INTRODUCTION

TOP ABSTRACT INTRODUCTION CONCLUSION DISCUSSION REFERENCES

The path leading to potent and selective cysteinyl leukotriene (cysLT) receptor antagonists began when slow-reacting substance of anaphylaxis (SRS-A) was identified by its ability to elicit a slowly developing and long-lasting contraction of guinea pig ileum and shown to be distinct from histamine (1). The subsequent identification by scientists at Fisons Pharmaceuticals Laboratories of FPL 55712, a chromone carboxylic acid, as a prototype SRS-A antagonist provided medicinal chemists with a starting point in structure-based drug design and gave pharmacologists a tool to probe the bioactivity of SRS-A (2). Then, in 1979 SRS-A was identified as a family of lipid mediators, termed "leukotrienes," a name that was derived from their cell source (leukocytes) and their unusual conjugated triene chemical structure (3). Three members of the leukotriene family, known as LTC₄, LTD₄, and LTE₄, and collectively as cysLTs, accounted for the original bioactivity of SRS-A.

	MEDICINAL CHEMISTRY APPROACHES

With the ability of SRS-A and, subsequently, synthetic cysLTs to mimic many features of asthma, numerous laboratories began the race to find potent and selective cysLT receptor antagonists, with the expectation that they would become novel, effective anti-asthma drugs. This hypothesis was based on the ability of SRS-A and synthetic cysLTs to contract airway smooth muscle, enhance vascular permeability, and stimulate mucous secretion (4). The importance of inflammation in asthma was not fully appreciated at that time. In 1980, three approaches were available to medicinal chemistsleukotriene structural analogs, FPL 55712 analogs, or other compounds found during screens of corporate compound banks.

Early Rational Structure-based Approaches

At Zeneca Pharmaceuticals, for example, early analysis of the structures of LTD₄ and FPL 55712 led to two hypotheses: (1) the hydroxyacetophenone region of FPL 55712 was mimicking the olefinic region of LTD₄; and (2) the chromone carboxylic acid segment of FPL 55712 was mimicking one of the other two regions of LTD₄, either the backbone C₁-C₅ carboxylic acid region or the peptidic component (Figure 1). In order to test these hypotheses, four hybrid molecules were synthesized that combined each region of FPL 55712 with surrogates for either the peptidic or C₁-C₅ region of LTD₄. An aliphatic acid structure was one of the first compounds identified that exhibited modest cysLT antagonist activity. In order to reduce its conformational flexibility, an aryl group was incorporated into the structure with a resulting increase in potency. An exhaustive structure-activity relationship (SAR) analysis concluded that addition of a methoxy group to the aryl moiety was associated with a modest increase in potencythe best compound was approximately fourfold more potent and 2.5-fold more selective than FPL 55712 (7).

View larger version (17K): [in this window] [in a new window]   Figure 1.   Structures of LTD4 and FPL 55712: starting points in the design of leukotriene receptor antagonists. Analogs of FPL 55712 containing surrogates for either the peptidic or C1-C5 region of LTD4 were synthesized. An aliphatic acid structure exhibited modest cysLT antagonist activity. An exhaustive structure-activity relationship analysis was performed after conformational flexibility was reduced by incorporating an aryl group. The best compound developed was approximately fourfold more potent and 2.5-fold more selective than FPL 55712. Reprinted with permission from P. R. Bernstein, 1997. The challenge of drug discovery: developing leukotriene antagonists. In S. T. Holgate and S. E. Dahlén, editors. SRS-A to Leukotrienes. Blackwell Scientific Publications, Boston. 171-187.

Similar approaches at other pharmaceutical companies revealed other FPL 55712-derived compounds that were then clinically evaluated (8). These included YM-16638, Ro 23-3544 (ablukast), L-649,923, and LY 171,883 (tomelukast). Of these, tomelukast was the most important for several reasons. First, it demonstrated that replacement of a carboxylic acid group with a biomimetic tetrazole may result in large increases of in vitro and in vivo potency. Second, the results from clinical studies with tomelukast were promising (9); however, they demonstrated that even further increases in potency would be required. Although subsequently withdrawn from clinical study due to toxicity, the tomelukast experience spurred additional efforts in the search for cysLT receptor antagonists.

Random Screening of Compound Banks

The discovery of pranlukast (ONO 1078) may be traced back to a series of carboxylic acids that were probably identified through random screening (10) (Figure 2). During the optimization of these early leads, two intervening compounds were made that demonstrated links to other chemical series. This "cross-fertilization" of ideas enhanced cysLT receptor antagonist drug discovery efforts. First, restricting rotational freedom via incorporation of a ring-containing substructure led to the discovery that the chromone carboxylic acid segment of FPL 55712 resulted in a 90-fold increase in potency. Second, replacement of the carboxylic acid with tetrazole, as in the tomelukast example, led to a further increase in potency. Optimization of the lipid backbone then led to pranlukast, the first cysLT receptor antagonist approved for marketing (11). Pranlukast is currently sold in Japan.

View larger version (22K): [in this window] [in a new window]   Figure 2.   Identification of leads by random screening: the path to pranlukast. The development of pranlukast may be traced to a series of carboxylic acids. Two intervening compounds that demonstrate links to other chemical series were made during series optimization. Restricting conformational flexibility led to incorporation of the chromone carboxylic acid structure of FPL 55712 and increased potency. The carboxylic acid was replaced with the tetrazole of tomelukast in order to improve bioavailability. Optimization of the lipid backbone then led to pranlukast. Adapted from P. R. Bernstein, 1997. The challenge of drug discovery: developing leukotriene antagonists. In. S. T. Holgate and S. E. Dahlén, editors. SRS-A to Leukotrienes. Blackwell Scientific Publications, Boston. 171-187. Reprinted with permission.

A second series of cysLT receptor antagonists, the oxymethylquinolines, were also discovered by chance. An early analog, Rev-5901, was prepared as a 5-lipoxygenase inhibitor, then found to be a weak cysLT receptor antagonist. Several compounds with improved potency and oral bioavailability were identified and entered into clinical development. These included WY-48,252, SR2640, and RG 12525; their development, however, appears to have been suspended for unknown reasons (12).

Leukotriene Analogs

Efforts to design LT receptor antagonists based solely on leukotriene structure faced two major hurdles: replacing the inherently unstable triene-containing chain and converting cysLT agonist activity into antagonist activity. Based on nuclear magnetic resonance (NMR) (13) and molecular modeling results, initial efforts explored replacing the unstable triene with a set of aryl-containing groups. This led to the examination of homo-cinnamyl-containing LT mimics (14). These compounds acted as agonists, but subsequent variation of the peptidyl chain led to compounds that were partial agonists or weak antagonists (15). One of the acid moieties was then deleted to simplify the structure and remove the potential for agonist activity. The resulting compound was a pure antagonist, although less potent than FPL 55712.

A number of other cysLT receptor antagonists based on the leukotriene structure were identified and advanced into clinical trials. These include BAY x7195, LY170680 (sulukast), SKF 104353 (pobilukast), CGP 45715A, and MK-0476 (montelukast sodium) (8). The Bayer group incorporated two aspects from other chemical series into its compoundthe homo-cinnamyl triene replacement pioneered at Zeneca, and shortened links to the carboxylic acids, a modification pioneered at SmithKline in the development of pobilukast. CGP 45715A is especially interesting, because it incorporates both the hydroxyacetophenone and chromone carboxylic acid motifs from FPL 55712 into a leukotriene-based lipid backbone. Of these cysLT analog antagonists, montelukast sodium is the most clinically advanced.

The starting point in the development of montelukast appears to be a quinoline-containing structure, likely identified as a weak random screening lead (Figure 3). The Merck group hypothesized that this molecule was mimicking the olefin backbone of cysLTs, and that the addition of mimics for the acid and peptide regions of LTD₄, might improve its potency. As a first step, the dithioacetal linkage first seen in some SmithKline compounds was incorporated; this led to a compound with greatly increased in vitro potency but poor oral bioavailability. When one of the carboxylic acids was replaced by an amide, forming MK-571, the new antagonist had even greater potency and good efficacy following oral administration. The enantiomers were resolved to yield MK-679 (verlukast), a compound with better clinical effects than MK-571, but whose clinical development was stopped for safety reasons. Further structure-activity relationship studies led to the development of montelukast (16), an antagonist that appears free of the safety concerns plaguing earlier members of this series.

View larger version (20K): [in this window] [in a new window]   Figure 3.   Evolution of montelukast sodium. The starting point in the identification of montelukast appears to be a quinolone-containing structure. The dithioacetal linkage first seen in some SmithKline compounds was incorporated and one of the carboxylic acids was then replaced by an amide to form MK-571. The enantiomers were resolved to yield verlukast. An extensive structure-activity relationship analysis led to the development of montelukast. Reprinted from Reference 8 by permission.

	DEVELOPMENT OF ZAFIRLUKAST

As described above, based upon the cysLT structure, the Zeneca group identified lipid-acid analogs that were weak receptor antagonists. In order to increase potency, an attempt was made to restrict these compounds to a bioactive conformer by reducing their flexibility. An indazole ring was used as the initial template to test this hypothesis. Further synthetic work involved indole substitution for the indazole, and by analogy to the hydroxyacetophenone work done earlier, a 3-methoxy group was added to the acidic aryl group. These changes produced a compound with potency comparable to FPL 55712, but with fivefold greater selectivity (17).

To rapidly explore the structure-activity relationships surrounding this lead structure, changes were made simultaneously in three regions of the molecule: the lipid-like tail, the heterocyclic body, and the acidic head (Figure 4). In the lipid-like tail region, branching at the - or -carbons led to increased in vitro potency and selectivity, but most importantly, to increased oral efficacy in animal models. A preferred substitution was a carbocyclic moiety, which resulted in a compound that had 100-fold greater potency than the original indazole lead. The structure-activity relationship studies demonstrated that the amide link could be replaced by other carbonyl-containing groups, such as carbamate.

View larger version (12K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   View larger version (36K): [in this window] [in a new window]   Figure 4.   Evolution of zafirlukast. Initial structure-activity relationship analyses on the lipid-acid analogs described in Figure 1 led to the fabrication of an indole-containing lead compound (A). Modifications were simultaneously made on three regions of this molecule: (B) the lipid-like tail region, (C ) the acidic head region, and (D) the indole backbone. These efforts led to the identification of ICI-198,615, a potent and selective leukotriene receptor antagonist having weak oral bioavailability. An inverted indole template was incorporated, yielding zafirlukast (ICI 204219, Accolate). Adapted from P. R. Bernstein, 1997. The challenge of drug discovery: developing leukotriene antagonists. In S. T. Holgate and S. E. Dahlén, editors. SRS-A to Leukotrienes. Blackwell Scientific Publications, Boston. 171-187. Reprinted with permission.

A broad exploration of the acidic head region was undertaken, based in part upon the Lilly group's results with tetrazoles in the discovery of tomelukast. Various links to the indole group and the nature of the acidic group itself were both explored. Structure-activity relationship studies demonstrated that a preferred linkage occurred with a 3-methoxy aryl group; however, replacement of the carboxylic acid head group with phenylsulfonimide produced a 100-fold increase in potency. More importantly, this change was additive to other alterations made in the amide region of the tail. The in vitro potency of this agent was now more than 1,000-fold greater than FPL 55712. The increased potency, as well as a longer duration of action, were observed in vivo, as the new lead compound could be evaluated in animal models using a 3-h pretreatment time and an oral dose of 30 µmol/kg (18).

Variations of the indole backbone were explored simultaneously. While a number of variants were tolerated in vitro, these substitutions had a dramatic effect on oral bioavailability and therefore on oral potency (19). An inverted indole template was associated with better in vivo properties (20). One of the most potent compounds synthesized was ICI 198,165. It had a pK_B of 10.3 when evaluated as an antagonist of LTE₄-induced contractions of isolated guinea pig trachea (21). However, in the guinea pig, it had an oral/intravenous ratio of 333, and oral bioavailability of less than 1% in rats and dogs (22).

Although the inverted indole template exhibited better bioavailability properties, it was initially not readily available from a synthetic vantage. Thus, most synthetic work exploring sulfonamide substitutions was done in the normal indole series. After observing that sulfonamide substitutions, such as 2-methyl or 2-chloro groups, had a dramatic beneficial effect on oral activity (23), they were applied to the inverted indole template, forming zafirlukast. In its initial biological screening, zafirlukast provided a pK_B of 9.7 in inhibiting LT-induced contraction of guinea pig trachea and an in vivo ED₅₀ of 0.52 µmol/kg against LTD₄-induced dyspnea in conscious guinea pigs (ED₅₀ = dose producing 50% inhibition of dyspnea). Its bioavailability was 68% in rats and 67% in dogs. The synthesis of zafirlukast is depicted in Figure 5.

View larger version (20K): [in this window] [in a new window]   Figure 5.   Synthesis of zafirlukast.

	CONCLUSION

TOP ABSTRACT INTRODUCTION CONCLUSION DISCUSSION REFERENCES

The synthesis of a new compound is merely the first step in a series of hurdles that, more likely than not, preclude a therapeutic agent from reaching the market. Identification of metabolites and degradation products, formulation and manufacturing issues, and, of course, safety and efficacy evaluations may lead to termination of a drug's development at any point.

The approval of zafirlukast in a number of countries, pranlukast in Japan, and the fact that additional leukotriene receptor antagonists are now poised to enter the market in the foreseeable future bear witness to the fulfillment of a monumental intergenerational, cross-disciplinary effort. From the first discovery of SRS-A to the marketing of these agents took almost 60 years of effort by many dedicated people. Now, those dedicated people have the opportunity to help those in need benefit from a new, safe, and effective treatment for asthma.

	DISCUSSION

TOP ABSTRACT INTRODUCTION CONCLUSION DISCUSSION REFERENCES

Drazen: Was the guinea pig ileum used as your primary biological assay?

Bernstein: No. We didn't use guinea pig ileum as a regular assay. When we first started, our studies were done on guinea pig trachea using LTD₄ as the agonist. We measured the ability of our compounds to inhibit LTD₄-induced contraction. Then Dr. Carl Buckner showed that the effects of LTE₄ in guinea pigs more closely resembled the effects of LTD₄ on human bronchi. We then changed over to using LTE₄ as the agonist in guinea pig trachea. Later in the project, after Dr. David Aharony joined the team, we used a binding assay that he developed. The binding assay came into use at about the time that we discovered ICI 198,615 and zafirlukast. These compounds were found by exploring their ability to block agonist activity on guinea pig trachea.

Dahlén: I think that the development of this class of antagonists is a tremendous achievement, and the fact that some have minimal liver enzyme induction properties is an important point to stress.