International Standard Serial Number ISS (1)
International Standard Serial Number (ISSN): 2319-8141
International Journal of Universal Pharmacy and Bio Sciences 2(4): July-August 2013
Pharmaceutical Sciences
Research
Article……!!!
Received: 01-08-2013; Accepted: 05-08-2013
DIAZOTIZATION OF CARISOPRODOL AS SKELETAL MUSCLE
RELAXANT BY DERIVATIZATION SPECTROSCOPY
Patwekar Mohamadazhar A. *, Salunkhe V. R.
Rajarambapu College of Pharmacy, Kasegaon, Dist: Sangli (M.S.).
KEYWORDS:
Carisoprodol,
Derivatization,
Diazotization,
Spectroscopy.
For Correspondence:
Mr. Patwekar
MohamadAzhar A.*
Address: Rajarambapu
College of Pharmacy,
Kasegaon-415404,
Maharashtra, India.
Email:
azharp4u@gmail.com
474
ABSTRACT
Carisoprodol is skeletal muscle relaxant. Chemically carisoprodol is
N-isopropyl-2-methyl-2-propyl-1,3-propanediol-dicarbamate.
Carisoprodol doesn’t having chromophores which are essential for
absorption of UV rays and hence not detectable in UV region. UV
method development of drug is most essential analytical technique
which can be used as a basis for chromatographical and
spectroscopical methods. Derivatization spectroscopy can be used for
preparation of new derivatives from original one which can be detect
in UV region. Methods like nitration, sulphonation, methylation,
esterification, acetylation and diazotization was used for formation of
new derivative which can be detected in UV region. Different
reactions of diazotization were used for getting a new and novel
derivative of carisoprodol. Physiochemical properties, TLC, UV, IR
and NMR analysis of carisoprodol and newly obtained derivatives of
carisoprodol was studied and it showed that there was change in color,
odour, taste, melting point, solubility pattern of original drug and
derivatives. UV analysis showed no any absorption of UV rays by
carisoprodol and not detected by UV but other derivatives showed
absorbance for D-1 224nm, D-2 203nm, D-3 206nm and D-4 213nm.
Also IR analysis showed different peaks of carisoprodol in its
fingerprint region with respect to their functional group. While new
derivatives shows extra peaks of “N=N” at 1468cm-1. NMR study
showed peaks for NH2 at 3.39 values while derivatives do not
showed peaks at 3.39 value.
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International Standard Serial Number (ISSN): 2319-8141
INTRODUCTION [1]:
Carisoprodol is a widely used skeletal muscle relaxant and analgesic and is available as a
prescription drug and by blocking interneuronal activity in the descending reticular formation and
spinal cord and as an analgesic; it is available as a prescription drug in 250 mg capsules and 350 mg
tablets, with or without additional analgesics such as aspirin. A single oral dose of 350 mg
carisoprodol, with the onset of action occurring at 0.5 hours and duration of 4 to 6 hours. The peak
serum concentration is 4 to 7 g/mL; the half-life is 8.0 hours. Carisoprodol is rapidly absorbed
from the gastrointestinal tract. A single gavage dose of carisoprodol produced a peak blood
concentration in approximately 30 to 60 minutes in rats and approximately 15 minutes in mice.
DERIVATIZATION SPECTROSCOPY: [2]
Derivatization is the process by which a compound is chemically changed, producing a new
compound that has properties more amenable to a particular analytical method. Compounds that
have poor volatility, poor thermal stability, or that can be adsorbed in the injector will exhibit
nonreproducible peak areas, heights, and shapes. Other compounds that respond poorly on a specific
detector may need to be “tagged” with a different functional group to improve detection. Although
most organic compounds have UV/VIS chromophores, derivatization is still important and
sometimes necessary, for UV/VIS detection in capillary electrophoresis. Very often, UV/VIS
detection of the chromophores does not give a satisfactory response owing to the very short light
path length (50–100µm) in capillary electrophoresis.
DIAZOTIZATION:
The nitrosation of primary aromatic amines with nitrous acid (generated in situ from sodium
nitrite and a strong acid, such as hydrochloric acid, sulfuric acid, or HBF4) leads to diazonium salts,
which can be isolated if the counterion is non-nucleophilic [3]. Diazonium salts are important
intermediates for the preparation of halides (Sandmeyer Reaction, Schiemann Reaction), and azo
compounds. Diazonium salts can react as pseudohalide-type electrophiles, and can therefore be used
in specific protocols for the Heck Reaction or Suzuki Coupling [4]. The intermediates resulting from
the diazotization of primary, aliphatic amines are unstable; they are rapidly converted into
carbocations after loss of nitrogen, and yield products derived from substitution, elimination or
rearrangement processes [5].
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EXPERIMENTAL WORK:
MATERIAL:
Instrument used was an UV/Visible double beam spectrophotometer, SHIMADZU model 1800
(Japan) with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched
quartz cell was used to measure absorbance of all the solutions. An electronic analytical balance was
used for weighing the sample. Melting point taken on thiele’s tube apparatus and are uncorrected.
Thin layer chromatography was used to assess the course of reaction and the purity of the
intermediates and the final compounds.
Chemical:
All the chemicals and solvents were used are of A.R. grade; Carisoprodol was procured as gift
sample from Watson Pharmaceutical, Goa.
METHODS:
1] Diazotization:
D-1 product: [6]
Carisoprodol was placed in a 250 ml round bottom flask fitted with a mechanical stirrer,
thermometer and flask was surrounded by an ice salt bath. The compound was dissolved in 15 ml of
concentrated hydrochloric acid and cooled to 0-5 0C. Sodium nitrite was dissolved in water and
chilled to 0-50C and added slowly to the reaction flask with stirring. The temperature was
maintained between 0-50C. The stirring was continued for 10 minutes more.
D-2 product: [7]
Carisoprodol was dissolved in 5 mL of 2 M hydrochloric acid. The solution was then cooled to 05 C in an ice-bath and maintained at this temperature. Sodium nitrite (5 mmol, 0.345 g) solution in
water (5 mL) was then added dropwise. Stirring was continued for 30 min to produce diazonium
salts at the same temperature.
D-3 product: [8]
200 mg of carisoprodol was placed in a flask. Add 60 mg of anhydrous sodium carbonate and 2-ml
of water. Mixture was warmed gently on the sand bath to dissolve the solids and then, cooled the
solution to room temperature. 85mg of sodium nitrite was added. Stirred to dissolved and placed in
an ice-water bath to cool. While this solution was cooled, 2 gm of ice with six drops of concentrated
hydrochloric acid was mixed in a reaction tube. Poured this mixture into the flask and stirred
thoroughly. The diazonium salt was begun to form within 2-5 minutes.
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International Standard Serial Number (ISSN): 2319-8141
D-4 product [9]:
D-4 product was prepared by the addition of one mole of carisoprodol in three moles of hydrochloric
acid (one mole to form salt with carisoprodol, one mole to liberate nitrous acid, one mole to keep the
reaction mixture acidic) and cooled to 00C in ice bath.
2] Sulphonation: [10]
1gm of carisoprodol was placed in a round bottom flask and 1.85ml of concentrated sulphuric acid
in small portions was added, swirled the mixture gently during the addition and keeped it in cold
water. Supporting the flask in an oil bath and heated the mixture at 180-1900C for about 5 hrs.
Sulphonation was completed when a test portion was completely dissolved by 3-4ml of 2M sodium
hydroxide solution without leaving the solution cloudy. The product was allowed to cool and poured
it with stirring into cold water dryed between sheets of filter paper.
3] Nitration: [11]
1 gm of carisoprodol was dissolved in 1.2 ml of concentrated sulphuric acid in a flask equipped with
a reflux condenser. 0.4 ml of fuming nitric acid was added (caution) flask was shaked well and
cooled in ice water during addition, heat was evolved and a clear yellow solution resulted. Few
fragment of porous porcelain was added and mixture was heated on a water bath to 1000C during 45
minutes. Mixture was maintained at 1000C for 15 minutes with occasional shaking and then it was
transferred to an oil bath at 1000C, raising the temperature to 130-1400C for 1 hour. Flask was
allowed to cool, a crystal was separated at about 900C. When cold, the reaction mixture was poured
into ice water, filtered the separated crystals, washed with water and dryed.
4] Methylation: [12]
In round bottom flask mixture of 1 gm of carisoprodol, 2 ml of absolute methanol and 0.050 ml of
concentrated sulphuric acid was placed. A small chip of porcelain was added, reflux condenser was
attached and mixture was boiled for four hours. Excess of alcohol was distilled off on water bath and
allowed to cool. Residue was poured into water containing separatory funnel and flask was rinsed
with a few ml of water which was also poured into the separatory funnel. 10-15 ml of carbon
tetrachloride was added and mixed with mixture in the funnel, upon standing the heavy solution of
methyl benzoate in the carbon tetrachloride separated by separatory funnel. Upper aqueous was
rejected, returned the methyl benzoate to the funnel and shaked it with a strong solution of sodium
hydrogen carbonate until all free acid was removed and no further evolution of carbon dioxide
occurred. Washed once with water and dried.
5] Esterification [13]:
100 mg of carisoprodol was added in 3 ml boron trifluoride in methanol or boron trifluoride in nbutanol was added and heated to 600C for 5 to 10 minutes. Heating temperature and time may vary
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International Standard Serial Number (ISSN): 2319-8141
i.e, used 900C for 10 minutes for the process. This was followed by cooling and transferring the
mixture to a separating funnel with 25 ml hexane. Further, the sample was washed two times with
saturated sodium chloride solution, and then dried over anhydrous sodium thiosulphide.
EVALUATION OF PRODUCTS:
Carisoprodol as original drug and the derivatives obtained through reactions have been evaluated by
physiochemical characteristic, TLC, UV spectral analysis, IR studies and NMR interpretation. Color
was observed visually while taste was evaluated organoleptically and solubility behavior of all
derivatives was studied using different solvent.
Melting point determination:
A drug was placed in the closed end of the capillary tube by tapping it on the table. Capillary tube
was attached to a thermometer. Tube was placed in thiele’s tube assembly. Carefully heated the
thiele’s tube. Temperature was noted at which drug melted.
Thin layer chromatography:
Thin layer chromatography was done by using glass plate. Plate was activated in hot air oven at
1100C for 30 min. Different mobile phase was used. Application of sample was done by capillary
tubes. Plate was placed 450 in chromatographic tank. Spot was identified by placing in iodine
chamber. Evaluation was done by quantitative method.
Mobile phase: Acetonitrile: Pyridine (5:5)
Fig 1: TLC OF D-1 PRODUCT
478
Mobile phase: Acetonitrile: Pyridine (8:2)
Fig 2: TLC OF D-2 PRODUCT
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Mobile phase: Acetonitrile: Toluene (5:5)
Mobile phase: Acetonitrile: n-hexane (6:4)
Fig 3: TLC OF D-3 PRODUCT
Fig 4: TLC OF D-4 PRODUCT
Ultraviolet (UV) spectrophotometer determination:
Carisoprodol and its derivatives (D-1 to D-4) were analyzed by UV spectrophotometer SHIMADZU
model 1800 (Japan). The stock solution of derivative was obtained diluted and detected by UV
spectrum. The different sample were runned from 200-400nm for determination of max. Different
concentration of sample was analyzed by using methanol as solvent that obeyed Lambert’s-Beer
law.
Fig 5: UV OF CARISOPRODOL
Fig 7: UV OF D-2 PRODUCT
479
Fig 6: UV OF D-1 PRODUCT
Fig 8: UV OF D-3 PRODUCT
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Fig 9: UV OF D-4 PRODUCT
Infrared (IR) determination:
IR for carisoprodol and its derivatives was carried out in Appasaheb Birnale College of Pharmacy;
Sangli (M.S.) The infrared spectrum was recorded by passing a beam of infrared light through the
sample. Examination of the transmitted light reveals how much energy was absorbed at each
frequency. This can be achieved by scanning the wavelength range using a monochromator and then
a transmittance or absorbance spectrum is generated. Analysis of intensity of peaks in this spectrum
reveals details about the molecular structure of the sample.
480
Fig 10: IR OF CARISOPRODOL
Fig 11: IR OF D-1 PRODUCT
Fig 12: IR OF D-2 PRODUCT
Fig 13: IR OF D-3 PRODUCT
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International Standard Serial Number (ISSN): 2319-8141
Fig 14: IR OF D-4 PRODUCT
Table 1: IR spectrum of carisoprodol and its derivatives.
Sr. No.
Peak No.
IR spectrum of Carisoprodol :
1
5
2
7
3
8
4
9
IR Spectrum D -1 Product:
1
2
2
6
3
9
4
10
5
12
IR Spectrum D -2 Product:
1
1
2
5
3
8
4
9
5
11
IR Spectrum D -3 Product:
1
9
2
13
3
16
4
17
5
20
IR Spectrum D -4 Product:
1
1
2
5
3
7
4
8
5
11
481
Value (cm-1)
Indicator
3450
2966
1698
1606
N-H
O-H
C=N
C=O
3450
2966
1698
1606
1468
N-H
O-H
C=N
C=O
N=N
3451
2966
1696
1606
1469
N-H
O-H
C=N
C=O
N=N
3450
2966
1692
1606
1468
N-H
O-H
C=N
C=O
N=N
3450
2966
1698
1606
1468
N-H
O-H
C=N
C=O
N=N
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Nuclear magnetic resonances (NMR) determination:
NMR for carisoprodol and its derivatives was carried out in IIT, Powai, Mumbai. NMR was
recorded by preparing the sample in NMR tube. Spectrometer was switch on. In PNMR, deuterium
oxide sample was runned to optimized spin rate and shim magnet. Shim and gain was adjusted to
sample. Field offset frequency was set up so tetra methyl saline was at 0 PPM. Data for NMR
sample was collected, 1H or 13C. Base line correction (BC) was applied and 1H NMR spectrum was
integrated and assigned integrated value. Peaks were picked and chemical shift was labeled and
spectrum was plotted.
Fig 15: NMR OF CARISOPRODOL
Fig 17: NMR OF D-2 PRODUCT
Fig 16: NMR OF D-1 PRODUCT
Fig 18: NMR OF D-3 PRODUCT
Fig 19: NMR OF D-4 PRODUCT
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Table 2: NMR signals of carisoprodol and its derivatives.
Sr. No.
Value ( ppm)
Indicator
NMR spectrum of Carisoprodol:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.39
s, 2 H, NH2
6
3.59
m, 1H, NH
7
3.79
d, 4 H, CH2
NMR spectrum of D-1 product:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.59
m, 1H, NH
6
3.79
d, 4 H, CH2
NMR spectrum of D-2 product:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.59
m, 1H, NH
6
3.79
d, 4 H, CH2
NMR spectrum of D-3 product:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.59
m, 1H, NH
6
3.79
d, 4 H, CH2
NMR spectrum of D-4 product:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.59
m, 1H, NH
6
3.79
d, 4 H, CH2
RESULTS AND DISCUSSION:
Derivatization of Carisoprodol was done by using different diazotization methods. Physiochemical
property of diazotization product formed where compared with carisoprodol. The color of
carisoprodol is white where as D-1 is pale brown, D-2 and D-3 is white and D-4 is pale green, which
showed that there is change in color of derivatized product as compared to carisoprodol. The odour
of carisoprodol is odourless and the entire derivatized product is odourless. Taste of carisoprodol is
bitter while D-1, D-3 and D-4 are tasteless and D-2 is bitter in taste, this also showed that there is
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change in taste as compared to carisoprodol. Carisoprodol and all derivatized product are soluble in
alcohol. Melting point of carisoprodol is 92-930C where as D-1 it was 80-820C, D-2 74-750C, D-3
116-1180C and D-4 it is 104-1050C, which showed that there is change in melting point of
derivatized products compared to carisoprodol. Rf value of carisoprodol is 0.78 whereas of D-1 is
0.91, D-2 is 0.72, D-3 is 0.82 and D-4 is 0.88 [Fig.1-4] which confirms that there is changes in
derivatized products obtained by diazotization methods.
Carisoprodol does not showed wavelength in UV spectrum, while the derivatized products produced
showed different absorbances at different wavelength, it might be possible due to addition of
chromophores by diazotization method. The wavelength of D-1 product was found to be 224 nm, D2 203 nm, D-3 206 nm and D-4 213 nm in UV spectrum using methanol as solvent[Fig.5-9]. IR
analysis of carisoprodol was done, showing different peaks with respect to their functional groups.
While new derivatized products i.e. D1, D2, D3 and, D4 showed extra peak of “N=N” at 1468 cm-1
[Fig.10-14] which confirms that there was changes in derivatized products obtained by diazotization
methods. NMR analysis of carisoprodol was done, showing
(ppm) value at 3.39 for NH2 group
while new derivatized product i.e. D1, D2, D3 and D4 does not showed the signal at 3.39 [Fig.1519] which confirms that there was changes in derivatized products obtained by diazotization
methods.
Derivatization of carisoprodol was done by sulphonation but the reaction was failed and no findings
were obtained. Also the derivatization of carisoprodol was also done by methylation, nitration,
esterification but the result obtained was not as effective as diazotization method.
Table 3: Results of evaluation parameter of carisoprodol and its derivatized product.
Drug.
Parameter
Color
Odour
Taste
Solubility
Melting point
TLC (Rf
value)
UV ( max)
IR (cm-1)
N=N
NMR (No. of
proton)
484
Carisoprodol
D-1
D-2
D-3
D-4
White
Odourless
Bitter
Alcohol
92-930C
0.78
Pale brown
Odourless
Tasteless
Alcohol
80-820C
0.91
White
Odourless
Bitter
Alcohol
74-750C
0.72
White
Odourless
Tasteless
Alcohol
116-1180C
0.82
Pale green
Odourless
Tasteless
Alcohol
104-1050C
0.88
-----
224
1468
203
1469
206
1468
213
1468
24
22
22
22
22
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CONCLUSION:
Derivatization offer an excellent method in producing a product which can be used to overcome the
analytical problem occurs during the analysis of drug. Derivatization proves advantageous for many
polar compounds and samples that are not suitable for chromatographic analysis due to their
physical and chemical properties. Derivatization promises to be a potential approach for many
compounds. So this study highlights the need of derivatization for the compounds which shows
problems in analysis. Derivatization of carisoprodol was conducted by using diazotization,
sulphonation, nitration, and methylation and esterification process. Some methods of derivatization
were conducted but diazotization was preferred for formation of derivatization. Physiochemical
properties, TLC, UV spectral analysis, IR and NMR analysis of carisoprodol and derivatized product
obtained of carisoprodol was studied and it was found there was change in color, odour, taste,
melting point, TLC, UV spectrum, IR and NMR analysis pattern of original drug and derivatized
product. So this study highlights that diazotization is most suitable methods for derivatization of
carisoprodol.
ACKNOWLEDGMENTS:
Authors are thankful for Watson Pharmaceutical, Goa for providing the carisoprodol as a gift
sample. We are also thankful to Dr.S.K.Mohite for their kind cooperation to carry out the present
research work.
REFERENCES:
1. Franz DN, (1975), Drugs for Parkinson’s disease; centrally acting muscle relaxants. In The
Pharmacological Basis of Therapeutics (L.S. Goodman and A. Gilman, Eds.), 5th ed., New
York: MacMillan Publishing Company; 227-244.
2. Neil D. Danielson, (2000), Chemical reagent and derivatives procedure in Drug Analysis.
Encyclopedia of Analytical Chemistry, 7042-7076.
3. Backett AH, Stenlake JB. Practical Pharmaceutical Chemistry. Vol. II, 4th ed.,144, 300-301.
4. Furniss BS, Smith PWG., (2005), Vogel textbook of practical organic chemistry. 5th ed.,
1077.
5. Arun Bahl, Bahl BS.,(2010), Advanced Organic Chemistry. 1st ed., 1255-1256.
6. Bapu.R.Thorat, Ramesh V. Nawathye, (2012), Synthesis of Diazo compound from amino
benzopyrano thiazoles. IJCSA ISSN-0976-2590; 3:356-365.
7. Bullent Kirkan and Ramazan Gup, (2008), Synthesis of New Azo Dyes and Copper
Complexes Derived from Barbituric Acid and 4-Aminobenzoylhydrazone.Turk J Chem. 32;
9-17.
8. Jag Mohan, (2003), Organic Analytical Chemistry Theory and Practice, 498-499.
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International Standard Serial Number (ISSN): 2319-8141
9. Arun Bahl and Bahl BS., (2010), Advanced Organic Chemistry. 1st ed., 1031.
10. Furniss BS., (2005),Vogel textbook of practical organic chemistry. 5th ed., 912.
11. Furniss BS, Hannaford AJ., (2005), Vogel textbook of practical organic chemistry. 5th
ed.,1072.
12. Furniss BS, Hannaford AJ., (2005), Vogel textbook of practical organic chemistry.5th
ed.,1077.
13. Francis Orata., (2012), Derivatization Reactions and Reagents for Gas Chromatography
Analysis. 83-108.
486
Full Text Available On www.ijupbs.com
International Journal of Universal Pharmacy and Bio Sciences 2(4): July-August 2013
Pharmaceutical Sciences
Research
Article……!!!
Received: 01-08-2013; Accepted: 05-08-2013
DIAZOTIZATION OF CARISOPRODOL AS SKELETAL MUSCLE
RELAXANT BY DERIVATIZATION SPECTROSCOPY
Patwekar Mohamadazhar A. *, Salunkhe V. R.
Rajarambapu College of Pharmacy, Kasegaon, Dist: Sangli (M.S.).
KEYWORDS:
Carisoprodol,
Derivatization,
Diazotization,
Spectroscopy.
For Correspondence:
Mr. Patwekar
MohamadAzhar A.*
Address: Rajarambapu
College of Pharmacy,
Kasegaon-415404,
Maharashtra, India.
Email:
azharp4u@gmail.com
474
ABSTRACT
Carisoprodol is skeletal muscle relaxant. Chemically carisoprodol is
N-isopropyl-2-methyl-2-propyl-1,3-propanediol-dicarbamate.
Carisoprodol doesn’t having chromophores which are essential for
absorption of UV rays and hence not detectable in UV region. UV
method development of drug is most essential analytical technique
which can be used as a basis for chromatographical and
spectroscopical methods. Derivatization spectroscopy can be used for
preparation of new derivatives from original one which can be detect
in UV region. Methods like nitration, sulphonation, methylation,
esterification, acetylation and diazotization was used for formation of
new derivative which can be detected in UV region. Different
reactions of diazotization were used for getting a new and novel
derivative of carisoprodol. Physiochemical properties, TLC, UV, IR
and NMR analysis of carisoprodol and newly obtained derivatives of
carisoprodol was studied and it showed that there was change in color,
odour, taste, melting point, solubility pattern of original drug and
derivatives. UV analysis showed no any absorption of UV rays by
carisoprodol and not detected by UV but other derivatives showed
absorbance for D-1 224nm, D-2 203nm, D-3 206nm and D-4 213nm.
Also IR analysis showed different peaks of carisoprodol in its
fingerprint region with respect to their functional group. While new
derivatives shows extra peaks of “N=N” at 1468cm-1. NMR study
showed peaks for NH2 at 3.39 values while derivatives do not
showed peaks at 3.39 value.
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International Standard Serial Number (ISSN): 2319-8141
INTRODUCTION [1]:
Carisoprodol is a widely used skeletal muscle relaxant and analgesic and is available as a
prescription drug and by blocking interneuronal activity in the descending reticular formation and
spinal cord and as an analgesic; it is available as a prescription drug in 250 mg capsules and 350 mg
tablets, with or without additional analgesics such as aspirin. A single oral dose of 350 mg
carisoprodol, with the onset of action occurring at 0.5 hours and duration of 4 to 6 hours. The peak
serum concentration is 4 to 7 g/mL; the half-life is 8.0 hours. Carisoprodol is rapidly absorbed
from the gastrointestinal tract. A single gavage dose of carisoprodol produced a peak blood
concentration in approximately 30 to 60 minutes in rats and approximately 15 minutes in mice.
DERIVATIZATION SPECTROSCOPY: [2]
Derivatization is the process by which a compound is chemically changed, producing a new
compound that has properties more amenable to a particular analytical method. Compounds that
have poor volatility, poor thermal stability, or that can be adsorbed in the injector will exhibit
nonreproducible peak areas, heights, and shapes. Other compounds that respond poorly on a specific
detector may need to be “tagged” with a different functional group to improve detection. Although
most organic compounds have UV/VIS chromophores, derivatization is still important and
sometimes necessary, for UV/VIS detection in capillary electrophoresis. Very often, UV/VIS
detection of the chromophores does not give a satisfactory response owing to the very short light
path length (50–100µm) in capillary electrophoresis.
DIAZOTIZATION:
The nitrosation of primary aromatic amines with nitrous acid (generated in situ from sodium
nitrite and a strong acid, such as hydrochloric acid, sulfuric acid, or HBF4) leads to diazonium salts,
which can be isolated if the counterion is non-nucleophilic [3]. Diazonium salts are important
intermediates for the preparation of halides (Sandmeyer Reaction, Schiemann Reaction), and azo
compounds. Diazonium salts can react as pseudohalide-type electrophiles, and can therefore be used
in specific protocols for the Heck Reaction or Suzuki Coupling [4]. The intermediates resulting from
the diazotization of primary, aliphatic amines are unstable; they are rapidly converted into
carbocations after loss of nitrogen, and yield products derived from substitution, elimination or
rearrangement processes [5].
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EXPERIMENTAL WORK:
MATERIAL:
Instrument used was an UV/Visible double beam spectrophotometer, SHIMADZU model 1800
(Japan) with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched
quartz cell was used to measure absorbance of all the solutions. An electronic analytical balance was
used for weighing the sample. Melting point taken on thiele’s tube apparatus and are uncorrected.
Thin layer chromatography was used to assess the course of reaction and the purity of the
intermediates and the final compounds.
Chemical:
All the chemicals and solvents were used are of A.R. grade; Carisoprodol was procured as gift
sample from Watson Pharmaceutical, Goa.
METHODS:
1] Diazotization:
D-1 product: [6]
Carisoprodol was placed in a 250 ml round bottom flask fitted with a mechanical stirrer,
thermometer and flask was surrounded by an ice salt bath. The compound was dissolved in 15 ml of
concentrated hydrochloric acid and cooled to 0-5 0C. Sodium nitrite was dissolved in water and
chilled to 0-50C and added slowly to the reaction flask with stirring. The temperature was
maintained between 0-50C. The stirring was continued for 10 minutes more.
D-2 product: [7]
Carisoprodol was dissolved in 5 mL of 2 M hydrochloric acid. The solution was then cooled to 05 C in an ice-bath and maintained at this temperature. Sodium nitrite (5 mmol, 0.345 g) solution in
water (5 mL) was then added dropwise. Stirring was continued for 30 min to produce diazonium
salts at the same temperature.
D-3 product: [8]
200 mg of carisoprodol was placed in a flask. Add 60 mg of anhydrous sodium carbonate and 2-ml
of water. Mixture was warmed gently on the sand bath to dissolve the solids and then, cooled the
solution to room temperature. 85mg of sodium nitrite was added. Stirred to dissolved and placed in
an ice-water bath to cool. While this solution was cooled, 2 gm of ice with six drops of concentrated
hydrochloric acid was mixed in a reaction tube. Poured this mixture into the flask and stirred
thoroughly. The diazonium salt was begun to form within 2-5 minutes.
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D-4 product [9]:
D-4 product was prepared by the addition of one mole of carisoprodol in three moles of hydrochloric
acid (one mole to form salt with carisoprodol, one mole to liberate nitrous acid, one mole to keep the
reaction mixture acidic) and cooled to 00C in ice bath.
2] Sulphonation: [10]
1gm of carisoprodol was placed in a round bottom flask and 1.85ml of concentrated sulphuric acid
in small portions was added, swirled the mixture gently during the addition and keeped it in cold
water. Supporting the flask in an oil bath and heated the mixture at 180-1900C for about 5 hrs.
Sulphonation was completed when a test portion was completely dissolved by 3-4ml of 2M sodium
hydroxide solution without leaving the solution cloudy. The product was allowed to cool and poured
it with stirring into cold water dryed between sheets of filter paper.
3] Nitration: [11]
1 gm of carisoprodol was dissolved in 1.2 ml of concentrated sulphuric acid in a flask equipped with
a reflux condenser. 0.4 ml of fuming nitric acid was added (caution) flask was shaked well and
cooled in ice water during addition, heat was evolved and a clear yellow solution resulted. Few
fragment of porous porcelain was added and mixture was heated on a water bath to 1000C during 45
minutes. Mixture was maintained at 1000C for 15 minutes with occasional shaking and then it was
transferred to an oil bath at 1000C, raising the temperature to 130-1400C for 1 hour. Flask was
allowed to cool, a crystal was separated at about 900C. When cold, the reaction mixture was poured
into ice water, filtered the separated crystals, washed with water and dryed.
4] Methylation: [12]
In round bottom flask mixture of 1 gm of carisoprodol, 2 ml of absolute methanol and 0.050 ml of
concentrated sulphuric acid was placed. A small chip of porcelain was added, reflux condenser was
attached and mixture was boiled for four hours. Excess of alcohol was distilled off on water bath and
allowed to cool. Residue was poured into water containing separatory funnel and flask was rinsed
with a few ml of water which was also poured into the separatory funnel. 10-15 ml of carbon
tetrachloride was added and mixed with mixture in the funnel, upon standing the heavy solution of
methyl benzoate in the carbon tetrachloride separated by separatory funnel. Upper aqueous was
rejected, returned the methyl benzoate to the funnel and shaked it with a strong solution of sodium
hydrogen carbonate until all free acid was removed and no further evolution of carbon dioxide
occurred. Washed once with water and dried.
5] Esterification [13]:
100 mg of carisoprodol was added in 3 ml boron trifluoride in methanol or boron trifluoride in nbutanol was added and heated to 600C for 5 to 10 minutes. Heating temperature and time may vary
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i.e, used 900C for 10 minutes for the process. This was followed by cooling and transferring the
mixture to a separating funnel with 25 ml hexane. Further, the sample was washed two times with
saturated sodium chloride solution, and then dried over anhydrous sodium thiosulphide.
EVALUATION OF PRODUCTS:
Carisoprodol as original drug and the derivatives obtained through reactions have been evaluated by
physiochemical characteristic, TLC, UV spectral analysis, IR studies and NMR interpretation. Color
was observed visually while taste was evaluated organoleptically and solubility behavior of all
derivatives was studied using different solvent.
Melting point determination:
A drug was placed in the closed end of the capillary tube by tapping it on the table. Capillary tube
was attached to a thermometer. Tube was placed in thiele’s tube assembly. Carefully heated the
thiele’s tube. Temperature was noted at which drug melted.
Thin layer chromatography:
Thin layer chromatography was done by using glass plate. Plate was activated in hot air oven at
1100C for 30 min. Different mobile phase was used. Application of sample was done by capillary
tubes. Plate was placed 450 in chromatographic tank. Spot was identified by placing in iodine
chamber. Evaluation was done by quantitative method.
Mobile phase: Acetonitrile: Pyridine (5:5)
Fig 1: TLC OF D-1 PRODUCT
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Mobile phase: Acetonitrile: Pyridine (8:2)
Fig 2: TLC OF D-2 PRODUCT
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Mobile phase: Acetonitrile: Toluene (5:5)
Mobile phase: Acetonitrile: n-hexane (6:4)
Fig 3: TLC OF D-3 PRODUCT
Fig 4: TLC OF D-4 PRODUCT
Ultraviolet (UV) spectrophotometer determination:
Carisoprodol and its derivatives (D-1 to D-4) were analyzed by UV spectrophotometer SHIMADZU
model 1800 (Japan). The stock solution of derivative was obtained diluted and detected by UV
spectrum. The different sample were runned from 200-400nm for determination of max. Different
concentration of sample was analyzed by using methanol as solvent that obeyed Lambert’s-Beer
law.
Fig 5: UV OF CARISOPRODOL
Fig 7: UV OF D-2 PRODUCT
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Fig 6: UV OF D-1 PRODUCT
Fig 8: UV OF D-3 PRODUCT
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Fig 9: UV OF D-4 PRODUCT
Infrared (IR) determination:
IR for carisoprodol and its derivatives was carried out in Appasaheb Birnale College of Pharmacy;
Sangli (M.S.) The infrared spectrum was recorded by passing a beam of infrared light through the
sample. Examination of the transmitted light reveals how much energy was absorbed at each
frequency. This can be achieved by scanning the wavelength range using a monochromator and then
a transmittance or absorbance spectrum is generated. Analysis of intensity of peaks in this spectrum
reveals details about the molecular structure of the sample.
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Fig 10: IR OF CARISOPRODOL
Fig 11: IR OF D-1 PRODUCT
Fig 12: IR OF D-2 PRODUCT
Fig 13: IR OF D-3 PRODUCT
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Fig 14: IR OF D-4 PRODUCT
Table 1: IR spectrum of carisoprodol and its derivatives.
Sr. No.
Peak No.
IR spectrum of Carisoprodol :
1
5
2
7
3
8
4
9
IR Spectrum D -1 Product:
1
2
2
6
3
9
4
10
5
12
IR Spectrum D -2 Product:
1
1
2
5
3
8
4
9
5
11
IR Spectrum D -3 Product:
1
9
2
13
3
16
4
17
5
20
IR Spectrum D -4 Product:
1
1
2
5
3
7
4
8
5
11
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Value (cm-1)
Indicator
3450
2966
1698
1606
N-H
O-H
C=N
C=O
3450
2966
1698
1606
1468
N-H
O-H
C=N
C=O
N=N
3451
2966
1696
1606
1469
N-H
O-H
C=N
C=O
N=N
3450
2966
1692
1606
1468
N-H
O-H
C=N
C=O
N=N
3450
2966
1698
1606
1468
N-H
O-H
C=N
C=O
N=N
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Nuclear magnetic resonances (NMR) determination:
NMR for carisoprodol and its derivatives was carried out in IIT, Powai, Mumbai. NMR was
recorded by preparing the sample in NMR tube. Spectrometer was switch on. In PNMR, deuterium
oxide sample was runned to optimized spin rate and shim magnet. Shim and gain was adjusted to
sample. Field offset frequency was set up so tetra methyl saline was at 0 PPM. Data for NMR
sample was collected, 1H or 13C. Base line correction (BC) was applied and 1H NMR spectrum was
integrated and assigned integrated value. Peaks were picked and chemical shift was labeled and
spectrum was plotted.
Fig 15: NMR OF CARISOPRODOL
Fig 17: NMR OF D-2 PRODUCT
Fig 16: NMR OF D-1 PRODUCT
Fig 18: NMR OF D-3 PRODUCT
Fig 19: NMR OF D-4 PRODUCT
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Table 2: NMR signals of carisoprodol and its derivatives.
Sr. No.
Value ( ppm)
Indicator
NMR spectrum of Carisoprodol:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.39
s, 2 H, NH2
6
3.59
m, 1H, NH
7
3.79
d, 4 H, CH2
NMR spectrum of D-1 product:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.59
m, 1H, NH
6
3.79
d, 4 H, CH2
NMR spectrum of D-2 product:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.59
m, 1H, NH
6
3.79
d, 4 H, CH2
NMR spectrum of D-3 product:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.59
m, 1H, NH
6
3.79
d, 4 H, CH2
NMR spectrum of D-4 product:
1
0.9
s, 6 H, CH3
2
1.01
d, 6 H, CH3
3
1.23
s, 4 H, CH2
4
2.50
s, 1 H, CH
5
3.59
m, 1H, NH
6
3.79
d, 4 H, CH2
RESULTS AND DISCUSSION:
Derivatization of Carisoprodol was done by using different diazotization methods. Physiochemical
property of diazotization product formed where compared with carisoprodol. The color of
carisoprodol is white where as D-1 is pale brown, D-2 and D-3 is white and D-4 is pale green, which
showed that there is change in color of derivatized product as compared to carisoprodol. The odour
of carisoprodol is odourless and the entire derivatized product is odourless. Taste of carisoprodol is
bitter while D-1, D-3 and D-4 are tasteless and D-2 is bitter in taste, this also showed that there is
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change in taste as compared to carisoprodol. Carisoprodol and all derivatized product are soluble in
alcohol. Melting point of carisoprodol is 92-930C where as D-1 it was 80-820C, D-2 74-750C, D-3
116-1180C and D-4 it is 104-1050C, which showed that there is change in melting point of
derivatized products compared to carisoprodol. Rf value of carisoprodol is 0.78 whereas of D-1 is
0.91, D-2 is 0.72, D-3 is 0.82 and D-4 is 0.88 [Fig.1-4] which confirms that there is changes in
derivatized products obtained by diazotization methods.
Carisoprodol does not showed wavelength in UV spectrum, while the derivatized products produced
showed different absorbances at different wavelength, it might be possible due to addition of
chromophores by diazotization method. The wavelength of D-1 product was found to be 224 nm, D2 203 nm, D-3 206 nm and D-4 213 nm in UV spectrum using methanol as solvent[Fig.5-9]. IR
analysis of carisoprodol was done, showing different peaks with respect to their functional groups.
While new derivatized products i.e. D1, D2, D3 and, D4 showed extra peak of “N=N” at 1468 cm-1
[Fig.10-14] which confirms that there was changes in derivatized products obtained by diazotization
methods. NMR analysis of carisoprodol was done, showing
(ppm) value at 3.39 for NH2 group
while new derivatized product i.e. D1, D2, D3 and D4 does not showed the signal at 3.39 [Fig.1519] which confirms that there was changes in derivatized products obtained by diazotization
methods.
Derivatization of carisoprodol was done by sulphonation but the reaction was failed and no findings
were obtained. Also the derivatization of carisoprodol was also done by methylation, nitration,
esterification but the result obtained was not as effective as diazotization method.
Table 3: Results of evaluation parameter of carisoprodol and its derivatized product.
Drug.
Parameter
Color
Odour
Taste
Solubility
Melting point
TLC (Rf
value)
UV ( max)
IR (cm-1)
N=N
NMR (No. of
proton)
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Carisoprodol
D-1
D-2
D-3
D-4
White
Odourless
Bitter
Alcohol
92-930C
0.78
Pale brown
Odourless
Tasteless
Alcohol
80-820C
0.91
White
Odourless
Bitter
Alcohol
74-750C
0.72
White
Odourless
Tasteless
Alcohol
116-1180C
0.82
Pale green
Odourless
Tasteless
Alcohol
104-1050C
0.88
-----
224
1468
203
1469
206
1468
213
1468
24
22
22
22
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CONCLUSION:
Derivatization offer an excellent method in producing a product which can be used to overcome the
analytical problem occurs during the analysis of drug. Derivatization proves advantageous for many
polar compounds and samples that are not suitable for chromatographic analysis due to their
physical and chemical properties. Derivatization promises to be a potential approach for many
compounds. So this study highlights the need of derivatization for the compounds which shows
problems in analysis. Derivatization of carisoprodol was conducted by using diazotization,
sulphonation, nitration, and methylation and esterification process. Some methods of derivatization
were conducted but diazotization was preferred for formation of derivatization. Physiochemical
properties, TLC, UV spectral analysis, IR and NMR analysis of carisoprodol and derivatized product
obtained of carisoprodol was studied and it was found there was change in color, odour, taste,
melting point, TLC, UV spectrum, IR and NMR analysis pattern of original drug and derivatized
product. So this study highlights that diazotization is most suitable methods for derivatization of
carisoprodol.
ACKNOWLEDGMENTS:
Authors are thankful for Watson Pharmaceutical, Goa for providing the carisoprodol as a gift
sample. We are also thankful to Dr.S.K.Mohite for their kind cooperation to carry out the present
research work.
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