International Standard Serial Number (ISSN): 2319-8141 .... RPA1300162.pdf · methyl benzoate in the carbon tetrachloride separated by separatory funnel. Upper aqueous was rejected,

International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 2(4): July-August 2013

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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:

[email protected]

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 δ values while derivatives do not

showed peaks at 3.39 δ value.

474 Full Text Available On www.ijupbs.com

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 µg/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].



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 0-

5◦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.



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 130-140

0C 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 n-

butanol was added and heated to 600C for 5 to 10 minutes. Heating temperature and time may vary



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) Mobile phase: Acetonitrile: Pyridine (8:2)

Fig 1: TLC OF D-1 PRODUCT Fig 2: TLC OF D-2 PRODUCT



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 λmax. Different

concentration of sample was analyzed by using methanol as solvent that obeyed Lambert’s-Beer

law.

Fig 5: UV OF CARISOPRODOL Fig 6: UV OF D-1 PRODUCT

Fig 7: UV OF D-2 PRODUCT Fig 8: UV OF D-3 PRODUCT



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.

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



Fig 14: IR OF D-4 PRODUCT

Table 1: IR spectrum of carisoprodol and its derivatives.

Sr. No.

Peak No.

Value (cm-1

)

Indicator

IR spectrum of Carisoprodol :

1 5 3450 N-H

2 7 2966 O-H

3 8 1698 C=N

4 9 1606 C=O

IR Spectrum D -1 Product:

1 2 3450 N-H

2 6 2966 O-H

3 9 1698 C=N

4 10 1606 C=O

5 12 1468 N=N


1 1 3451 N-H

2 5 2966 O-H

3 8 1696 C=N

4 9 1606 C=O

5 11 1469 N=N


1 9 3450 N-H

2 13 2966 O-H

3 16 1692 C=N

4 17 1606 C=O

5 20 1468 N=N


1 1 3450 N-H

2 5 2966 O-H

3 7 1698 C=N

4 8 1606 C=O

5 11 1468 N=N



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 16: NMR OF D-1 PRODUCT

Fig 17: NMR OF D-2 PRODUCT Fig 18: NMR OF D-3 PRODUCT

Fig 19: NMR OF D-4 PRODUCT



Table 2: NMR signals of carisoprodol and its derivatives.

Sr. No. Value (δ 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


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


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


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



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 80-82

0C, D-2 74-75

0C, D-3

116-1180C and D-4 it is 104-105

0C, 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, D-

2 203 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 δ (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.15-

19] 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

Carisoprodol D-1 D-2 D-3 D-4

Color White Pale brown White White Pale green

Odour Odourless Odourless Odourless Odourless Odourless

Taste Bitter Tasteless Bitter Tasteless Tasteless

Solubility Alcohol Alcohol Alcohol Alcohol Alcohol

Melting point 92-930C 80-82

0C 74-75

0C 116-118

0C 104-105

0C

TLC (Rf

value)

0.78 0.91 0.72 0.82 0.88

UV ( λ max) --- 224 203 206 213

IR (cm-1

)

N=N

--- 1468 1469 1468 1468

NMR (No. of

proton)

24 22 22 22 22



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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International Standard Serial Number (ISSN): 2319-8141 .... RPA1300162.pdf · methyl benzoate in the carbon tetrachloride separated by separatory funnel. Upper aqueous was rejected,