inferred that the yield and purity obtained in the
present study was almost similar (Table-2). In addition, the
present synthetic process involves simple and user friendly
methodology for the deprotection benzoyl, acetyl group. So that
this approach could be applied for the large scale production.
12. F. Ishikawa, A. Nomura, T. Ueda, M. Ikehara and Y. Mizuno, Chem.
Pharm. Bull., 8, 380 (1960);
https://doi.org/10.1248/cpb.8.380.
13. S.S.K. Madana, V. Sethuraman and N. Gupta, Asian J. Chem., 28, 429
(2016);
TABLE-2
YILED AND PURITY OF FINAL PRODUCT
Silylating
reagent
Temp.
(°C)
Time
(min)
Yieldd
(%) of 4
Entries
Catalyst
https://doi.org/10.14233/ajchem.2016.19409.
1
2
3
4
5
HDMS
HDMS
HDMS
HDMS
BSA
125
125
125
125
80
TMSCl
(NH4)2SO4
I2
TMSI
–
180
180
180
180
180
60
65
58
50
60