Perspectives in Nonsteroidal Anti-inflammatory Agents

Perspectives in Nonsteroidal Anti-inflammatory Agents

By T. Y. Shen[*]

Among numerous nonsteroidal anti-imflammatory agents synthesized in the past few years, various analogs of indomethacin, phenylacetic acid and heteroarylacetic acid have reached the stage of clinical evaluation. Their biochemical mechanisms of action are exemplified by the broad activity profile of indomethacin which includes inhibition of mediators and enzymes, effects on cell membranes, and, most recently, inhibition of prostaglandin biosynthesis. The importance of pharmacodynamic properties to clinical efficacy was clearly demonstrated in some cases. Several candidates were eliminated because of their side-effects. A group of a- methylarylacetic acids showed a high degree of stereospecificity in their potency and meta- bolisms in uiuo, as well as inhibition of prostaglandin synthetase and albumin binding in uitro. Extrinsic Cotton effect provides a sensitive technique in the study of interactions of these drugs with biopolymers. Competitive binding and antagonistic interactions between nonsteroidal drugs, particularly salicylate, were observed in uitro and in uiuo. Progress in salicylate research was marked by the synthesis of flufenisal as a new derivative with enhanced potency and longer duration of action. Several fenamate analogs and new chemical types have shown promise in preliminary clinical trials. Various immunological approaches are under investigation for the treatment of rheumatoid arthritis. Newer concepts are still needed to achieve more effective control of arthritic disorders.

1. Introduction"]

Despite the intensive and ever-increasing efforts in anti- inflammatory studies during the past decade, the treatment of rheumatoid arthritis remains a major challenge for bio- logists and medicinal chemists alike. A better understanding of the disease, particularly its etiology and pathogenesis, the development of more significant animal models, new medicinal chemical concepts and novel chemical structures are still much needed to achieve a major break- through in this difficult field.

As was noted in the Annual Report in Medicinal Chemistry for 1966['], a wide-spread interest in the search for new anti- inflammatory agents at that time was evidenced by the disclosure of more than one hundred groups of anti-inflammatory structures in the patent literature within one year. This feverish pace of screening and synthesis has apparently been maintained, if not accelerated. However, judging from the scientific literature, apparently only a small number of leads showed adequate potency and short- term tolerance to merit further consideration. An even smaller fraction of these eventually qualified as preclinical candidates after chronic toxicity studies. This process of elimination of anti-inflammatory compounds is certainly a costly and frustrating one.

Since the general background of arthritis research, e. g., clinical aspectd- 6], pharmacological studies" lo] and new active structures[' '*"], is regularly reviewed by many excellent articles, the main emphasis of this review will be on the current status of a selected group which has advanced to the clinical stage or beyond. The selection is necessarily limited by the scarcity of clinical data on new drugs under

[*] Dr. T. Y. Shen Merck Sharp & Dohme Research Laboratories Rahway, New Jersey 07065 (USA)

development, but it may help to focus the attention of medicinal chemists on a few significant compounds among a maze of literature claims. It is also hoped that by examin- ing the case history of these examples, the success of very few, the near-miss of many, the limitation of their clinical efficacy and, most of all, the tremendous amount of time and effort invested by so many will illustrate some critical problems in the field of arthritis research and underscore the need for realignment of our strategy and biological approaches.

2. Arylacetic Acids

2.1. General

Interest in arylacetic acids as nonsteroidal anti-inflammatory agents was developed independently in several laboratories about 15 years ago. Although not explicitly considered by the investigators at that time, it is of interest to note that two plant growth regulators, indolylacetic acid and phenoxyacetic acid served as the basic structure for the earlier studies at Boots['31 and at Mer~k' '~]. Hydro- phobic substitutions, so effectively employed as activity- enhancing groups in many pharmacological agents, in the form of aryl, aralkyl or aroyl, readily converted these plant growth regulators into derivatives highly active in animal assays such as foot-edema, UV erythema, granuloma pellet, and adjuvant arthritis assays['5! More recently, in uitro cytotoxicity and moderate in uiuo anti-inflammatory activity have also been observed with analogs or derivatives of several other plant regulants[16!

The predominance of arylacetic acids in anti-inflammatory research is reminiscent of the association of antihistaminic activity with diarylalkylamines or central nervous activity

460 Angew. Chem. internat. Edit. Vol. I 1 (1972) 1 No. 6

with phenothiazinylalkylamines. Not surprisingly, the optimal aryl moieties seem to be different in each case. Several attempts to interchange the dialkylamino side- chain with an acetic acid in each series resulted in mediocre analogs o ~ I Y [ ~ ~ ] .

2.2. Biochemical Modes of Action of Indomethacin

The primary modes of action of anti-inflammatory aryl acids are still being elucidated, but it seem likely that they exert a multitude of actions on the complex inflammatory process. Their broad activity-profiles are probably the basis for their rather nonspecific clinical efficacy and also for some potential side effects.

The introduction of indomethacin (I)[“* ‘‘I appeared to coincide with the recent interest in biochemical pharma-

R

( I ) , R = OCH3 (Indomethacin) (21, R = N(CH,),

c1

cology of nonsteroidal anti-inflammatory agents. As a new reference compound it has been evaluated in various biochemical systems. Although the significance of some observations remains to be substantiated, a selection of its biochemical properties reported in the literature is listed in Table 1. As noted before, most of these properties are commonly shared with other anti-inflammatory aryl acids.

Table 1. The biochemical profile of indomethacin

I. 1.1. Inhibition of proteolytic enzymes, e. g., trypsin, a-chymotrypsin

1.2. Inhibition of histidine decarboxylase in oitru 1.3. Inhibition of lymph node permeability factor (LNPF) 1.4. Marginal effect on kinin systems, Hageman factor and fibrinolysis 1.5. Antagonism of slow reacting substances (SRS) 1.6. Uncoupling of oxidative phosphorylation 1.7. Inhibition of mucopolysaccharide biosyntbesis 1.8. Stabilization of serum protein 1.9. Inhibition of prostaglandin synthetase mol/l) 2. Effect on Membrane Structures - mol/l) 2.1. Stabilization of erythrocytes 2.2. Inhibition of platelet functions

2.3. Inhibition of neutrophil participation in Shwartzman Reaction 2.4. Inhibition of fibroblast-leukocytes interaction in v i m 2.5. Inhibition of leukocytes migration

Chemotaxis and PMN motility 2.6. Stabilization of human skin lysosomes L‘S. histamine

-_ ~ ~ ~_____--______ Effect on Enzymes and Mediators (10-4-10-’ mol/l)

and “tissue proteases”

Aggregation and release of mediators

The biochemical profile of indomethacin may be broadly described in two parts : inhibition ofenzymes and mediators, and effect on cellular membranes. The latter, in particular, seems to be the basis of many recent observations. As shown in Table 1, indomethacin, along with other anti-inflammatory aryl acids, inhibits several proteolytic enzymes

in uitroat 10-4-10-3 m01/1[193201. Some ofthose proteases may be involved in tissue degradation, immunoglobulin metabolism, and the activation of inflammatory mediators‘”’. The possible roles of collagenase12’-’41 and of a lysosomal enzyme, cathepsin Dcz5], in the degeneration of arthritic joints have received much attention The recognition of several mammalian collagenases with different pH optima and sensitivity to serum inhibitors suggests caution in interpreting the data obtained from model in uitro studies[26!

Interest in histamine as the prime mediator of inflammation has decreased with time. It was found earlier that indomethacin inhibits fetal histidine decarboxylase in vitro at a concentration of 0.4 mm~l/l[*’~. However, in uico. the effect seems to vary with the tissue origin of the enzyme128~291. Conceptually, the inhibition of histidine decarboxylase was further complicated by the postulation of an “induced enzyme[30]. Lymph node permeability factor (LNPF) is an inflammatory mediator derived from lymphocytes and associated with a variety of phenomena involved in delayed hyper~ensitivity[~ ‘I. The factor produces an increase in vascular permeability, leukocyte emigration, and fibrinoid deposition. The vasoactive effects of LNPF are antagonized by indomethacin. On the other hand, indomethacin has little or no effect on the formation and activity of bradykinin[32.331. It has marginal effect on the activation of the Hageman factor in the dog knee jointL331 and it has very weak activity in fibrinolysis in ~ i t r o ‘ ~ ~ ] .

Indomethacin antagonizes the ability of “sIow reacting substances” (SRS-A, SRS-C), arachidonic acid, and bradykinin, to increase the resistance of guinea-pig lungs to in- f l a t i~n ’~~! This drug blocks directly the spasmogenic effect of SRS-CL361 and its possible release of an aorta-contract- ing substance[371.

Most nonsteroid anti-inflammatory agents uncouple oxidative phosphorylation and inhibit the biosynthesis of mucop~lysaccharide~~~~. The correlation of these properties with anti-inflammatory actions, however, was weakened by the lack of structure specificity and by the pharmacodynamic requirements for in uiuo activities. The effect of these drugs on the ATP level do not seem to parallel the effect on 35S incorporation[391. There appears to be no direct correlation between these two actions.

The binding of serum protein by many anti-inflammatory aryl acids and their mutual interference have been well investigated. The &-amino group of lysine, tryptophan, and tyrosine groups in serum albumin are possibly involved in the three strong binding sites[401. As a result of the binding, the denaturation of protein is inhibited. Whether the free SH-disulfide exchangeL4 ‘I is actually involved in pathological protein denaturation in uiuo remains speculative. Some anti-inflammatory agents displace albumin-bound warfarin and interfere with its anticoagulant actionL421, but the alleged effect of indomethacin was refuted[431. In the clinic, indomethacin does not seem to have any effect on blood clotting in patients undergoing anticoagulant therapy.

The stabilization of protein denaturation was originally proposed by Mitsushima as a simple screening assay for compounds with potential anti-inflammatory properties[44]. At concentrations of 0.4 mrnol/l indomethacin in-

Angew. Chem. inremat. Edit. / Vul. I 1 /1972/ / Nu. 6 461

hibits the heat denaturation of serum albumin[44]. It has been suggested that similar interaction of anti-inflammatory agents with membrane proteins may be the basis of their multiple effects on cells. Indomethacin at 0.05-0.3 mmol/l inhibits the lysis of human and animal erythrocytes induced by heat or h y p o t o n i ~ i t y [ ~ ~ . ~ ~ ! It inhibits the in uitro aggregation of erythrocytes induced by gelatin or fibrinogen[47]. Indomethacin and salicylate inhibit the aggregation of platelets induced by collagen, epinephrine, ADP, e t ~ . [ ~ ' * 491.

A transient but demonstrable effect was produced by a single oral dose of indomethacin in man[491. At concentrations of 5 pmol/l indomethacin is one of the most potent compounds to inhibit the release of serotonin and other mediators by platelets in the presence of connective tissues, ADP, or other stimulant^^^^^. It also inhibits the function, though not the migration, of neutrophils in the Shwartzman reaction in rabbitsf5 'I and the interaction of fibroblasts and leukocytes in ~ i t r o [ ~ ' I . The membrane effect is further re- flected in the chemotactic or migratory properties of cells. Indomethacin decreases the motility of polymorpho- nuclear leukocytes induced by crystalline urate[' 31, the migration of bone-marrow derived monocytes to the inflamed site in and the infiltration of neutrophils in inflamed rat The effect of anti-inflammatory drugs on the membrane of intracellular granules was demonstrated with the aid of an integrated microdensitometry tech- n i q ~ e ' ~ ~ ] . At a concentration of 5 x mol/l indomethacin inhibits the histamine-induced rupture of lysosomes in human skin culture1571. Unlike steroids, it has no effect on the histamine-induced increasing permeability of the mitochondria.

The stabilization of lysosomal membrane has received a great deal of attention as a potential antirheumatic approach. Recent in uiuo studies[58. 591 with chloroquine showed that daily doses of 120 mg/kg stabilize lysosomes in rat liver us. the damaging effect of hyperoxia and neutral red. It also produces myeloid body formation in liver and heart tissue, presumably by reducing the membrane permeability and thus interfering with the formation of primary lysosomes. Other nonsteroidal anti-inflammatory agents have no pronounced effect on isolated lysosomes at low concentrations[601. Some confusion in in vitro re- sultsr6',621 was probably caused by the sensitivity of some experiments to varying pH, temperature, media, and drug concentrations. Progress has been made in the development of artificial lysosome models, but the validity of studying lysosome rupture in an artificial system outside its cytoplasmic environment may require careful consideration.

Complement fixation is a recognized process in arthritic joints[63. 641. In addition to its roles in antigen-antibody reaction and hemolysis, it releases in a sequential manner anaphylatoxin, chemotactic factor and prostaglandins. Available anti-inflammatory agents are generally ineffec- tive in controlling this important pathological process.

The anti-inflammatory effect of indomethacin was originally evaluated in the carrageenin foot-edema assay in rats. In searching for the primary mode of action, it was hy- pothesized recently that the observed reduction of foot- swelling by indomethacin may be attributable to the sup-

pression of migration of monocytes to the inflamed site, thus preventing the subsequent activation of complement or enzymes, formation of prostaglandins, permeability increase, e t ~ . ' ~ ~ ] .

Recently, Vane and his colleagues reported that indomethacin and aspirin inhibit the biosynthesis of prostaglandins from arachidonic acid in several tissue homo- genates['601, human platelets['61i, as well as isolated, per- fused, intact spleen of the dog['621. Prostaglandins have been implicated as mediators of inflammation in several experimental systems including cutaneous inflammation in man[1631 and carrageenin foot-edema in rats['641.

The inhibition of their biosynthesis by these two drugs in uitro and in uiuo at levels below their physiological concentrations thus appears to be an attractive mechanism of action['65i. As an extension of these studies we have found that the S-flUOrO analog of indomethacin is a competitive inhibitor (Ki=8 x mol/l) of arachidonic acid ( K , = 2 x mol/l) in our in uitro system[1661. A large number of nonsteroidal anti-inflammatory agents such as phenylbutazone, indomethacin analogs ( 2 ) - (5 ) , phenylacetic acids ( 1 1 ) and (12), ibuprofen (19) , alclofenac (22) , naproxen (24), metiazinic acid (26) , flufenisal (33), fenamates"], nifluril (38), azapropazone (41) , and several non- acidic compounds (e. g., indoxole and thiabendazole), strongly inhibit the conversion of labeled arachidonic acid into prostaglandin E2 at 0.1 to 10 pg/ml. The magnitude of in uitro activities are approximately parallel with the potency of these compounds in the carrageenin foot-edema assay.

The specificity of the prostaglandin synthetase assay was further demonstrated by the findings that slow-acting antirheumatic drugs, D-penicillamine and chloroquine, and many other drugs such as benemid, disodium cromoglycate, glyvenol, colchicine, etc., are inactive at 10 pg/ml or higher.

Of particular interest is the observation that the high degree of stereospecificity for the anti-inflammatory action of several pairs of a-methylarylacetic acids in uiuo (see below) is also true for their inhibitory effect upon prostaglandin synthetase. In all cases examined, e.g., (3), ( 4 ) , and (II), the dextrorotatory enantiomers are much more potent than the levorotatory ones. To our knowledge this is the first stereospecific correlation between an in uitro enzymatic effect and the in uiuo activity of anti-inflammatory arylacetic acids.

The ability of nonsteroidal anti-inflammatory agents to inhibit prostaglandin biosynthesis has many implica- t i o n ~ [ ' ~ ~ ] . The central issue seems to be the selective effects of these agents upon different tissues. Drug localization, sensitivity of various prostaglandin synthetases and the changing ratio of prostaglandins E and F may explain the safety and the primary actions of these agents in uiuo. A better understanding of the basis for tissue specificity is also essential for the broader application of compounds related to these types of structures to other prostaglandin-mediated problems such as asthma, cholera, fertility control['681, etc .

[*] Fenamates IS the name given to compounds such as mefenamic acid and flufenamic acid.

462 Angew. Chem. internal. Edit. / Vol. 11 (1972) 1 No. 6

2.3. Indomethacin Analogs

In our study of indole derivatives, two dextrorotatory N-p-substituted x-(l-benzyl-5-methoxy-2-methyl-3-indol- y1)propionic acids ( 3 ) and ( 4 ) were first investigated as clinical candidates. The demonstration of their clinical

efficacy in man, albeit accompanied by gastrointestinal irritation at 1-2.5 g daily doses, established a reassuring correlation of their animal activity with clinical efficacy. Further study in this series led to the discovery of a more potent analog, indomethacin ( I ) [I4]. Subsequently, the 5-dimethylamino analog (2 ) [651 and an indene isoster (5)'6h1 were found to be 1/3-1/2 as potent as indometh-

c H 3 0 ~ c H 2 c 0 2 H

CH3

DCH ( 5 ) c1 '

acin in animal assays, but with somewhat different metabolic and distribution characteristic^[^^^ 681. Both analogs showed efficacy in clinical tests, but were less well tolerated. Other congeners of indomethacin are still under investigation.

Meanwhile several analogs [ (6) , (7), (S), and (911 have been investigated by other laboratories. A position isomer ( 6 ) is described in a patent taken out by G e i g ~ ' ~ ~ ] and in some recent reviews, but no testing data are available. According to our previous studies some substituted indole-

c -Ar

C H, C OzH '-CH3 C=O CHzCOzH

7)

c H 3 0 ~ c H 2 c 0 2 H H3 OZ

Pi CH, I I

2-acetic acids appeared to be metastable and readily under- went thermal decarboxyIati~n[~~! Another group of position isomers, the 3-aroyl-I-acetic acids (7) , was studied by us and independently at the firms Rous~e l [~ ' I , Geigy["] and others. In general, they are less potent than analogs in the indomethacin series.

The Sumitomo laboratory in Japan has been very active in developing novel chemical processes for making indoles as well as in searching for newer analogs. The cinnamoyl analog (8) has a potency one-sixth that of indomethacin in the carrageenin edema assay and one third in some other assays[731. The pharmacology of another Sumitomo compound, ID-955 (9), the piperonylic acid analog, has also been described in detail[741. It is about one-third as active as compound (8) .

The Bristol analog, Intrazole (BL-R 743) ( lo ) , has a potency of 1/15--1/20th that of ind~methacin[~~]. Interest- ingly, the replacement of carboxyl by an acidic tetrazole moiety (pK, = 6) circumvents the metabolic formation of

6 (10)

c1

acyl glucuronide and thus prolongs its serum half-life~'h! Intrazole does not have the 2-methyl and 5-methoxy substituents which were found to be activity-enhancing in investigations of the indomethacin series. Intrazole appeared to be less irritating in the clinic but its overall assessment probably was less promising than expected.

The continued interest in developing new indomethacin analogs is presumably based on the assumption that better- tolerated, even though less potent, derivatives should have clinical utility. This is an attractive approach but is complicated by the poor correlation of animal data with clinical efficacy and by the uncertainty of relating acute animal toxicity to long-term safety in man. The ED,,"] of indomethacin is 2.5 mg/kg in the carrageenin foot-edema assay but only 0.5 mg/kg in the adjuvant arthritis assay. In man, the average daily dose is about I-lSmg/kg. Although indomethacin is 20-80 times more potent than phenylbutazone in the above two assays, in the clinic its potency advantage is only 3-4-fold.

We have demonstrated previously that the toxicity and, to some extent, the metabolism of indomethacin are highly species specific[67! In the selection of a new drug candidate any derivative which is less potent in animals tmt has higher plasma and tissue levels or other improved pharmacodynamic properties in man, may indeed be more effective in the clinic. However, the same metabolic differences may render its long-term tolerance in man even less predictable from toxic data of small animals. A remarkable instance of _____ [*] ED,,=Dosage required to produce 50"/, response in the animal assak.

Angew. Chem. inrernat. Edit. Val. I1 11'972) ! N o . 6 463

species difference in tissue accumulation and the resulting chronic toxicity was described recently with metazamider7'. (See Section 5.1.)

2.4. Phenyl Aliphatic Carboxylic Acids

Among many active phenylacetic acids, the most potent compound is probably (S)-( +)-3-chloro-4-cyclohexyl-a- methylphenyl acetic acid (11) with a potency of 20 times that of indomethacin in the carrageenin foot-edema assay[779781. Unfortunately, further development of (11)

(s)-(+) W Z : C O z H (11)

Cl

was discouraged by its narrow therapeutic ratio with respect to gastrointestinal irritation and renal papillary necrosis. No significant therapeutic advantage was obtained by replacing the cyclohexyl ring with 4'-fluorophenyl (12) or by reducing the acid (11) to its alcohol derivative

Cl

(13) [781. Promising anti-inflammatory activities were also demonstrated by phenylbutyric acid derivatives such as (14)c781 and (15) (BDH 7538)[79.801 but no clinical data are available.

OH -CH,CHCH,CO,H I (15)

2.5. Development of Ibuprofen

The development of ibuprofen (19) (Brufen@) by Boots' laboratories dates back to their initial interest in p-alkyl- phenoxyacetic acids 15 years A total of 600 com-

pounds have been examined. A notable feature in the Boots' study is their persistent attitude, undoubtedly for good reasons, in testing a series of candidates in the clinic. It was stated thatr8 '1 the first candidate, p-butylphenylacetic acid (16), produced skin-rash in 50% of patients receiving doses of 2 gJday ; the p-cyclohexyl analog (1 7) exhibited the same effect. Ibufenac (18) did not produce skin-rash and seemed less irritating to the G. I. tract than aspirin, but its clinical application was terminated by hepatotoxicity such as SGOT[*I elevation at high dosesrs2]. Finally, the a-methyl analog, ibuprofen (19) [831, was successfully developed as a mild and well tolerated antiarthritic agent at a daily dose of 0.8-1.6 gr84, 851. It is currently under evaluation by Upjohn laboratories in the U.S. (Further comments on ibuprofen's being a racemic mixture are included in Sec- tion 3.)

D L

Fen~profen[~~"] , ~~-a-(3-phenoxyphenyl)propionic acid (19a), is a m-substituted analog developed by the Lilly group. It is highly potent in the UV erythema assay in guinea pigs (ED,,=0.5--1.0 mg/kg) but much less active in the rat foot-edema assay (ED5,-50mg/kg) and in febrile rats (at 25 mg/kg). The analgesic effect of (+)-prop- oxyphene was reported to be potentiated by fenoprofen and also by several other anti-inflammatory acids. The major metabolic conversion of ( I 9 a ) is hydroxylation at the 4'-position. Its clinical trial is in progress.

An o-substituted phenoxyacetic acid with structure features resembling an open-chain analog of indomethacin is clamidoxic acid (20) [I6].

A OCHz COZH

It is equivalent to phenylbutazone in acute anti-inflammatory assays and is now undergoing clinical evaluation.

2.6. Bufexamac and Alclofenac

A generally assumed requirement for nonsteroid anti-inflammatory agents is that their mode of action should not be mediated by pituitary-adrenal stimulation and the release of endogenous cortico hormones. Whether a moderate degree of stimulation should always be avoided is controversial among some investigators. The hydrox- amic derivative of p-butoxyphenylacetic acid, bufexamac (21) (Droxarym), represents such an example. Its anti-inflammatory effect in animals is, at least in part, attributable

[*I SGOT= Serum Glutamo-oxaloacetic Transaminase.

Angew. Chem. internot. Edit. / Vol. I 1 (1972) 1 No. 6 464

to adrenal stimulation’861. In rheumatic patients it is used at a dose rate of 4 x 250 mg during the day and a 1 g sup- pository overnight[8 71. Unlike most aryl acids it significantly decreases the abnormal sedimentation rate of erythrocyte in arthritic patients. It would be of interest to ascertain the long-term tolerance of this drug. The free acid of bufexamac is very weakly active in animal assays.

In our study of phenylacetic acids, a rn-chloro substituent was found to be a~tivity-enhancing[’~! In addition to the example of i I I ) , the 3-chloro derivatives of 4-isobutyl- and 4-cyclohexyl-phenylacetic acid are also more potent than their parent compounds. By combination of the 3-chloro- with the 4-allyloxy-substituents, Buu-Hoi et al. obtained alclofenac (22) (Mervan A marked species difference in the metabolism and toxicity was again demonstrated by

0

A

a l~ lofenac[~~] . Its LD,, is only 45 mg/kg in rats but 300 to 500mg/kg in dogs and monkeys. The blood level, metabolism, and routes of excretion, e. g . urinary us. biliary, also vary drastically with species. Compared with the metabolism of 4-alkylphenylacetic acids the presence of a p - allyloxy group apparently favors urinary excretion. Indica- tions are that the allyloxy group is an active metabolic site leading to the formation of several metabolites.

The activity of alclofenac is comparable to phenylbutazone in acute inflammatory assays[881. However, in osteo- arthritic patients it was used at 1.5 g to 3 g daily to match the efficacy of 300 mg p h e n y l b u t a ~ o n e [ ~ ~ ~ ~ ~ ! The higher dosage required may be attributed to its low plasma level and short half-life in man.

2.7. Naphthylacetic Acid Derivatives

An oc,ci-disubstituted ci-naphthylacetamide, naphthypra- mide (23) (DA-992), has been introduced as a mild analgesic anti-inflammatory agent with moderate muscle- relaxant activity as a useful adjunct‘92, 931.

More recently ( + )-ci-(6-methoxy-2-naphthyl)propionic acid (24) (Naproxen) and its (-)-ethanol analog (25) (Naproxol) were found by the Syntex group to be potent

anti-inflammatory a g e n t ~ [ ~ ~ . ~ ’ ! Naproxen possesses an anti-edema activity of ca. 1/3 that of indomethacin with similar G. I. irritation properties. Both (24) and (25) were reported to be in clinical trial.

2.8. Metiazinic Acid

Consideringphenothiazine as a fused-ring aromatic moiety, its acetic acid derivative, metiazinic acid (26) (Soripal@) was found by the RhGne-Poulenc group to be a clinically

useful Several years ago, in order to compare the optimal aryl moieties in CNS and antihistamine drugs with those in anti-inflammatory agents, we investigated a number of phenothiazinyl-N-carboxylic acids (27) and were discouraged by their weak anti-inflammatory activities. In

investigations by the RhGne-Poulenc’s group, metiazinic acid was one of the first members of this series to be synthesized ; the anti-inflammatory activity was not improved by the evaluation of some 300 analogs. It was tested in 6000 clinical cases over a period of 5 years. The daily doses were 3-6 capsules, each of 250 mg; several minor side- effects were 981.

One may recall that the phenothiazine analog of flufenamic acid (28) was investigated by Smith, Kline and French a few years ago, but no clinical findings were reported[991.

2.9. Fenclozic Acid and Related Compounds

A less fortunate venture in the field of heteroarylacetic acids was 1.C.l.’~ fenclozic acid (29) (Myalex@)[lOO. ‘O1I ,

Fenclozic acid showed early promise as a moderately active compound with a wide-range of tolerance. In short- term assays, such as the carrageenin foot-edema assay, fenclozic acid is comparable to phenylbutazone with an ED,, of ca. 90 mg/kg. It is four times more effective than phenylbutazone in the adjuvant arthritis assay, both against developing and established arthritis. In rats it was well tolerated at 70 mg/kg for 3 months. A species difference in serum half-life, in favor of man, was also noticed. In fact,

Angew. Chem. internat. Edit. / Vol. I I (1972j i No. 6 465

the half-life turned out to be longest in rheumatoid arthritis patients (46 hrs), almost twice that in normal volun- t e e r ~ [ ’ ~ ~ * ’ ~ ~ ] . It was excreted in the urine mainly as un- changed drug or ester conjugates. The clinical efficacy of 300-400 mg of fenclozic acid was comparable to 3.6 g aspirin[’04! However, further development of this drug was terminated by the occurrence of hepatotoxicity[’ O5]. 1.C.1.’~ workers[’061 found that the corresponding oxazole, isoxazole, pyridine, and pyrimidine analogs are less active.

It is of interest to note that the potency of fenclozic acid, on a weight basis, is higher in man than in animals. It is comparable to other arylacetic acids, e. g. ibuprofen, metiazinic acid and alclofenac, in animals, yet its effective clinical dose is only 1/4--1/5 times the others. Perhaps it is the pharmacodynamic advantage of fenclozic acid which enhances its potency and, unfortunately at the same time, toxicity in man.

A related compound [2-phenyl-4-(p-chlorophenyl)-5-thi- azolyl]acetic acid (30) is undergoing clinical study by the Wyeth group in

3. Stereospecificity of Arylacetic Acids

Generally speaking, arylacetic acids constitute a broad anti-inflammatory lead. As we have seen, moderate to good activities are readily found with many combinations of heteroaryl and substituted phenyl groups bearing an acetic acid side-chain. A partial dissociation of activity and toxicity, in terms of its tolerance in man, has been achieved by the fortuitous selection of some aryl moieties.

The object of the game is to find less toxic aryl moieties, but unfortunately not enough data are available to guide a rational approach. The preferred geometry of the aryl groups, their interaction with aromatic amino acids, such as tryptophan and tyrosine, in albumin or membrane proteins, their metabolic stability, their distribution and excretion characteristics etc., have been examined as possible factors. During the past few years we have looked into some properties of the acetic acid side-chain, which is a constant feature of all arylacetic acids, particularly the stereochem- istry of the a-methyl group.

3.1. Potency Correlation

$H3 CH3

I CH,

X D C H

Y (3), x = c1 X

(4) . X = SCH,

Scheme 1. Examples of(S)-( +) isomers with higher activity than (R)-( - ) isomers.

3.2. Preferential Metabolism

In the case of ibuprofen (19), Boots’ use the DL mixture; presumably similar activity was found with both optical isomers[831. Interestingly, a partial analysis of the metabolic mixture showed that only dextrorotatory metabolites had been formed[’081. In metabolite (31) the only asymmetric center is present in the acetic acid side-chain. a-Alkylphenyl acetic acid generally gives a plain positive

Scheme 2. Preferential metabolism of a racemic mixture of iboprufen

ORD curve[1og! As the sign of rotation is not much affected by p-alkyl substitutions, one may assume that this metabolite has the same (S)-( +) configuration as the compounds in Scheme 1. With metabolite (32) assignment of absolute configuration is complicated by the presence of the second asymmetrical center in the molecule. The stereo- selective metabolism of a racemic mixture has been observed in other cases[’1o1. Further characterization of metabolite (32) may shed some light on the specificity of side- chain oxidation of arylacetic acids.

In several series the activity of an arylacetic acid is enhanced several-fold by the introduction of an &-methyl group. In most cases, the dextrorotatory isomer, with ( S ) configuration, seems to possess higher, if not exclusive, activity in uivo[2]. A recent example is Naproxen (24 ) (see Scheme I).

.3.3- Biochemical Comparisons

In order to see whether the observed difference in in vico activity of optical enantiomers is really attributable to selective “receptor site” binding, their activities in several

466 Angew. Chem. internat. Edit. 1 Vol. I 1 (1972) 1 NO. 6

in vitro biochemical systems were examined. With several pairs of substituted indolyl- and phenyl-a-methylacetic acids, no significant difference was observed in erythrocyte membrane stabilization["'], protein denaturation, and uncoupling of oxidative phosphorylation["21. Both isomers showed inhibitor activity at 10-4-10-5 mol/l, roughly parallel with the in uioo potency of the dextrorotatory enantiomer. So far, the only demonstration of stereospecificity is the inhibition of prostaglandin synthetase by dextrorotatory enantiomers (see Section 2.2).

10

a -

6 -

3.4. Serum Albumin Binding

a I \

-

/ ', I I \ ; /ZE l+HSA l l

I :, I I

Most arylacetic acids have strong affinity for serum proteins, a property which has obvious pharmacodynamic, or even mechanistic, implications. We recently became interested in using the induced Cotton effect to study drug biopolymer interactions. Cotton effect denotes the anomalous change of optical rotation at the wavelength corresponding to the UV maximum of an asymmetrical molecule. A drug such as phenylbutazone is not asymmetric and shows no optical rotation. However,-as aptly described by Chignall" 1 3 * ' 14]

-when phenylbutazone is bound to serum albumin, the interaction between the Iigand chromophore and an asymmetric locus at the binding site perturbs the chromophore in an asymmetric manner and induces optical activity. The rotatory dispersion curve of the mixture shows a Cotton effect near the UV maximum of phenylbutazone, this is referred to as an induced, or extrinsic, Cotton effect.

Chignall investigated the comparative binding of phenylbutazone analogs["41 and flufenamic acid analogs[4o1. Each group shows three strong binding sites on human serum albumin. The molar ellipticities of drug-albumin complexes are enhanced by hydrophobic interactions and by rigidity of the complex. The change in ellipticity also provides a rapid method for estimating association con-

The binding of drugs such as dicoumarol and warfarin to serum albumin quenches the native tryptophan fluorescence of the protein. The light energy absorbed by the tryptophan groups and nonradioactively transferred to bound warfarin is re-emitted by the drug as fluorescent light. The mean effective transfer distance between the two groups estimated in this way gives an indication of the receptor site

Hypothetical "receptor sites" derived from structure activity relationships have often been postulated as working models by medicinal chemists. Until a real receptor site is actually isolated, as in the case of estradiol["'], or possibly, corticosteroids" la], the significance of postulated receptor contours can only be measured by their usefulness in sug- gesting new and active structure equivalents. For a drug like indomethacin, which possibly executes a multitude of primary actions, one would assume that the optimal structural features are the best fit for several similar but non- identical binding sites. Since strong serum protein binding is a common characteristic for nonsteroid anti-inflammatory aryl acids, one might use serum albumin as a model to probe the "receptor site" interaction; thrombocyte and erythrocyte membrane preparations[119.'201 may serve as other models.

1 I I 1 I

260 270 280 290 300 310 320 1187911 h I n m l --3

Fig. 1. CD spectra of indomethacin ( I ) , its indene isoster is), and flufenaminic acid (28) in the presence of human serum albumin ; HSA = serum albumin alone (phosphate buffer pH 7.4).

Phenylacetic acid derivatives do not produce significant extrinsic Cotton effects in the presence of human serum albumin. The circular dichroic spectra of indomethacin and its indene analog are shown in Fig. 1[1211. The optical activities are in general less pronounced than that produced by flufenamic acid. We are particularly interested in the binding of their a-methyl analogs which have an asymmetric acid side-chain. As mentioned above the stereospecificity of the a-methyl side-chain in in uiuo activity was not found in a number of in uitro assays. If albumin binding involves the acetic acid side-chain in a stereospecific manner one might detect the difference in binding in the circular dichroic spectra of those optical enantiomers. As shown in Fig. 2, the spectra of HSA plus an optical isomer do indeed vary according to the configuration of the a-carbon atom. The difference spectra in Fig. 3 seem to suggest two dis- tinctive groups of binders. The influence of p-substituents

- lo ' 260 2;O 280 2;O 3d0 3;O 320 3iO rn h Inml-

Fig. 2. CD spectra of the enantiomers of u-[l-(p-methylthiobenzyl)-5- methoxy-2-methyl-3-indolyl]propionic acid (4) with and without addition of human serum albumin (HSA).

Angew. Chem. internat. Edit. Vol. 11 (1972) / No. 6 467

I I

260 270 280 290 300 310 320 330 1)17931 h lnml-

Fig. 3. Difference CD spectra of the enantiomers of a-[I-@-methylthiobenzyl)-5-methoxy-2-methyl-3-indolyl]propionic acid (4 ) and the p-methoxy derivative (44) with and without addition of human serum albumin (HSA): [Observed (compound + HSA)]-(spectrum of compound)-(spectrum of HSA).

on binding is also observable. As the theoretical implications of an asymmetric side-chain on the extrinsic Cotton effect of a chromophore have not been analyzed, more detailed interpretation of these spectra was not attempted. Further investigations to elucidate the number of binding sites for each group of enantiomers and the competition of common binding sites between enantiomers and among different analogs are in progress. The sensitivity of this technique to steric and electronic environment and the small amount of sample required provide us with a valuable tool for studying drug-biopolymer interaction in solution.

4. New 'Salicylate Derivatives

4.1. Objectives and Structure-Activity Relationship

The search for a novel salicylate analog as a superior antiarthritic drug has been an attractive yet highly elusive goal for many years. In general, the objectives are three-fold : (i) to increase potency, (ii) to reduce gastric irritation, and (iii) to provide a longer duration of action. While various sustained-release and antiacid formulations of aspirin have been developed in recent years to improve the last two properties, no single chemical derivative of salicylate has been uncovered as a superior aspirin analog. In our broad- based research on nonsteroidal anti-inflammatory agents a determined effort was launched several years ago to evaluate a large number of substituted salicylic acid derivatives by the granuloma-pellet and carrageenin foot-edema assays, both of which seemed to be responsive to the anti- inflammatory activity of nonsteroidal drugs such as phenylbutazone, indomethacin, and other experimental agents.

The structure-activity relationships established by previous studies of sali~ylates[ '~~ have shown that replacement of the 0-acetyl group by other acyl or alkyl groups and deriva- tization of the carboxyl group, although beneficial in certain aspects, are generally detrimental to the overall anti-inflammatory-analgesic-antipyretic profile of aspirin. Ring- substitution of salicyclic acid with chloro, hydroxy, methoxy, amino, alkyl groups etc., either diminished activity or

increased both potency and toxicity in a nonspecific manner. Similar conclusions were readily verified in our assays with more than 300 salicylates including many newly synthesized analogs.

With salicylates, the establishment of a definitive structure- activity relationship was complicated by the low order of activity of many analogs which were very sensitive to biological variations, and by the apparent lack of additive effects of different activity-enhancing groups. Obviously, with a small molecule like salicylic acid the overall physico- chemical and metabolic properties become easily influenc- ed by multiple substitutions. Advantages sometimes occur- red in the case of 0-acetylation of less hydrophobic salicylic analogs, presumably due to an improvement of absorption or distribution. Perhaps more significant was the apparent trend of hydrophobic substitutions, e. g. phenyl, 4'-chlorophenyl, and benzyloxy, at the 5-position to en- hance the activity of salicylic acid. Interestingly, similar preference of 5-alkyl~'zz' and 5 - h a l 0 ~ ' ~ ~ ' substituents was recently observed in the inhibition of several enzyme systems by salicylic acid derivatives.

4.2. Synthesis of Flufenisal

The activity-enhancing effect of a p-fluorophenyl group in anti-inflammatory agents was well demonstrated by the potency of p-fluorophenylpyrazolo [ 3,2-c] steroids" 241

and ~-(4'-fluorobiphenyl)propionic acid ( 12) which were studied previously in our Laboratories. The 5-(4-fluorophenyl) derivative of aspirin, flufenisal (33), was synthesized as shown in Scheme 31'251:

'COzH

j33) COzH

Scheme 3. Synthesis of flufenisal ( 3 3 ) .

Other aryl and heteroaryl analogs substituted in position 5['261 as well as two aza isosters of flufenisal (34) and (35)['*'] were also investigated.

4.3. Biological Properties of Flufenisal

Compared to aspirin, flufenisal (33) is 4-5 times more potent in the carrageenin foot-edema and cotton pellet

468 Angew. Chem. infernat. Edit. / Vol. I 1 j1972) / No. 6

granuloma assays. In acute gastric hemorrhage test in the rat the ED,, for flufenisal was ca. 200mg/kg, almost 15 times the ED,, of 14 mg/kg for aspirin. Its analgesia in the yeast-induced hyperesthesia assay was estimated as twice that of aspirin during the first 2-3 hours; but more interestingly, the duration of effect was much longer. Flufenisal is present in the plasma as its deacetyl derivative which is 98-99.5% bound to the plasma protein in equili- brium dialysis experiments. It produces a higher and more prolonged plasma level than aspirin in rats and in dogs. The longer duration of analgesic action has been confirmed in the clinic['281. Single doses of 300 or 600 mg of flufenisal appeared equal in effectiveness to 600-1200 mg of aspirin in relieving episiotomy pain ; further, the duration of action was double that of aspirin.

Aspirin has a spectrum of side-effects which are widely recognized but also accepted as reasonable risks by clinicians and patients alike['*']. An up-to-date pharmacological analysis of aspirin summarized its many actions in terms of depression of defensive reactions[130! Some subtle biological properties, such as potential renal papillary necrosis'1311 and in uiuo acetylation of platelet membrane[13'] and serum proteins, have attracted a certain amount of attention. The need to re-evaluate the safety of aspirin has also been suggestedr129!

The difficulty in quantifying the anti-inflammatory effect of nonsteroids and aspirin-like compounds has been well documented in recent clinical literature. To improve the time-honored aspirin therapy by a new derivative may well require large-scale and long-term comparative studies. Nevertheless, with flufenisal we have both the encourage- ment and a new direction for further exploitation of this grand old lead in the arthritis field.

5. Other Chemical Compounds

5.1. Anti-inflammatory Agents

Several members of the fenamate family (mefenamic acid, flufenamic acid), such as meclofenamic acid (36) and Sch 10304 (37), are still under clinical evaluation. The propensity of N-arylanthranilic acids to produce papillary necrosis in animals has been noted recently['33! In clinical use, the aza analog of flufenaminic acid, Nifluril (38), seems to be more irritating to the gastrointestinal tract than fenamates.

(7fH3 c1

aco:H NH

Q,,;, Two benzophenone derivatives['341, triflumidate (39) and diflumidone (40) bearing certain structural resem- blance to fenamates, showed some promise as effective but less irritating anti-inflammatory agents. The fluorinated methanesulfonylamino groups constituted novel variations of the carboxy group in anti-inflammatory aryl acids.

,S02CF3 H N -SO,CHF,

d ' . 0 2 C 2 H 5 c) (391 (40)

A new antiarthritic drug with a partial structure of phenylbutazone (or generally of 3,5-dioxopyrazolidines) is the azapropazon (MI 85, Apazone@) (41) [1351. This compound is generally comparable to phenylbutazone but is more active in the UV erythema assay. The daily dose for man is 0.6-1.2 g, i.e. about 2 4 times that for phenylbutazone[' '1.

\

CH3

Several aryl lactam derivatives, e. g. metazamide (42)[6.1371, tesikam (43)[13** 13'], nimazone (44)["01, and substituted pyridones (45) [1411, showed activity in animals, apparently

with activity-profiles different from aryl acids ; however, their further development was curtailed by unexpected toxicities encountered in the clinic or in animals.

A novel carbohydrate derivative, tribenoside (Glyvenol@, Ciba 21401-Ba) (46), was reported to be antiallergic, antiexudative, and capable of inhibiting antigen-antibody

qH2OR

reaction and capillary stabilizationr1421. It was not very effective in the treatment of rheumatoid arthritis pat- i e n t ~ [ ' ~ ~ ] .

5.2. Antirheumatic Agents

Some renewed interest in D-penicillamine (47) 14'1

and gold therapy has been noted among rheumatologists

Angew. Chem. internat. Edit. Vol. 11 (1972) / No. 6 469

in Europe and in the USA. Both agents have a slow onset, but sustained objective improvements in rheumatic patients are often observed after 3-6 months’ treatment. Their clinical application is still somewhat limited by various side effects. Unfortunately, no animal assay is available to aid the development of a superior agent with similar clinical efficacy. The mode of action of D-penicillamine remains speculative. There is not enough evidence to decide whether any of its biochemical properties considered previously, e. g. increase of tissue sulfhydryl groups[’481, dissociation of macroglobulins[1491, metal chelation, pyrid- oxine antagonism, and inhibition of collagen biosynthesis, play a major role in its antirheumatic action[1441.

Immunosuppressants, such as c y t ~ x a n [ ’ ~ ~ ] and imuran, have been used effectively in severe cases of rheumatoid arthritis, but with anticipated toxicities. The possible application of the less cytotoxic cinanserin (48)[15 was precluded by its hepatotoxicity. Contrary to the general

F[3C, r) ,C-CH-C02H

Sk AH,

(47 )

CI-I,

c=o I

CH (48) SH

assumption, no direct correlation could be made between the clinical efficacy of cyclophosphamide and its depression of several immunological parameters such as rheumatoid factor, serum immunoglobulins, and in citro lympho- cyte response etc>Ln‘x.The aciidty oj ‘immunosuppressive agents may be enhanced if potential antibody-forming cells are first stimulated by antigen. When administered with antigen the drug suppresses both the primary response and immunological memory. Specific tolerance induced in this way may last for several months[’531. It would be of great interest if the autoimmune component in rheumatoid arthritis could be suppressed by a similar strategy.

Recent interests in the immunological aspects of rheumatoid arthritis have focused attention on abnormal immunoglobulins, complement fixation, degradative enzymes, mediators of delayed hypersensitivity, and deranged metabolic activities of synovial cells in arthritic jointst3’. The current fervor in immunological research may suggest additional approaches to curtail the pathogenesis process of arthritis. Undoubtedly, a new series of non-cytotoxic and narrow-spectrum inhibitors or immunoregulants should emerge from many laboratories in the years to come. Their clinical applications, either alone or in conjunction with conventional anti-inflammatory therapy, may bring forth a new era of antirheumatic treatment.

6. Drug Combinations and Interactions

As indicated above, a major concern in the current search for new antiarthritic drugs is to minimize their side effects. The clinical efficacy of some drugs may be limited not by

their intrinsic activity but by their low ceiling of tolerable dosage. A logical approach to circumvent the side effects commonly associated with broad-spectrum nonspecific anti-inflammatory drugs would be the development of a group of narrow-spectrum agents, each acting specifically at one of the many facets of the complex inflammatory process, such as lysosomal enzymes, inflammation mediators, etc. A “cocktail” mixture comprising several such inhibitors has been suggested as an ultimate This futuristic approach of combination therapy is worthy of consideration as we become more proficient in deline- ating the pathogenesis of various arthritic disorders and in prescribing the optimal combination for individual cases. On the other hand, many adverse problems associated with drug interactions must be very carefully investigated. Perhaps a case in point is the growing awareness of the possible interaction between aspirin and other nonsteroid anti-inflammatory agents. Salicylate is often used concurrently with other drugs in the therapy of arthritis, either by design or unintentionally. In two independent animal studies[”5, 1561 no cumulative anti- inflammatory effect was observed with combinations of salicylate and other agents. A dramatic antagonism was observed in certain adjuvant arthritis protocol[’5h1, the inhibitor actions of indomethacin, phenylbutazone, and others were markedly diminished by low doses of aspirin given in combination. The mechanism of this antagonism is not clear, although it is reminiscent of the observation that the anticoagulant effect of warfarin is potentiated by aspirin or phenylbutazone through interference with serum protein binding of warfarin[157, 1581. A profound influence of salicylate, but not phenylbutazone, on the metabolism distribution and excretion of indomethacin by rats has recently been Possible factors involved are inhibition of intestinal absorption, alteration of tissue deposition, competitive glucuronide conjugation, and serum protein binding. Whether these laboratory observations are also true in the clinic remains to be verified.

7. Conclusion

In conclusion, the extensive chemical efforts on arylacetic acids, fenamates, and salicylates in recent years have gradually produced a small group of “second generation” clinical candidates of the usual anti-inflammatory-analgesic-antipyretic category. Nevertheless, better tolerated and more effective agents of this type are still needed. Progress towards the elucidation of mechanisms of action of known drugs and the understanding of their potential side effects, such as gastrointestinal irritation, hepatotoxicity, and papillary necrosis, should help in the selection of a safer antiarthritic candidate. It would be interesting to see whether some newer types of active structures under study in many laboratories will lead to the discovery of superior drugs having distinct biological profiles. From the pathogenesis point of view, immunological control seems to offer a new direction with many promising possibilities, but further knowledge and more convincing clinical verification of some concepts are still needed to establish a rational approach with a high degree

470 Angew. Chem. internat. Edit. 1 Val. 11 (1972) I N o . 6

of selectivity. To regulate the dynamic network of the chronic inflammatory process, advances in membrane chemistry, immunotolerance, and host resistance, may offer fresh perspectives.

[A 879 IE] German version: Angew. Chem. 84, 512 (1972)

Received: December 3,1971

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