Synthesis and characterization of novel oxime prodrug of G liclizide

Article abstract of DOI:10.14233/ajchem.2014.17642

Diabetes has emerged as a major healthcare problem throughout the world. Gliclizide is a widely used sulphonyl urea compound to treat diabetes. Gliclizide is categorized under biopharmaceutical classification system class II drug which do have poor solubility. Drug molecules with limited aqueous solubility are becoming increasingly prevalent in the research and development portfolios of discovery focused pharmaceutical companies. Prodrugs are an established concept to overcome barriers like poor solubility. Aqueous solubility is an important parameter to enhance the bioavailability of drug. Hence the present study aims to enhance aqueous solubility and in turn bioavailability by synthesis of novel oxime prodrug of gliclizide. The prepared prodrug was characterized by IR, NMR, Mass and DSC. in vitro chemical hydrolysis profiles revealed that the synthesized oxime derivatives of gliclizide are chemically stable in simulated gastric fluid pH 1.2. Decrease in log P value indicates the increase in hydrophilic property of synthesized oxime derivatives of gliclizide.

Full text of DOI:10.14233/ajchem.2014.17642

Asian Journal of Chemistry; Vol. 26, No. 20 (2014), 6989-6992
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.17642
Synthesis and Characterization of Novel Oxime Prodrug of Gliclizide
1
,*
2
2
3
S. VIJAYARAJ , S. ANITHA , B. OMSHANTHI and K.P. SAMPATH KUMAR
1
2
3
Department of Pharmaceutical Sciences, NIMS University, Jaipur-303 121, India
Department of Pharmaceutical Analysis, Sree Vidyanikethan College of Pharmacy, Tirupathi-517 101, India
Department of Pharmaceutical Sciences, Coimbatore Medical College, Coimbatore-641 014, India
*
Corresponding author: Phone: +91-877-2272760, e-mail: vijaysurender@yahoo.co.in
Received: 21 March 2014; Accepted: 31 May 2014; Published online: 25 September 2014;
AJC-16052
Diabetes has emerged as a major healthcare problem throughout the world. Gliclizide is a widely used sulphonyl urea compound to treat
diabetes. Gliclizide is categorized under biopharmaceutical classification system class II drug which do have poor solubility. Drug molecules
with limited aqueous solubility are becoming increasingly prevalent in the research and development portfolios of discovery focused
pharmaceutical companies. Prodrugs are an established concept to overcome barriers like poor solubility.Aqueous solubility is an important
parameter to enhance the bioavailability of drug. Hence the present study aims to enhance aqueous solubility and in turn bioavailability
by synthesis of novel oxime prodrug of gliclizide. The prepared prodrug was characterized by IR, NMR, Mass and DSC. in vitro chemical
hydrolysis profiles revealed that the synthesized oxime derivatives of gliclizide are chemically stable in simulated gastric fluid pH 1.2.
Decrease in log P value indicates the increase in hydrophilic property of synthesized oxime derivatives of gliclizide.
Keywords: Synthesis, Characterization, Oxime Prodrug, Gliclizide.
preparation of prodrug in solubility enhancement of gliclizide.
A huge sum of money has been invested in new drug research
and it will take around 15 years to develop a new chemical
entity whereas prodrug strategy is aimed to enhance the
effectiveness of existing drug hence it is economic and time
saving approach. Therapy enhancement via successful delivery
of a therapeutic agent is the principal goal of drug delivery
research.Achieving therapeutic efficacy of any pharmaceutical
dosage form mainly depends upon the availability of drug with
INTRODUCTION
Diabetes mellitus (DM) is the most common endocrine
disorder. It is estimated that more than 200 million people
worldwide have diabetes mellitus and by 2025 the amount
1
may rise to 300 million . Gliclizide (GZ), chemically 1-[(4-
methylbenzene) sulfonyl]-3-{octa-hydrocyclopenta [c]pyrrol-
2-yl} urea (Fig. 1) is a second generation sulphonyl urea which
2
acts as a hypoglycemic agent . Gliclizide belongs to Biophar-
maceutical classification system (BCS) class-II drug which
do have low aqueous solubility. Compounds with poor solu-
bility influences both pharmacokinetic and pharmacodynamic
properties of the compound. Although numerous strategies
exist for enhancing the bioavailability of drugs with low
aqueous solubility, the success of these approaches is not yet
able to be guaranteed and is greatly dependent on the physical
9
desired concentration to the target site .The bioavailability of
poorly water-soluble drug like Gliclizide is a well-known
difficulty to be coped with during drug delivery. The current
research aims to resolve aforementioned issue by prodrug
approach. Oxime prodrug of gliclizide was prepared and the
1
synthesized prodrug was investigated by FT-IR, 1H NMR,
Mass and DSC studies. Aqueous solubility studies were
performed to ensure the enhancement in solubility.
3
and chemical nature of the molecules being developed .
Prodrug is an efficient technique to enhance solubility of drugs.
Prodrugs are defined as a biologically inactive derivative of a
parent drug molecule that usually requires a chemical or
enzymatic transformation within the body to release the active
drug and possess improved delivery properties over the parent
EXPERIMENTAL
Melting points were determined on a Differential Scanning
Calorimeter (DSC) apparatus. The IR spectra were recorded
in KBr discs on an FT-IR Bruker IFS 55 spectrophotometer
4
molecule . Literature survey reveals that pH change approach,
inclusion complex with β-cyclodextrin, solid dispersion
techniques and have been reported for enhancement of
-1
1
and wavenumbers are reported in cm . The 1H NMR spectra
were obtained on a Bruker DRX-300 spectrometer (75 MHz)
5
solubility of gliclizide . There are no reported method for
-8
3
in CDCl . Chemical shifts were recorded in ppm (δ) relative

6
990 Vijayaraj et al.
Asian J. Chem.
Microscopical characterization: Morphology of the
gliclizide and prepared oxime prodrug were studied by optical
microscopy at magnification of 45X.
HN
O
N
HN
Spectral and thermal characterization: Pressed pellet
technique was adapted for FT-IR analysis, drug admixed with
KBr were made in to disc and was analyzed in spectral range
O
S
O
-1
1
of 4000 to 400 cm and IR spectrum was recorded. H NMR,
Mass and DSC studies were carried out for the prepared
prodrug.
11
Partition coefficient : The partition coefficient of product
was determined in n-octanol/water system (10:10) by standard
technique. Product (drug or prodrug) was accurately weighed
H C
3
(
10 mg) and added to 10 mL each of n-octanol and aqueous
phase. The mixture was shaken using mechanical shaker for
4 h until equilibrium was reached. Phases were separated by
Fig. 1. Structure of gliclizide (GZ)
2
to TMS as an internal standard. High resolution mass spectra
were recorded on an Agilent 1100 series LC-MSD-TRAP-SL
system using electro spray ionization technique. Aluminum
sheets coated with silica gel 60F254 were used for TLC chroma-
tography.All chemicals used were of analytical grade procured
from SD fine, Himedia and E. Merck while standard drug of
Gliclizide was purchased fromYarrow Chem products Limited,
Mumbai.
separating funnel and aqueous phase was analyzed for amount
of product after appropriate dilution. Procedure was performed
13
in triplicate .
[
solute]o
Co
K
=
=
d
[
solute]aq Caq
is partition coefficient
= Concentration of solute distributed organic phase
aq = Concentration of solute distributed in aqueous phase
where K
d
10
C
o
Synthesis of oxime prodrugs
C
Preparation of N-[N-(hexahydrocyclopenta[c]pyrrol-
12
Aqueous solubility : Equilibrium solubility was deter-
13
mined by a "shake-flask" method . The aqueous solubility of
2(1H)-yl)-N′-hydroxycarbamimidoyl]-4-methylbenzene
sulfonamide: Accurately weighed quantity of 1.5 g of gliclizide
compound was determined by adding excess amount of drug
beyond its saturation limit in sealed conical flask containing
was taken in a round bottom flask and is diluted with 8 mL of
9
5 % ethanol. To the above solution 0.6 g of hydroxylamine
1
0 mL of water. This conical flask is placed in a mechanical
hydrochloride (97 % pure), along with 25 mL of deionized
water was added.At the end 15 mL of a 10 % by weight aqueous
solution of sodium hydroxide was added. The solution was
refluxed at 100 °C. Completion of the reaction is marked by
the formation of large crystals that float on the surface of the
reaction mixture. The mixture was cooled in an ice/water bath,
collected and then the recrystallised by using 95 % ethanol.
Prepared prodrugs were physically chara-cterised by TLC
method by using mixture of toluene: ethyl acetate in the ratio
of 1:1 (v/v) as the mobile phase (Fig. 2).
shaker for 48 h (This duration was previously tested to be
sufficient to reach equilibrium). The solvent was filtered
through Whatmann filter paper No. 42 and the portion of the
filtrate was suitably diluted with water. Solutions were analyzed
by using UV spectrophotometer at 227 nm, which was the
absorption maxima and drug concentrations were calculated.
13
Chemical hydrolysis study : The rate of chemical hydro-
lysis of the prodrug was determined in Simulated Gastric Fluid
(
pH 1.2) at 37 °C. Solution of 10 mg of prodrug was prepared
in 90 mL of SGF. An aliquot of 15 mL of this solution was
withdrawn repeatedly and kept in test tubes maintained at 37 ±
HN
O
N
0
.5 ºC. At a definite interval of time (0.5, 1, 2 up to 8 h), an
HN
aliquot was withdrawn from different test tubes and was
transferred to micro centrifuge tubes followed by addition of
methanol to make up the volume. The tubes were placed in
freezing mixture in order to arrest further hydrolysis, followed
by vortexing at high speed for 5 min.After vortexing, the tubes
were centrifuged at high speed 3000 rpm for 5 min. A 5 mL of
clear supernatant obtained from each tube was measured on
UV spectrophotometer for the amount of free drug released
after the hydrolysis of prodrug in SGF at 227 nm.
O
S
O
Gliclizide
3
H C
2
NH OH·HCl
Reflux 70-80 °C
Hydroxylamine
hydrochloride
HN
N
HN
RESULTS AND DISCUSSION
O
S
NOH
O
TLC analysis reveals R values of gliclizide and its oxime
f
prodrug to be 0.52 and 0.21 cm, respectively.
Microscopical characterization shown morphological
difference between gliclizide and oxime prodrug of gliclizide
Oxime derivative of gliclizide
3
H C
(
Fig. 3).
Fig. 2. Chemical scheme for synthesis of oxime prodrug of gliclizide

Vol. 26, No. 20 (2014)
Synthesis and Characterization of Novel Oxime Prodrug of Gliclizide 6991
patterns. In the first fragmentation pattern, from oxime of
gliclizide m/z 337, (C15
fragmented apart giving the characteristic line at m/z 271
). Two separate ring systems are formed namely
methyl benzene of m/z 91 (C ) and azabicyclo carba-
midoxime of m/z 181 (C O). From this azabicyclo
carbamidoxime, the carbamidoxime gets fragmented and gives
the characteristic line at m/z 73 (CH O), which undergoes
further fragmentation to give the characteristic line at m/z 60
, base peak). In the second fragmentation pattern,
azabicyclo carbamidoxime fragmented in to azabicyclic amine
ring of m/z 128 (C ). Azabicyclic amine ring undergoes
further fragmentation by the elimination of NH followed by
N to form stable species of m/z 68 (C ). In the third
fragmentation pattern, from methylbenzene sulphonyl com-
19 3 3 2
H N O S) sulphonyl group (SO ) is
15 19 3 2
(C H N O
7
H
7
8
15 4
H N
3 3
N
Fig. 3. Photomicroscopic Image of a) gliclizide b) oxime prodrug of
gliclizide
9 19 2
(C H N
9
19 2
H N
The FTIR spectrum of oxime prodrug of gliclizide (Fig. 4)
-1
represents characteristic peaks at 1601.4 cm and 1479.1 (C=N
-1
C
2
H
5
5 8
H
ring stretch of C=NOH), 1091.3 cm (N-O stretch of NOH),
3
-1
661.0 cm (O-H stretch) (Fig. 4).
+
pound m/z 155, (C
7 7 2 2
H SO ), (SO ) is fragmented apart giving
+
the characteristic line at m/z 91 (C
Methyl benzene (parent tropylium ion) undergoes further
fragmentation by eliminating C H to produce stable simple
7 7
H ) of methyl benzene.
8
8
8
8
8
8
8
7
7
7
7
7
7
7
5.8
Gliclizide prodrug
5.0
4.0
2
2
3.0 3890.5
2.0
5 5
tropylium ion of m/z 65 (C H ) (Fig. 6).
3
789.7
2810.4
861.0
1479.7
779.1
11.6
1.0
0.0
9.0
8.0
7.0
6.0
5.0
4.0
2.8
556.8
541.1
2
8
3
661.0
1061.0
879.2
100
6
0
2
937.4
90
80
70
60
73
6
75.0
1
091.3
1
534.2
2
71
1
311.0
3
268.7
1242.1 1122.1
5
4
3
0
0
0
4000
3000
2000
1500
1000
450
Fig. 4. FT-IR spectrum of oxime prodrug of Gliclizide
83
91
128
2
70
1
1
29
52
2
73
9
7
1
The H NMR of oxime prodrug of gliclizide (DMSO) δ
20
77
181
127
9
8
151
242257
1
0
0
153
135 155
306
1
85
337
in ppm: 1.5(m, 3H, cyclopentane), 2.4 (m, 4H, Pyrrolidine
protons), 2.90 (s, 3H, -CH ), 3.4 (s, 1H, - NOH), 7.18 (d, 1H,
aromatic protons), 7.65 (d, 1H, aromatic protons) (Fig. 5).
274 309
3
100
200
300
400
500
600
Fig. 6. Mass spectra of oxime prodrug of gliclizide
DSC experiments were carried out to study the thermal
behaviour of the synthesised prodrug in relation to the indi-
vidual drug. DSC study of gliclizide shows endothermic peak
at 176.28 °C, (Fig. 7) while DSC study of oxime prodrug shows
sharp endothermic peaks at 266.41 °C (Fig. 8), respectively.
The sharp endothermic values of synthesized prodrug and the
0.00
8
7
6
5
4
3
2
1
0
ppm
–2.00
1
.00 1.03 0.44
1.03 3.31 3.97
3.17
1
Fig. 5. H NMR Spectra of oxime prodrug Gliclizide
–4.00
–6.00
The mass spectrum of Gliclizide represents characteristic
lines at m/z 339 (C15 S) which is parent ion and also
the base peak of gliclizide. Mass spectrum of oxime prodrug
21 3 3
H N O
100
200
Temperature (°C)
300
of gliclizide represents characteristic lines at m/z 337 (M-2
Fig. 7. DSC spectra of gliclizide
19 3 3
parent ion peak, C15H N O S). It has shown three fragmentation
TABLE-1
PARTITION COEFFICIENT VALUES OF DRUG AND PRODRUGS
Compound
Gliclizide
Concentration in octanol (a)
Concentration in water (b)
K (a/b)
332.3
37.4
Log P
2.52
9.97
9.74
0.03
0.26
Oxime prodrug
1.57

6
992 Vijayaraj et al.
Asian J. Chem.
TABLE-2
CHEMICAL HYDROLYSIS STUDY
Hydrolysis (%)
-
1
Compound
pH
1.2
Kobs (min )
t1/2 (min)
81.33
1
5 min
30 min
79.19
45 min
80.81
60 min
81.53
90 min
82.43
120 min
83.60
-
3
Oxime prodrug
78.65
8.52*10
In chemical hydrolysis study, rate of hydrolysis of oxime
prodrug in buffer solutions at 37 °C was found to be 83.6 % at
0.00
1
20 min (Table-2).
ACKNOWLEDGEMENTS
The authors are thankful to IICT, Hyderabad for their
–5.00
–10.00
financial assistance in spectral studies. Thanks are also due to
the Management of SreeVidyanikethan College of Pharmacy,
Tirupathi for providing necessary laboratory facilities to carry
out the work.
50
100
150
Temperature (°C)
200
250
Fig. 8. DSC spectra of oxime prodrug of gliclizide
REFERENCES
individual drug agreed with the measured melting range in
the melting point determination. The thermal profiles of synthe-
sized prodrugs were distinct, with a different melting transition
from that of individual drug. This indicates the formation of
novel prodrug.
1. B. Salim, Int. J. Diabetes Metab., 13, 111 (2005).
2. Pubchem Compound, Gliclizide-Compound Summary (CID 3475)
(
2005).
3
4
.
.
J. Alsenz and M. Kansy, Adv Drug Delivery Rev., 59, 546 (2007).
H. Bundgaard, Design of Prodrug, Elsevier, Amsterdam, Chapter 1,
pp. 3-79 (1985).
Log P value was calculated from partition coefficient studies
and was found to be 2.52 and 1.57 for drug and prodrug,
respectively. Table-1
5. R. Talari, J. Varshosaz, A. Mostafavi and A. Nokhodchi, J. Pharm.
Pharmaceut. Sci., 12, 250 (2009).
6
.
Y. Ozkan, T. Atay, N. Dkmen, A. Isimer and H.Y. Aboul-Enein, Pharm.
Acta Helv., 74, 365 (2000).
7. B.D. Shewale, R.A. Fursule and N.P. Sapkal, Int. J. Health Res., 1, 95
Aqueous solubility of gliclizide and oxime prodrug was
calculated in mg/mL and was found to be 1.012 and 148.7 for
gliclizide and prodrug of gliclizide, respectively (Fig. 9).
(2008).
S. Biswal, J. Sahoo, P.N. Murthy, R.P. Giradkar and J.G. Avari, AAPS
PharmSciTech, 9, 563 (2008).
8
9
.
.
A.B. Shargel and B.C.Andrew,Applied Biopharmaceutics and Pharma-
cokinetics, edn. 4, pp. 47-66, 129-154 (2007).
1
1
0. G.P. Arun, Asian J. Pharm. Clin. Res., 5, 50 (2012).
1. OECD Guidelines for the Testing of Chemicals, Partition Coefficients,
Vol. 107 (1981).
1
50
00
1
12. E. Baka, J.E.A. Comer and K. Takacs-Novak, J. Pharm. Biomed. Anal.,
6, 335 (2008).
3. M.F. Mahdi and H.N. Alsaad, Pharmaceuticals, 5, 1080 (2012).
4
1
50
0
Gliclizide
Prodrug of gliclizide
Fig. 9. Solubility of gliclizide and oxime prodrug of gliclizide