Analytical Study of Selected Anti-Inflammatory Drugs

Thesis Presented by

Enas Taha Abdelhamed M.Sc. in Pharmaceutical Sciences Pharmaceutical Chemistry Faculty of Pharmacy - Cairo University 2012

Submitted for The Degree of Doctor of Philosophy In Pharmaceutical Sciences (Pharmaceutical Chemistry)

Under the supervision of

Prof. Dr. Sonia Talat Hassib Professor of Pharmaceutical Chemistry Faculty of Pharmacy - Cairo University

Prof. Dr. Ghaneya Sayed Hassan Professor of Pharmaceutical Chemistry Faculty of Pharmacy - Cairo University

Prof. Dr. Asmaa Ahmed El-Zaher Professor of Pharmaceutical Chemistry Faculty of Pharmacy - Cairo University

Dr. Marwa Ahmed Fouad Associate Professor of Pharmaceutical Chemistry Faculty of Pharmacy - Cairo University

Faculty of Pharmacy Cairo University 2018

Abstract Four simple, accurate, sensitive and economic Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopic (ATR-FTIR) methods have been developed for the quantitative estimation of some non-steroidal anti- inflammatory drugs alone or in presence of related substances. The first method involves the determination of by direct measurement of the absorbance at 1716 cm-1. In the second method, the second derivative of the IR spectra of and its imperity (2-chlorobenzoic acid) was used and the amplitudes were measured at 1084.27 cm-1 and 1058.02 cm-1 for tolfenamic acid and 2-chlorobenzoic acid, respectively. The third method used the first derivative of the IR spectra of bumadizone and its reported degradation product, N,N-diphenylhydrazine and the amplitudes were measured at 2874.98 cm-1 and 2160.32 cm-1 for bumadizone and N,N-diphenylhydrazine, respectively. The fourth method depends on measuring the amplitude of at 1059.18 cm-1 and of rhein, its reported degradation product, at 1079.32 cm-1 in their first derivative spectra. The four methods were successfully applied on the pharmaceutical formulations by extracting the active constituent in chloroform and the extract was directly measured in liquid phase mode using a specific cell. Moreover, validation of these methods was carried out following International Conference of Harmonisation (ICH) guidelines. Synthesis of new derivatives through an esterification reaction between the carboxylic acid functional group in NSAIDs and a naturally occurring phenolic antioxidant, thymol, to give anew prodrug. This is one of possible approaches to solve GIT irritation problem. The suggested prodrugs to be prepared are Etodolac-thymol and Tolfenamic acid-thymol prodrugs. The new prodrugs structure should be confirmed by FT-IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis. After synthesis and structure confirmation, the prodrugs will be evaluated for their chemical stability in

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different temperatures and pHs. Moreover, a kinetic study in 20% liver homogenate and 10% buffered plasma would be carried based on RP- HPLC method for the separation and determination of the 2 new prodrugs and their related bioactive products. Key words: ATR-FTIR, NSAIDs, Etodolac, Diacerein, Bumadizone, Tolfenamic acid, Prodrugs, RP- HPLC.

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Aim of the Work NSAIDs represent one of the most important classes used for the treatment of many inflammatory diseases and possessing analgesic and antipyretic activity. The aim of this work is to develop simple, accurate, precise and, most importantly, sensitive methods for the determination of some NSAIDs either alone or in combination with their related substances. The work involves the analysis of these drugs in their pure form or in commercially used pharmaceutical dosage forms using ATR-FTIR spectroscopic technique. Drugs cited in this part are etodolac, diacerein, bumadizone and tolfenamic acid. The pharmacopoeial methods described for the analysis of etodolac and tolfenamic acid depend on titrimetric method in pure form. In literature, numerous analytical methods have been reported for the determination of etodolac and diacerein, while few methods were reported for bumadizone and tolfenamic acid. Till date, no reported ATR-FT-IR method appears to be available for the determination of the cited drugs. Derivative IR-spectroscopy has been suggested for resolving spectral overlap displayed by Diacerein and its degradation product rhein, bumadizone and its degradation product, N,N-diphenylhydrazine, finally tolfenamic acid and its impurity, 2-chlorobenzoic acid. In addition, method validation and statistical analysis was included to compare the obtained results of the proposed methods with those of reference or pharmacopoeial methods. Gastric irritation and ulcerogenic effect of the acidic NSAIDs are one of the most challenging problems in designing novel anti-inflammatory agents.

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Therefore, prodrug approach can give an opportunity in medicinal chemistry to improve some undesirable properties hindering the clinical usefulness of a drug. The work was aimed to include synthesis of new derivatives through an esterification reaction between the carboxylic acid functional group in NSAIDs and a naturally occurring phenolic compound, thymol, to give a new prodrug. Thymol was selected to get a safer promoiety, a target which is always challenging in designing prodrugs. These types of promoieties were traditionally in use for their medicinal as well as flavoring properties with well documented safety profiles, thus do not involve the risk of unwanted effects after they are hydrolyzed. This is one of possible approaches to solve GIT irritation problem. The suggested prodrugs prepared were etodolac-thymol tolfenamic acid- thymol prodrugs. The new prodrugs structure were confirmed by FT-IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis. After synthesis and structure confirmation, the prodrugs were evaluated for their chemical stability in different temperatures and pHs. Moreover, a kinetic study in 20% liver homogenate and 10% buffered plasma were carried out based on a new developed RP-HPLC method for the separation and determination of the new prodrugs and their related bioactive products.

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Non-Steroidal Anti-Inflammatory Drugs (NSAIDS).

The term NSAID indicates a class of drugs known as non-steroidal anti- inflammatory drugs. This is an important therapeutic class of drugs which in addition to their anti-inflammatory effects, they may possess both analgesic and antipyretic activities. (1) The mechanism of action of NSAIDs involves inhibition of (COX), enzymes that initiate the formation of .(2) There are 3 subtypes of COX enzymes: COX-1(constitutive), COX-2 (inducible in inflammatory processes), and isozyme COX-3.(3) To be an effective competitive inhibitor of that binds to COX, (Figure- 1), a drug must possess both high lipophilic and acidic properties to mimic the natural substrate. Some NSAIDs inhibit the lipoxygenase pathway, which may itself result in the production of algogenic metabolites.(4)

Figure-1: Arachidonic acid cascade.(4)

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Classification of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). NSAIDs can be classified based on their chemical structure into: 1. Salicylate and derivatives:

This group of drugs was among the first introduced into medicine, and is the prototype of this group. (3) Most of these drugs are either marketed as salts of salicylic acid, ester or amide derivatives. Salicylate derivatives may inhibit both forms of COX by reversible or irreversible mechanism (5) (Figure- 2a). is a moderately potent inhibitor of biosynthesis; it does not have appreciable effect on platelet aggregation and does not significantly produce gastric or intestinal bleeding (6) (Figure-2b).

O

O a. Aspirin O

HO HO O

HO F b. Diflunisal

F Figure-2: Salicylate and salicylic acid derivatives.

2. derivatives: i) Phenylacetic acid derivative is used to treat painful conditions such as arthritis, sprains and strains, gout, migraine, dental pain, and pain after surgical operations. It decreases pain and inflammation (7) (Figure-3). Diclofenac potassium salt was developed to increase absorption rate with rapid onset of pain relief. (5)

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HO O

Cl NH

Cl Figure-3: Chemical structure of diclofenac. ii) Aryl and heteroaryl carboxylic acid derivatives Indomethacin is more potent COX inhibitor than aspirin, but patient intolerance generally limits its use to short-term dosing (5) (Figure-4a). , (Figure-4b) is a non-steroidal agent with potent analgesic and moderate anti-inflammatory activity. It is administered orally, intramuscularly, intravenously, and as topical ophthalmic solution.(8) , (Figure-4c) is a prodrug which is metabolized to a pharmacologically active sulfide derivative. This metabolite potently inhibits COX. Sulindac is also metabolized to the inactive sulfone derivative. (9) Etodolac, (Figure-4d) is an acetic acid derivative, which is primarily used in the treatment of rheumatic diseases and postoperative pain. It is rapidly metabolized in the liver, followed by renal elimination as the primary route of excretion. (10)

O

O OH a. Indomethacin N Cl O O O b. Ketorolac N OH

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F O HO S c. Sulindac O

HO H N d. Etodolac O O

Figure-4: Aryl- and heteroaryl carboxylic acid derivatives.

3. derivatives: Propionic acid derivatives are nonselective COX inhibitors, although there are considerable variations in their potency as COX inhibitors, they are approved for use in the symptomatic treatment of rheumatoid arthritis and osteoarthritis. Some are also approved for pain, ankylosing spondylitis, acute gouty arthritis, tendinitis, bursitis, migraine and for primary dysmenorrhea. (11) was the first member of this class of NSAIDs to come into clinical use (12) (Figure-5a). Some of the propionic acid derivatives, particularly , have prominent inhibitory effects on leukocyte function and some data suggest that naproxen may have slightly better efficacy regarding analgesia and relief of morning stiffness (13) (Figure-5b). , unlike NSAIDs, inhibits the synthesis of leukotrienes and leukocyte into inflammed joints in addition to inhibiting the biosynthesis of . Approximately 30% of patients experience mild GI side effects with ketoprofen, which decreased if the drug is taken with food or antacids (14) (Figure-5c). has similar pharmacological properties, adverse effects, and therapeutic uses to those of other propionic acid derivatives, but with rapid onset of action and prolonged duration of action (15) (Figure-5d).

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HO a. Ibuprofen O

b. Naproxen HO O O

c. Ketoprofen HO O O

O d. Oxaprozin O OH N

Figure-5: Propionic acid derivatives.

4. Anthranilic acid derivatives (Fenamates): The mechanism of anti-inflammatory of arylanthranilic acids is mainly potent inhibition of prostaglandin biosynthesis. (16) Tolfenamic acid belongs to the fenamates family and is used in both humans and animals for the management of pain and inflammation.(17) Tolfenamic acid is also effective in reducing the duration and intensity of migraine with less side-effects, especially nausea, than ergotamine.(16) Recently, it has gained tremendous popularity due to its anticancer activity against a variety of cancers and for slowing down the progression of Alzheimer’s disease (18)(Figure-6a). Meclofenamate sodium is used in the treatment of acute and chronic rheumatoid arthritis and primary dysmenorrea. The most significant side effects are the gastrointestinal effects, including diarrhea (19) (Figure-6b).

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O OH H a. Tolfenamic acid N Cl

O OH Cl H b. N

Cl Figure-6: Fenamates.

5. is an example of pyrazolones that has classically been used as an anti-inflammatory drug in ruminants (14) (Figure-7a). Bumadizone calcium is reported to be metabolized to phenylbutazone and . Its use was limited due to the risk of agranulocytosis and other haematological adverse effects (19) (Figure-7b).

N N O NH N O O O OH

a. Phenylbutazone b. Bumadizone Figure-7 6. The group of NSAIDs has emerged as a highly effective class of drugs against various arthritic conditions and post-operative inflammation.(20) , and are the most famous members of this group. Piroxicam (Figure-8a), is a derivative of oxicams with a benzene ring, while tenoxicam this benzene ring replaced with thiophene (Figure-8b) and in lornoxicam (Figure-8c) with chloro atom at position 8. (21) is has been shown to have a favorable gastrointestinal (GIT) tolerability profile compared with standard NSAIDs (22) (Figure-8d).

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OH H a. Piroxicam N O S N N O O

S OH H b. Tenoxicam O S N N O O N Cl S OH c. Lornoxicam H N S O N N O O OH O N

d. Meloxicam N S H N S O O Figure-8: Oxicams derivatives.

7. Diaryl heterocyclic derivatives (COX-2 selective): The first NSAIDs to be introduced as selective COX-2 inhibitors were and (Figure-9a&9b). In place of the carboxyl group of the conventional non-steroid anti-inflammatory acids, the structure of celecoxib contains a sulfonamide group and that of rofecoxib contains a methylsulfone group. The sulfur-containing phenyl rings of these drugs bind into the side pocket of the catalytic channel of COX-2 but interact weakly with the active site of COX-1.(23) Rofecoxib was voluntary worldwide withdrawn because of the increased risk of cardiovascular thrombotic, myocardial infarction, and stroke.(24)

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H2N O S O

a. Celecoxib N N F F F

O S b. Rofecoxib O O O Figure-9: Diaryl heterocyclic derivative. 8. Sulfonanilides. has been reported to inhibit COX-2 more selectively than COX-1 in low doses.(25) It has been observed that the analgesic effect is partially related to its inhibition of cytokines while the antipyretic effect via inhibiting the prostaglandin (PG) synthesis.(26) In experimental studies, nimesulide was found to suppress acute and chronic phases of inflammation, and decrease the capillary permeability increased in the inflammatory area. It has also been found to have antioxidative properties (27) (Figure-10).

O -O N+ NH O O S O Figure-10: Nimesulide 9. Anthraquinones: Diacerein is a slow-acting medicine of the anthraquinone class used to treat joint diseases such as osteoarthritis (swelling and pain in the joints). After absorption, the drug is metabolized to its active metabolite rhein. (28) Diacerein and rhein are anthraquinone compounds that ameliorate the course of osteoarthritis (19) (Figure-11).

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O O OH O OH O O O

HO HO O O O O Diacerein Rhein Figure-11: Chemical structure of diacerein and rhein.

Common side effects of NSAIDs: The use of NSAIDs can be associated with variable side effects such as renal dysfunction, cardiovascular adverse events, triggering asthma and can significantly increase clotting times (29) Also, it has been demonstrated that NSAIDs exhibit adverse effects on the gastrointestinal tract including nausea, vomiting and diarrhea. Ulcerogenic properties of NSAIDs stem from the organic acid they contain, which is responsible for irritating the gastric mucosa.(5) (PGE2) and (PGI2) are the major prostaglandins synthesized by the gastric mucosa. PGE2 can prevent gastric injury by cytoprotective effects that include stimulation of mucin and bicarbonate secretion and increased mucosal blood flow.(30) The gastric mucus and the alkaline stomach secretion creates a pH gradient across the adherent mucus gel and represents the first line barrier against acid damage. Thus, inhibition of PGs (mainly by inhibition of COX-1) by NSAIDs leads to GI disorder and bleeding. A correlation has been found between the ulcerogenic activity of NSAIDs and their ability to inhibit the synthesis of mucus (31 )(Figure- 12).

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Figure-12: Pathogenesis of NSAID-induced gastric injury and bleeding. (31)

Upon using NSAIDs, we should look for the lowest effective dose and patients should take the the shortest period of time and as much as possible, only a single NSAID should be prescribed. (32) A minority of patients are intolerant of all NSAIDs. They may benefit from the co-administration of a proton pump inhibitor, a H2-receptor blocker or the , . To address this problem, some NSAIDs are presented in combination with misoprostol, e.g. diclofenac with misoprostol (Arthrotec) and naproxen with misoprostol (Napratec). (33) The protective properties of prodrugs from coupling of NSAIDs and NO or

H2S (NO or H2S donating) in the GI tract make them attractive candidates which can reduce their toxicity and boost efficacy.(34) Ester prodrugs for NSAIDs containing carboxylic acid group can be helpful to depress the GIT irritation and bleeding.(35) Esterification of the acidic moiety would suppress gastro-toxicity without adversely affecting their anti-inflammatory activity.(36)

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Summary Analytical study of selected anti-inflammatory drugs This thesis includes the following: ATR-FT-IR methods for the determination of some NSAIDs drugs, in pure and dosage forms have been introduced in this thesis. These drugs are etodolac, diacerein, bumadizone and tolfenamic acid. The work also involves the synthesis of new prodrugs through an esterification reaction between the carboxylic acid functional group in NSAIDs and a naturally occurring phenolic compound thymol. The thesis consists of the following sections: Part I: Introduction It includes: 1. A review about NSAIDs, classification and activity of different drugs used in the treatment inflammation, and the pharmacological and side effects of different classes. 2. Literature review about the official and reported methods for the quantitative determination of the drugs under investigation.

Part II: Aim and basis of the work In this section, the aim of this work and the basis on which the proposed methods are chosen, have been clarified. Part III: Experimental and Discussion This section is further divided into two parts:

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Part I-Quantitative ATR- FT-IR spectroscopic analysis: In this part an introduction about the quantitative infrared, ATR crystal instrument and factors affecting the liquid sample measurements.  Four simple, accurate, sensitive and economic Attenuated Total Reflectance- Fourier Transform Infrared Spectroscopic (ATR-FTIR) methods have been developed for the quantitative estimation of some non-steroidal anti- inflammatory drugs. The first method involves the determination of etodolac by direct measurement of the absorbance at 1716 cm-1. In the second method, the second derivative of the IR spectra of tolfenamic acid and its impurity (2- chlorobenzoic acid) was used and the amplitudes were measured at 1084.27 cm-1 and 1058.02 cm-1 for tolfenamic acid and 2-chlorobenzoic acid, respectively. The third method used the first derivative of the IR spectra of bumadizone and its reported degradation product, N,N-diphenylhydrazine and the amplitudes were measured at 2874.98 cm-1 and 2160.32 cm-1 for bumadizone and N,N-diphenylhydrazine, respectively. The fourth method depends on measuring the amplitude of diacerein at 1059.18 cm-1 and of rhein, its reported degradation product, at 1079.32 cm-1 in their first derivative spectra.  The four methods were successfully applied on the pharmaceutical formulations Etodolac® tablets, Diacerein® capsules, Octomotol® tablets and Clotam® Rapid tablets by extracting the active constituent in chloroform and the extract was directly measured in liquid phase mode using a specific Znse cell . Moreover, validation of these methods was carried out following International Conference of Harmonisation (ICH) guidelines.  Statistical comparison between the results obtained by the proposed method and that of the reference methods for each drug showed no significant difference between them concerning accuracy and precision.

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Part II: Synthesis and kinetic study of new prodrugs: It includes:  Introduction about classification and clinical application of different prodrugs.  Synthesis of new derivatives through an esterification reaction between the carboxylic acid functional group in NSAIDs and a naturally occurring phenolic compound, thymol, to give new prodrugs. Thymol was selected to get a safer promoiety, a target which is always challenging in designing prodrugs. This type of promoiety was traditionally in use for its medicinal as well as flavoring property with well documented safety profile, thus did not involve the risk of unwanted effects after it is hydrolyzed. This is one of possible approaches to solve GIT irritation problem.  The new prodrugs structure was confirmed by FT-IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analysis.  After synthesis and structure confirmation, the prodrugs were evaluated for their chemical stability in different temperatures and pHs. Moreover, a kinetic study in 20% liver homogenate and 10% buffered plasma was carried out based on RP- HPLC method for the separation and determination of the new prodrugs and their related bioactive products.

Section IV: English summary. Section V: References This section contain 155 references Section VI: Arabic summary.

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دراسة تحليلية ألدوية مختارة من مضادات اإللتهاب

رسالة مقدمة من إيناس طه عبد الحميد ماجستير العلوم الصيدلية - كيمياء صيدلية كلية الصيدلة - جامعة القاهرة 2102

للحصول على درجة دكتوراه الفلسفة فى العلوم الصيدلية - كيمياء صيدلية

تحت اشراف

أ.د. سونيا طلعت حسيب أستاذ الكيمياء الصيدلية كلية الصيدلة - جامعة القاهرة

أ.د. غنية سيد حسن أستاذ الكيمياء الصيدلية كلية الصيدلة - جامعة القاهرة

أ.د. أسماء أحمد الزاهر أستاذ الكيمياء الصيدلية كلية الصيدلة - جامعة القاهرة

د. مروة أحمد فؤاد أستاذ مساعد الكيمياء الصيدلية كلية الصيدلة - جامعة القاهرة

كلية الصيدلة جامعة القاهرة 1028

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