PHA 4110 - Immunology and Inflammatory Diseases Section

Medicinal Chemistry Tutorial

Instructor: Patrick M. Woster, Ph.D.

Optional reading assignment: Wilson and Gisvold, pgs. 656-673

Introduction

Non-steroidal antiinflammatory drugs (NSAIDs) are just that, drugs which act to relieve inflammation, but which are not structurally related to the corticosteroids.

Recall that NSAIDs have four major activities:

Analgesia - refers to the relief of pain by a mechanism other than the reduction of inflammation (for example, headache). These agents produce a mild degree of analgesia which is much less than the analgesia produced by opioid analgesics such as morphine.
Antipyretic - these drugs lower elevated body temperature by their action on the hypothalamus. However, they will not reduce normal body temperature.
Antiinflammatory - these drugs are used to treat rheumatoid disorders, and also in other inflammatory diseases and injuries. Their antiinflammatory activity is due to their ability to inhibit the cyclooxygenase activity of prostaglandin synthase, an enzyme which mediates the production of prostaglandins from arachadonic acid. These drugs were developed as an alternative to the corticosteroids and their analogues, which have many side effects.
Uricosuric - many of these agents cause the excretion of uric acid, and thus are useful in the treatment of gout.

There are also two inportant facts you should remember about NSAIDs in general: 1. Most of these compounds will potentiate the action of oral anticoagulants such as coumadin, by their effects on platelet aggregation, and 2. Most of these agents cause some degree of gastric upset.

Salicylates

Salicylates were first discovered when the observation was made that chewing willow bark could relieve pain. Some time later, the active constituent of willow bark, a glycoside of glucose and salicyl alcohol was found to be the active species. Later, methyl salicylate (found in Oil of Wintergreen) was found to have similar activity. Acetylsalicylic acid (aspirin) was first synthesized in 1898 and introduced as a pain reliever in 1899, at which time it was used in doses of 650 mg every 4 hours.

Aspirin has now been shown to function by acetylation of the cyclooxygenase active site, thereby reducing the production of the prostaglandins responsible for inflammation. It also acts at the hypothalamus, causing an increase in peripheral blood flow, sweating, and therefore cooling. Although most of its analgesic properties are due to relief of inflammation, it also has a low degree of true analgesia. It is uricosuric, but only in high doses.

Aspirin requires an acidic environment such as the stomach for optimal absorption, but is also poorly soluble under these conditions. Some aspirin products contain buffering agents, which raise the pH locally and aid in dissolution of the drug. Since aspirin and many other NSAIDs are weak acids, a fact which, as we will see, contributes to their activity.

Aspirin, as was mentioned above, inhibits cyclooxygenase by acetylation of the active site. Aspirin is metabolized (see below) by N-deacylation to salicylic acid, which is also active, acting as a reversible inhibitor of the enzyme. Salicylic acid is them metabolized primarily to two major inactive metabolites: salicyluric acid arises from conjugation of salicylic acid with glycine, and gentisic acid arises from p-hydroxylation mediated by cytochrome P450. These two metabolites are then excreted in the urine.

Salicylates Modified at the Carboxyl Group

Most carboxyl-modified salicylates are salts, and various salt forms of this drug are available. The sodium salt is somewhat unstable, and is not often used. The choline salt, shown below, is marketed as Trilisate¨. The counterirritant compound methyl salicylate, found in Oil of Wintergreen, is fairly toxic when taken internally, although it is used as a flavoring agent in small quantities. It is mainly used in topical preparations such as Ben-Gay¨. Another salicylate ester, phenyl salicylate, is an example of a "salol", since it is cleaved in the GI tract to salicylic acid and phenol, which acts as an antiseptic agent. Salols are generally covalently bonded combinations of two drugs that have undesirable qualities such as taste, GI upset, etc. When combined, the adverse effects of each drug is masked.

Salicylates Modified at the Hydroxyl Group

Although there are many hydroxyl-modified salicylates that have been synthesized, the best known in this class is aspirin (see above), which argueably is the most successful drug ever produced. Other forms of salicylic acid (sodium salicylate, choline salicylate, magnesium salicylate, salsalate) are not as effective as NSAID's, but they have a lower incidence of GI effects, and they have negligible effects on platelet aggregation. Aspirin is a common choice for treatment of mild analgesia and inflammation, and is a reasonable antipyretic, although many of the newer NSAID's are gaining popularity. Aspirin is also uricosuric in high doses (greater than 2g/day), although in lower doses it can actually induce the occurance of gout. It is now commonly used to prevent recurrance of myocardial infarction in low chronic doses due to its ability to inhibit platelet aggregation.

Salicylates Containing Nuclear Substitutions

Only two drugs in the salicylate class have been developed which contain nuclear substitutions. The first of these was flufenisal, which has twice the potency of aspirin, and a lower incidence of gastric irritation. However, the agent diflunisal (Dolobid¨) is three times more potent than aspirin, and can be dosed twice a day. For these reasons, it is the more clinically useful drug. As you will see later, newer NSAIDs are usually longer acting, so that they do not need to be given as often, reducing the incidence of gastric side effects.

Aniline Derivatives

The aromatic compound aniline, shown below, was found to have antipyretic effects at about the same time that aspirin was discovered. Unlike aspirin, however, aniline causes methemoglobinemia, a condition which may result in cyanosis and death. The related analogue p-aminophenolacetaminophen (Tylenol¨, Datril¨, Tempra¨, etc., etc.) is an excellent choice for the relief of mild to moderate pain. It is analgesic and antipyretic, but is not an antiinflammatory drug. The usual adult dose is 300-600 mg every 4 hours.

Acetaminophen Metabolism and Overdose

Aromatic compounds are metabolized mainly by the so-called mixed-function oxidases, producing highly reactive intermediates, as shown below. The mixed function oxidases react with aromatic rings to form an epoxide species, as shown, and this epoxide is then combined with glutathione (GSH) by the action of glutathione-S-epoxide transferase. Since the reactive epoxide reacts preferentially with GSH, it does not have a chance to react with hepatic tissue; thus GSH protects the liver from arylation by reactive intermediates. Aromatic glutathione conjugates are then further metabolised to the corresponding mercapturic acid derivative in a 3-4 step process, and the mercapturic acid is excreted.

Acetaminophen becomes a particularly toxic drug in acute overdose, and the major manifestation of this toxicity is hepatic in nature. Normally, acetaminophen is metabolized by N-deacetylation and glucuronidation, as shown below. However, in an acute overdose, a minor metabolic pathway takes precedence when the normal pathway is saturated, giving rise to a reactive species known as the "arylating intermediate". this intermediate reacts rapidly with GSH, resulting in a complete depletion of glutathione. This leaves the liver defenseless against reactive intermediates produced by the mixed function oxidases, and against the arylating intermediate itself. The result is a dramatic hepatotoxicity. This process may be reversed by administration of acetylcysteine, which preferentially reacts with reactive species and protects the liver until GSH can be resynthesized.

Acetaminophen Analogues

There are a number of acetaminophen analogues which have been synthesized, but none are superior to acetaminophen itself. The ethyl ether derivative phenacetin used to be a component of many over-the-counter preparations. However, the drug is more toxic than acetaminophen, and is immediately O-dealkylated to acetaminophen. For this reason, it was removed from all APC (aspirin, phenacetin and caffiene) products and is no longer marketed. The acetaminophen analogue phenetsal (p-acetaminophyl salicylate, Salophen¨) is a true salol, since it is metabolized to acetaminophen and salicylic acid.

Derivatives of Pyrazole

All of the drugs in this chemical category are structurally related to the aromatic compound pyrazole, shown below. If one of the double bonds in pyrazole is saturated, pyrazoline results, and if both are saturated, the compound is known as pyrazolidine.

Pyrazolinones

Pyrazolinones which are or were clinically useful have the general structure shown below, and are numbered as indicated. All useful compounds have a 5-keto functionality. The pyrazolinone antipyrene (Felsol¨) is an analgesic about equal in potency to aspirin, and is a good antipyretic (in fact, it will reduce body temperature to below-normal levels). It is not an antiinflammatory drug, however, and can cause sedation. The structurally related analogue aminopyrene (Pyramidon¨) has similar activity to antipyrene, but is more potent. This drug has a propensity to cause agranulocytosis. These drugs have been removed from the market due to adverse side effects and the availability of more efficacious agents.

Derivatives of Pyrazolidine

Derivatives in this class are 3,5-pyrazolidinediones with the general structure shown below, and they are numbered like the pyrazolinones. These compounds are analgesic, antipyretic, antiinflammatory (due to their weak acidity) and uricosuric at near toxic doses. The acidity in these molecules is due to the presence of an enolizable hydrogen in the 4 position, and is pKa-dependent. The most active analogues have a pKa near 4.5. At lower pKa values, uricosuric activity predominates. The most widely used analogues in this class are shown below. Phenylbutazone (Azolid¨, Butazolidin¨) is an excellent antiinflammatory drug which has no inherant analgesic properties (except for analgesia due to the relief of inflammation). This drug is quite toxic, however, and is contraindicated in patients with peptic ulcer disease, cardiac patients, patients with high blood pressure, and patients with renal dysfunction. It also causes blood dyscrasias much more readily than aminopyrene. For this reason, phenylbutazone is no longer available. Phenylbutazone has an active metabolite, oxyphenbutazone (Tandearil¨, Oxalid¨), which was marketed seperately, but has also been taken off the market. It has the same potency and toxicity of phenylbutazone itself.

The related analogue sulfinpyrazone (Anturane¨) has a strongly electron-withdrawing (-Is) phenylsulfoxide group on the alkyl sidechain at position 4, and thus the pKa of the enolic hydrogen is lower (2.8). For this reason, it is much less active as an antiinflammatory agent, but is an excellent uricosuric used in the treatment of gout. It retains the same side effects of phenylbutazone.

N-Arylanthranilic Acid Derivatives (Fenamates)

N-arylanthranilic acid derivatives, known as fenamates, have the general structure shown below. They are amino isosteres of the salicylates, and always contain an N-aryl substituent. If there are substitutions on the N-aryl group, they are most effective in the 2, 3 and 6 positions. If the NH group is replaced by O, S or CH2, there is a decrease in activity. Flufenamic acid (Arlef¨) contains a trifluoromethyl group in the 3-position of the N-phenyl substituent. It is an NSAID which has no intrinsic analgesia, and it is about 7 times as potent as mefanamic acid (Ponstel¨). Mefanamic acid, which has a 2 and 3 methyl substitution on the N-phenyl group, has many side effects, and is about 1/5 the potency of phenylbutazone. It is only used for short term therapy (7 days or less). The related analogue meclofenamic acid (Meclomen¨) is about 25 times as potent as mefenamic acid, and about 150 times as potent as aspirin.

Meclofenamic acid is illustrative of a general trend which may be applied to all NSAIDs. The halogen substituents on the N-phenyl group are large, and prevent the free rotation of the two aryl rings. This creats a situation where there are two flat aromatic systems which are slightly out of plane with respect to one another (see below). This situation, as you will see, results in a better fit to the NSAID receptor, and thus a greater potency.

Indole-3-Acetic Acid Derivatives

As was the case with the other chemical classes of NSAID, there have been many indole-3-acetic acids developed, but only two have progressed to the market. The representative analogue in this series is indomethacin (Indocin¨), which is shown below and numbered as indicated.The methylene group adjacent to the carboxyl moiety is termed alpha, and the carboxyl group has a pKa of 4.5. Indomethacin, developed in 1965, is one of the more potent NSAIDs, and is about 2-3 times more potent than phenylbutazone. It is a potent antiinflammatory agent, and is a good antipyretic. It has an incidence of gastric upset about equal to aspirin, but does cause headache, vertigo and blood dyscrasias in some patients.

The indole-3-acetic acids have the following structure/activity relationships:

The group at position 1 must be an aracyl group. Aralkyl substituents reduce activity. If the aracyl group has an electron withdrawing group in the para position, activity is optimal.
In position 2, a methyl group is optimal. This group restricts rotation of the aracyl moiety and keeps it in an out-of-plane orientation.
The group at position 3 may be either an acetyl group or a propionyl moiety in which the methyl is in the alpha position (attached at carbon 2).
Position 5 is the best place for substituents. Halogens, ethers, alkyl and alkylamino substituents all produce active analogues.

In terms of stereochemistry, the aracyl group should lie under the benzene portion of the indole ring (this is called the "cis" conformation, and is best for receptor fit). The aracyl group lies slightly out-of-plane, as was the case with meclofenamate.

The other useful compound in this chemical class is sulindac (Clinoril¨), an arylacetic acid with a pKa of 4.7. Note that the nitrogen in the indole ring of indomethacin has been replaced in sulindac by a double bond, a so-called indene isostere which has the same electronic character as the lone pair of the indole nitrogen. Because if the double bond, the aracyl substituent lies permanently in the cis configuration, ensuring good receptor fit. The electron withdrawing p-methylsulfoxide increases potency and also increases solubility of the drug.

Sulindac may be considered a pro drug due to its unusual metabolism. The parent sulfoxide form of the drug is active, and has a T1/2 of 7-8 hours. It is reversibly converted to the corresponding sulfide form, which has even greater activity and a T1/2 of 16.8 hours. The parent drug can also be irreversibly converted to the sulfone analogue, which is inactive and excreted. Thus, sulindac has a long overall half life, and can be dosed less frequently.

Other Arylacetic and Arylpropionic Acid Derivatives

There are a number of analogues in this class that have been marketed as non-steroidal antiinflammatory agents. These agents generally have three structural features of indomethacin that allow them to retain activity: an acidic carboxyl group, and out-of-plane phenyl group and a flat, nitrogen-containing ring system. Tolmetin sodium (Tolectin¨) is about equal to ibuprofen as an NSAID, but with low incidence of GI upset. Ibuprofen is marketed under a number of trade names, and is widely used both over-the-counter and as a legend drug. Note that it is an arylpropionic acid, and as such has an alpha methyl group, imparting stereochemistry to the molecule. In general, the S(+) isomer of any given NSAID is the active form, and the R(-) isomer is inactive. However, ibuprofen is administered as a racemate. Fortuitously, the R(-) isomer of most NSAIDs is converted to the corresponding S(+) isomer in vivo, so that the stereochemistry is not a concern in the synthesis and production of the drug. Ibuprofen is indicated for mild to moderate pain and inflammation, and for dysmenorrhea. Other examples of NSAIDs in this chemical class appear above. Note that the S(+) isomer of naproxen is marketed as Naprosyn¨, while the R(-) isomer is marketed as Anaprox¨).

A number of more recently marketed NSAIDs are shown in the Figure below. The agent diclofenac is used extensively worldwide, and contains structural features of the arylalkanoic acid and anthranilic acid structural classes. Diclofenac undergoes extensive metabolism in vivo, by hydroxylation of the aromatic ring bearing the chlorines at the 3,4 and 5 positions. Nabumetone (Relafen¨) was introduced in the US in 1992. Note that the drug contains no acidic functional group, and thus must be converted by oxidation to the corresponding arylacetic acid known as 6MNA. Since the non-acidic form is administered orally, it potentially can cause less gastric erosion. In addition, it has a very long half life, and thus can be dosed once a day. The pyranocarboxylic acid derivative etodolac (Lodine¨) is a representative of a new class of NSAID. Two additional recent agents are ketorolac (Toradol¨) and oxaprozin (Daypro¨). Ketorolac is especially useful, since it can be used parenterally.

Oxicams

Oxicams represent a new trend in NSAIDs, because they have extremely long half lives, and thus can be dosed once a day. The oxicam derivative piroxicam (Feldene¨) derives its activity from an enolic hydroxyl group which has a pKa of 4.3. Oxicams are analgesic, antiinflammatory and antipyretic, and are indicated for rheumatoid and osteoarthritis.

COX2 Inhibitors

It has now been shown that cyclooxygenase (COX) occurs in two forms, termed COX-1 and COX-2. The COX-1 isozyme is a constituative enzyme (non-inducible) that is responsible for the synthesis of endogenous prostaglandins, including those that mediate normal cellular processes such as gastroprotection, normal kidney function and thrombogenesis. The COX-2 form of the enzyme is highly inducible by a number of pro-inflammatory mediators, and appears to be responsible for the high level of prostaglandin synthesis that results in inflammation. Thus, a specific inhibitor of COX-2 could prove to be effective against inflammation, but would allow COX-1 to perform its normal physiological functions, including gastroprotection. Interestingly, some of the newer NSAIDs, notably nabumetone and etodolac, show a high ratio of COX-2 to COX-1 activity. However, there is an ongoing search for COX-2-specific inhibitors.

Click HERE to download an interesting article comparing the COX-1/COX-2 specificities of various NSAIDs.

Two such COX-2 inhibitors, celecoxib (Celebrex¨) and rofecoxib (Vioxx¨) have recently been marketed. The structures of these two analogues are shown in the figure above.

The NSAID "Receptor"

The NSAID "receptor" (actually a site on the active site of cyclooxygenase) has been determined to look like the diagram shown below. Note the 5 by 6 angstrom flat area (which binds an aromatic ring), the 7 angstrom "trough" (which binds an out-of-plane aromatic group) and the cationic site (which binds the ionized acid group of a given NSAID). Phenylbutazone is shown as it would be expected to binds to the receptor.

Miscellaneous Compounds

There are a number of miscellaneous compounds available which are used to treat various aspects of inflammation. These compounds are commonly known as DMARDs (disease-modifying agents for rheumatoid diseases), since they alter factors pertaining to the etiology of the disease, rather than alleviating disease-induced inflammation. Gold compounds are useful in some patients, although they are not considered first line therapy. Gold sodium thiomalate and aurothioglucose are used by injection, while auranofin can be used orally. Penicillamine can also be used for treatment of rheumatoid arthritis, since it improves lymphocyte function in the affected joints.

The prostaglandin analogue misoprostol (Cytotec¨) is a compound which mimics the action of prostaglandin PGE1, increasing the secretion of bicarbonate and mucus in the stomach, as well as increasing gastric blood flow. Together, these actions protect the stomach from damage caused by chronic administration of NSAIDs. The methyl ester functionality makes the analogue a prodrug, since the ester must be cleaved for activity. In addition, the 16 hydroxy (rather than the natural 15 hydroxy) results in a decreased rate of metabolism by oxidation.

Agents Used for the Treatment of Gout

Gout is a disease that arises from the deposition of uric acid crystals in joints, causing pain and inflammation. Uric acid is a normal product arising from the metabolism of DNA and RNA, as shown below. Cleavage of nucleic acids results in the formation of the purines adenosine and guanosine. Adenosine is metabolized to inosine, and then to the nitrogenous base hypoxanthine. Guanosine is metabolized directly to xanthine. The enzyme xanthine oxidase is critical to the metabolism of purines, since it converts hypoxanthine to xanthine, and then xanthine to uric acid.

Gout can arise from excess uric acid secondary to a number of causes:

Increased production of uric acid from dietary nucleic acids.
Increased breakdown of endogenous purines.
Increased xanthine oxidase activity.
Increased tubular reabsorption of uric acid.

A number of compounds are useful in the treatment of gout. The alkaloid colchicine is used for both acute attack and, less often, for prophylaxis. It has no effect on the excretion of uric acid, and appears to act on polymorphonuclear leukocytes. For an acute attack, the patient is given 1 mg every 2 hours until the pain subsides, or the patient develops diarrhea. The acidic compound probenecid (Benemid¨) acts as an excellent uricosuric, and increases the renal excretion of uric acid. Coincidentally, the compound also inhibits the renal excretion of penicillin, and as such is sometimes given in combination with these antibiotics to increase their half-life. The most widely used antigout agent is allopurinol (Zyloprim¨), a structural analogue of hypoxanthine which is a competitive inhibitor of xanthine oxidase. Interestingly, allopurinol is also a substrate for xanthine oxidase, and is converted slowly to alloxanthine, which also inhibits the enzyme. Allopurinol interferes with the metabolism of the anticancer drug 6-mercaptopurine, which is metabolized by xanthine oxidase. Thus, patients on allopurinol should have their 6-mercaptopurine dose reduced by 75% to compensate.

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