Synthesis And Evaluation of Mutual Prodrugs of Ibuprofen as Non- Steroidal Anti-Inflammatory Drugs with Antioxidants

The concept of “mutual prodrug” is relatively in medicinal chemistry, pharmaceutics and drug delivery. Mutual prodrug is from if prodrug in which two pharmacologically active agents are attached to each other in a way that each drug acts as a carrier for each other^{. [1,2]}.

Non-steroidal anti-inflammatory drugs (NSAIDs):

These are the mostly used drugs. According the public survey their side effects such as nephrotoxicity, haemorrhages and the most serious GIT ulceration^.[3] These side effects produced due to carboxylic acid group (-COOH) in the presence of the structure which are significantly ionized at physiological pH of stomach. The local generation of various ‘reactive oxygen species’ (ROS) play a significant role in the formation of gastric ulceration associated with NSAIDs therapy. These observations indicate that antioxidants may be used to prevent NSAIDs induced gastric ulcers.^[3]

All NSAIDs are believed to inhibit the biosynthesis of prostaglandins by inhibiting the group of enzymes called cyclooxygenases that is COX-I and COX-II. The COX-I enzyme is located in normal tissues and is cytoprotective, physiologically important for GI and renal functions. On the other hand, COX-II is pathological, found primarily in inflamed tissues. The NSAIDs cause inhibition of both the isoforms, producing GI and renal side effects due to inhibition of COX-I. while selective inhibition of COX-II could block the prostaglandins production at the side of inflammation without affecting the beneficial prostaglandins tissues in normal tissues such as stomach and kidneys.^[4]

Prodrugs: Drug design involves the development of analogues and prodrugs by some chemical modifications from the lead molecule or from a parent compound by modifying the carbon skeletal transformation or by the synthesis of compounds of the same nucleus with various substitutions. Analogues can also be synthesized by changing the position of substitution group.^[5]

For example: synthesis of trans-diethylstilbesterol by the modification of oestradiol produced better oestrogenic activity than the latter one^.[6]

Mutual Prodrug

Mutual prodrug, where the carrier used is another biologically active drug instead of some inert molecule. A mutual prodrug consists of two pharmacologically active agents coupled together so that each act as a promoiety for the other agent and vice versa. The carrier selected may have the same biological action as that of the parent drug and thus might give synergistic action, or the carrier may have some additional biological action that is lacking in the parent drug, thus ensuring some additional benefit. The carrier may also be a drug that might help to target the parent drug to a specific site or organ or cells or may improve site specificity of a drug. The carrier drug may be used to overcome some side effects of the parent drugs as well.^[7] ex. Release of chlorzoxazone and acetaminophen from its prodrug.

MATERIALS AND METHOD

All the melting points were determined in open capillary tube and may be uncorrected.[8] The λmax spectra recorded on SHIMADZU UV-1601 UV-visible spectrophotometer using methanol as solvent.^[9] The purity of compound was checked by TLC on silica gel coated glass plate using toluene: ethyl acetate: glacial acetic acid (8.5:6.5:0.5) solvent system for ibuprofen.^[10] Infra-red spectra were monitored in KBr palates on SHIAMADZU-8400 infra- red spectrophotometer.^{[11,12] 1}HNMR spectra were obtained on a Bruker ACF 300 spectrometer with tetra methyl silane as an internal standard. ¹HNMR spectra were recorded with CDCl₃ as a solvent and the chemical shift data were expressed as δ values to relative to TMS. Mass spectra were recorded on SHIMADZU GCMS-2010 mass spectrometer using the EI technique at 70ev.^[13]

Synthetic Procedures

Step-1: Synthesis of Antioxidants Chloroacetyl Derivative (15a-b)

A mixture of an appropriate antioxidant (0.01mol), trimethyl (0.01mol) in dichloromethane (25ml) was cooled in an ice salt mixture to -10⁰. To this reaction mixture Chloroacetyl chloride (0.01mol) in chloroform (25) was added drop wise with constant starring over a period of 1hr, maintaining the temperature constant. The reaction mixture was stirred further for 5hr at room temperature on the magnetic stirrer, washed with 5% HCL (3 x 50ml), 5% sodium hydroxide (3x50 ml), and finally with brine solution (2 x 25 ml). The organic layer was dried over anhydrous sodium sulphate, filtered and the solvent was removed under reduced pressure to obtain the corresponding antioxidant Chloroacetyl derivative. These derivatives were recrystalised from petroleum ether and ethyl acetate.

Step-2: Synthesis Of NSAIDs (Ibuprofen) with Antioxidant of Mutual Prodrugs (17a- b)

A mixture of an appropriate antioxidant Chloroacetyl derivative (0.01 mol), NSAIDs (Ibuprofen): (0.01mol) triethylamine (0.01 mol), sodium iodide (0.01 mol) in DMF (25ml) was stirred overnight at room temperature. The reaction mixture was poured into finely crushed ice with stirred and extracted with chloroform (4 x 25 ml). The combined organic layer was washed with 2% sodium thiosulphate (3 x 50 ml), 5%HCL (3 x 50 ml), 5% sodium hydroxide (3 x 50 ml) and finally with brine solution (2 x 25 ml). The organic layer was dried over anhydrous sodium sulphate, filtered and the solvent was removed under reduced pressure to obtain the semisolid residue. All the mutual prodrugs were synthesized by the above procedure. The final products were synthesized form petroleum ether and ethyl acetate.^[2]

Anti-Inflammatory Activity ^[14,15]

Carrageenan induced rat paw edema method of winter et al. was used for evaluation of Anti-inflammatory activity

Principle

 The Anti-inflammatory reaction readily produced in rats in the form of paw edema with thr help of irritants or inflammaging. Carrageenan induced paw edema is the most commonly used experimental method. Carrageenan is a sulphated polysaccharide obtained from seaweed (Rhodophyceae) causing the release of histamine, 5HT, bradykinin and prostaglandins. It produced inflammation and edema.

Anti-Inflammatory Screening Methods

Anti-inflammatory drugs have been evaluated by inflammatory responses produced in the animals by infecting foreign of noxious agents. These responses mostly comprise of the development of edema and the formation of exudates glaucoma. Drugs which suppress any of these components are designed as anti-inflammatory agents.

MATERIALS

1. Wister albino rats.

 2. Saline or tween 80 and distilled water.

 3. Std. drug: a suspension of Ibuprofen was prepared in 1% v/v solution of tween 80 and dose equivalent to 10mg/kg, 20mg/kg, 20mg/kg respectively.

4. Suspension of mutual prodrugs were prepared in 1% v/v solution of tween 80 and dose equivalent to 10mg/kg, 20mg/kg, 20mg/kg were taken for mutual prodrug respectively.

5. Feeding needles (oral dosing), syringes (1ml, 2ml), tuberculin syringe, 24 G needles, sampling tubes (to prepare suspensions of test compounds).

Selection of Animal Species and Storage Condition

The healthy Wistar albino rats of either sex of weight 100-200g were selected. Animals were housed in standard condition of temperature 220 C (±3⁰) and relative humidity (30%-70%) with 12:12 light: dark cycle. The animals were fed with standard diet (pellets) and water ad libitum.

Grouping of Animals

Six animals were randomly selected in each group and marked to permit individual identification. They were kept in the one animal/cage for five days prior to dosing.

Procedure

Male of female Wistar rats with a body weight between 100-200g were used. The animals were stared overnight. These animals were treated with control, standard and synthetic mutual prodrug (17a-b) orally. Thirty minutes later, the rats were challenged by a subcutaneous injection of 0.05ml of 1% solution of carrageenan into planner side of the left hind paw. The paw volume was measured by venire calliper scale immediately after injection i.e. 0 hrs and 5 hrs.

% inhibition=1-[a-x/b-y] x 100

Where- 

a. Paw volume of test group after 5hrs of injecting carrageenan

b. Paw volume of test group before injecting carrageenan

c. Paw volume of control group after 5hrs of injecting carrageenan

d. Paw volume of control group before injecting carrageenan

Ulcerogenicity Activity ^[15]

The ulcer index o synthesized prodrugs were recorded to observe the extent of gastrointestinal side effects. The ulcer index of prodrugs was found much lesser in comparison with the std. drug. The minimized side effects obtained in the prodrug might be due to the inhibition of direct contact of carboxylic acid group of the drug to the gastric mucosa which is mainly responsible for the damage.

Material

1. Anaesthesia i.e. ether

2. Wister albino rats.

3. Saline or tween 80 and distilled water.

4. Std. drug: a suspension of Ibuprofen was prepared in 1% v/v solution off tween 80 and dose equivalent to 10mg/kg, 20mg/kg, 20mg/kg respectively.

5. Suspension of mutual prodrugs were prepared in 1% v/v solution of tween 80 and dose equivalent to 10mg/kg, 20mg/kg, 20mg/kg were taken for Ibuprofen mutual prodrug respectively.

6. Feeding needles (oral dosing), syringes (1ml, 2ml), tuberculin syringe, 24 G needles, sampling tubes (to prepare suspensions of test compounds).

Selection of Animal Species and Storage Condition

The healthy Wistar albino rats of either sex of weight 100-200g were selected. Animals were housed in standard condition of temperature 22⁰C (±3⁰) and relative humidity (30%-70%) with 12:12 light: dark cycle. The animals were fed with standard diet (pellets) and water ad libitum.

Grouping of Animals

In these method albino rats were divided into10groups. Each group considered were administered. The animals were randomly selected in each group and marked to permit individual identification. They were kept in the one animal/cage for five days prior to dosing.

Procedure

The animals were dosed orally. The control, test and standard drug were administered orally for four days in 1% tween 80 solution and they were having free access to water and food during this period. On fifth day (24 hrs after dose the last dose) the rats were scarified after anaesthesia and stomach was removed. The stomach was filled with 1.5ml-2% buffer formalin for 10 min. and it was opened along the greater curvature, wash with warm water and examine under a 3- fold magnifier. The lengths of the longest diameter of the lesions were measured by scoring method.

The number of ulcers is noted and the severity recorded with the following:

· 0= no ulcer

· 1= superficial ulcer

· 2= deep ulcer

· 3= perforation

Evaluation

An ulcer index UI was calculated by the following formula:

UI= UN + US + UP x 10 – 1

Were,

UN= average of number of ulcers per animal

US= average of severity score

UP= percentage of animals with ulcers.

Drug profile: Ibuprofen (IBU)

It is propionic acid derivative:

IUPAC: (RS)-2-(4-Isobutylphenyl) propionic acid.

Description: A white or almost white, crystalline powder or colourless crystals, odour slight Molecular formula: C₁₃H₁₈O₂

Mol wt:206.28

Category: Anti-inflammatory, analgesic

Solubility: soluble in water, freely soluble in acetone, chloroform, ethanol, ether and in ethylene chloride

Pharmacokinetics: Bioavailability is 49 to 73%, 90 to 99% plasma protein binding, t^1/2 is 2hr,

Extensively metabolites, excreted by urine.

Pharmacology: MOA of action of IBU, inhibiting the enzyme cyclooxygenase (COX), which converts arachidonic acid to prostaglandins H₂ (PGH2).

Adverse effects: Nausea, dyspepsia, GI ulceration/bleeding, raised liver enzymes, diarrhoea, constipation, epistaxis, dizziness, priapism, rash, salt, and fluid retention, and hypertension.

Dose: 600mg to 1.2g daily, in divided dose, after food.

Use: it is use in the management of mild to moderate pain, fever, and headache including migraine and tension type headache, pain.^[16]

Antioxidants

It’s a cyclohexanol compound

IUPAC: 5-methyl-2-(propan-2-yl) cyclohexanol

Description: it is waxy, crystalline substance, clear or white colour which is solid at room temp. and melts slightly above.

Molecular formula: C₁₀H₂₀O

Molecular wt.: 156.27

Solubility: it is slightly soluble in water and extremely soluble in alcohols.

Use: local anaesthetic and counterirritant, short term relief of minor sore throat minor mouth and throat irritation ^[17]

Vanillin

It’s a phenolic compound.

IUPAC: 4-hydroxy-3-methoxybenzaldehyde

Description: it is white crystalline substance with floral and pleasant odor Molecular formula:

C₈H₈O₃ Molecular wt.:152.15

Solubility: extremely soluble in ethanol

Use: Largest use is as a flavouring agent, usually in foods, The ice-cream and chocolate industries together comprise 75% of the market for vanillin as a flavouring agent. Also used in fragrance industry, perfumes, to mask unpleasant odours or tastes in medicines, and cleaning products. ^[18]

RESULTS AND DISCUSSION

General scheme and mechanism ^[2]

Step 1: Synthesis of antioxidants Chloroacetyl derivative

Step 2: Synthesis of mutual prodrug of NSAIDs with antioxidant

Following table given the physicochemical properties of synthesized mutual prodrug

Physicochemical studies Infrared spectroscopy

2-Isopropyl-5-methylcyclohexyl-2-[2-(4-isobutylphenyl) propanoyloxy] ethanoate (17a) In FTIR spectra of Compound 17a the stretching at about 2960 cm^-1 indicates the presence of C-H bond in the aromatic ring. The stretching at about 2850 cm^-1 shows the presence of CH bond. Due to presence of ester O=C-O group in 17a stretching vibration is shown at 1746.42 cm^-1. The bending vibration at about 11453 cm^-1 indicates the presence of O-H bond. The C-O stretching vibrations is present at about 1150.46 cm^-1. A stretching vibration at 1044 cm^-1 indicates the presence of C-C group.  4-formyl-2-methylphenyl-2-[2-(4-isobutylphenyl) propanoyloxy] ethanoate (17b) In FTIR spectra of Compound 17b the stretching at about 3000 cm indicates the presence of C-H bond in the aromatic ring. The stretching at about 2150 cm shows the presence of C-H bond. Due to presence of ester O=C-O group in 17b stretching vibration is shown at 1846.42 cm. The bending vibration at about 1500 cm-1 indicates the presence of O-H bond. The C-O stretching vibrations is present at about 1600.12 cm^-. A stretching vibration at 1446 cm^-1 indicates the presence of C-C group.

¹HNMR spectroscopy

Compound shows broad doublet peak at 7.1-7.2ppm presence of 4protons of aromatic ring. A cluster of triplet peak at 4.7 – 4.8ppm shows the presence of –CH₂ protons of cyclohexane ring. A cluster of quartet peak at 3.8ppm indicates the –CH₂ protons of ester linkage. A doublet peak at 2.4-2.5ppm shows the presence of –CH₂ protons attached to the ring. a broad multiplate at 2.2-2.3ppm indicates the presence of –CH₂ protons attached to the cyclohexane ring. A broad triplet peak at 1.6-1.7ppm shows the presence of –CH protons adjacent to aromatic ring. a sharps doublet peak at 1.5ppm indicates –CH₂ protons adjacent to benzylic protons. A cluster of broad at 1.3-1.4ppm indicates –CH₃ protons attached to cyclohexane ring. a broad triplet peak at 1.1-1.2ppm shows the –CH₃ protons adjacent to cyclohexane ring. a broad multiplate peak at 0.9-1ppm shows the –CH₃ protons. A sharp multiplate at 0.8ppm again shows the –CH₃ protons. A broad multiplate peak at 0.7ppm shows the –CH₃ protons.

Mass Spectroscopy

Mass spectra of compound 17a show a small peak at m/z 426.3 which is molecular ion peak indicating the molecular weight of the compound. 426 is an even number which indicates the absence of N, S. a sharp peak at m/z 160.1 is the base peak.  Mass spectra of compound 17b show a small peak at m/z 516.1 which is molecular ion peak indicating the molecular weight of the compound. 516 is an even number which indicates the absence of N, S. a sharp peak at m/z 210.1 is the base peak.

The synthesized mutual prodrug 17a-b showed significant anti-inflammatory activity as compared to standard compounds. The ulcer index of synthesized mutual prodrugs were less compared to standard drugs indicates that all synthesized mutual prodrugs have less gastric irritation as side effects of NSAIDs as compared to parent molecules. Differentiation between the control, standard and mutual prodrugs show the ulceration covered.