Progress in the Design of Bioactive Molecules - American Chemical

Chapter 1 Progress in the Design of Bioactive Molecules John H. Block College of Pharmacy, Oregon State University, Corvallis, OR 97331-3507

The traditional search for drugs and agricultural chemicals has been based on observing the use of plants by human populations, dietary habits, general pharmacological screens, and chance observations. The discovery of many biologically active chemicals began with their isolation from natural products. These substances became the prototype molecules from which modifications were made. Other drug discoveries have been based on what could be called the shotgun approach and chance favors the prepared mind.

For the former, a

large number of compounds are synthesized or plant extracts are isolated and then subjected either to general or specific pharmacological screens. In today's economic and regulatory climate, this latter approach is very expensive because there are relatively few commercially successful products obtained from the thousands of compounds tested. Increasingly, a productive search for biologically active molecules requires a fundamental understanding of the disease for which the drug is targeted. Statistical techniques, conformational analysis, and receptor characterization will provide valuable information for the synthetic chemist to better tailor the molecular structure required for desired activity. Over the long history in the development of bioactive molecules, there have been many approaches used. These range from rational, carefully thought out hypotheses, to general and specific pharmacological screens used to identify compounds with desired biological activity, to serendipity. Listed below are different ways that commercially useful bioactive molecules have been discovered. Because of the author's background, there will be more examples from human medicine than from agrochemicals. At the same time, the principles discussed for humans will apply generally to mammals 0097-6156/89/O413-OO02$O7.00/0 © 1989 American Chemical Society

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Progress in the Design ofBioactive Molecules

including commercial livestock. In the broad sense, the development of chemicals used to maximize crop yields use the same approaches. NATURAL

PRODUCTS.

This has been and probably will remain one of the main means of discovering biologically active molecules. It is realized that there is a tremendous number of terrestrial plants that have never been screened and, unfortunately, may never be.QJ There is concern that, with the loss of the tropical rain forests, many plant species will become extinct before there is a chance to evaluate their chemical constituents. On the other hand, the examination of natural sources now includes marine species. The search for active compounds from natural product sources will continue because so little is known about the etiology of so many diseases that it is difficult to design potentially active molecules for these conditions. Some classic examples of successful drugs and agrochemicals derived from plant extracts and the results from attempts at chemically modifying their structures include the following. Cardiac glycosides: These are the drugs of choice in the treatment of congestive heart failure. The synthetic medicinal chemist has not produced a product superior to cardiac glycosides such as digoxin. Atropine: This is a classic example of the prototype drug from which the anticholinergic class of agents are derived. In contrast with digitalis, a wide variety of anticholinergics, chemicals which block the cholinergic receptors, which are superior to the parent alkaloid have been made and continue to be introduced into medicine. Cocaine: This drug, which has become such a pariah in our society, is the prototype for the local anesthetics. It is one of the success stories as evidenced by the wide variety of local anesthetics that have been made free of any abuse potential. Indeed, the structure of procaine, which is a simple benzoic acid ester, illustrates how the pharmacophore moiety can be abstracted from a more complex natural product. Penicillins, cephalosporins, tetracyclines, actinomvdns: These are examples of classes of antibiotics each from a microorganism producing cytotoxic agents. The first three classes are selectively cytotoxic to bacteria, and the fourth cytotoxic to mammalian cancer cells, unfortunately with poor selective toxicity. The penicillins and cephalosporins inhibit bacterial cell wall synthesis, and tetracyclines selectively block protein synthesis at the bacterial ribosome. The actinomycins intercalate in a relative nonselective manner the

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chromosomal DNA in both malignant and benign cells. They were initially screened in the 1940's for their antibacterial activity and, while active against bacteria, were found to be too toxic. Several years later they were found to be successful against selected cancers. While synthetic and semi-synthetic analogues of the antibiotics are continually being synthesized, evaluated and marketed, microorganisms are still being actively screened for new leads. Pyrethrins: This family of insecticides from plants of the genus Chrysanthemum were first used in the early 1800's and continue to be widely used to the present, particularly in household insecticide products because of their relatively low toxicity in humans. Classical compounds like the pyrethrins illustrate one of the more frustrating aspects in the design of bioactive molecules. Even though this chemical class has been used for nearly 200 years, little can be said regarding their mechanism of action. Further, it is doubtful that much basic research will be reported in the immediate future because there is little economic incentive in the private sector, and the government funding agencies likely will continue to award grants for projects that investigate more novel chemistry. Cyclosporin:

The history of this drug, isolated from the fungus

Cylindrocarpon ludidium and Trichoderma potysporum, shows how

perseverance of an individual scientist, Dr. Jean-Francoise Borel (Sandoz), has led to the marketing of a drug which has increased significantly the prognosis of patients receiving organ transplants. In 1978, the one year survival rate for was only 66 percent for transplanted hearts and 65 percent for transplanted kidneys. The most recent figures show the one year survival rates now to be 80 percent for hearts and 91 percent for kidneys. (2) The search for additional immunosuppressant drugs has led to a new natural product, FK-506, isolated from Streptomyces tsukubaensis No. 9993.(2) It appears that both compounds, as structurally diverse as they appear, possibly have the same mechanism of action. Using the newer techniques of conformational analysis, it will be interesting to determine if there is a common pharmacophore. The list of commercial products from natural sources could fill volumes. It must be emphasized that their discoveries range from systematic searches, to tradition to chance favors the prepared mind. The latter is

reemphasized in the discovery of the alkaloids from the periwinkle plant (vincristine and vinblastine) which were first screened for their hypoglycemic activity based on reports of their use by local groups in Madagascar. While the hypoglycemic response could not be confirmed under controlled laboratory conditions, an immunosuppressive effect due to

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drastic reduction of white cells was seen. This led to the introduction of two effective agents used to treat leukemias and lymphomas. (4,5) BIOCHEMICALLY

ACTIVE

MOLECULES:

The neurotransmitters and hormones are good examples based on the approach of starting with a biochemically active substance as the prototype molecule. This group of compounds combine with specific receptors, and therefore, provide the basic structure for synthetic modification in order to obtain more specific activity or even antagonistic response. The following will serve as examples of this approach at developing bioactive molecules. Acetylcholine: Both anticholinergics (antagonists) that block the cholinergic receptor and acetylcholinesterase inhibitors (potentiate acetylcholine) are in use today and are based on the acetylcholine structure. Because of the efficiency of acetylcholinesterase, it has proved more productive to inhibit the enzyme that hydrolyzes this neurotransmitter rather than develop cholinergic agonists. Acetylcholinesterase inhibitors has been a productive approach in the design of insecticides starting with the phosphate esters such as malathion and continuing on to carbamate esters such as carbaryl. For the latter, leads came from two carbamate reversible cholinesterase inhibitors used in medicine, the natural product physostigmine and the synthetic derivative, neostigmine. The history of the development of the organophosphate acetylcholinesterase inhibitors as insecticides is a classic example of examining the early literature and systematically synthesizing a large group of compounds. Contrary to popular opinion, this group of insecticides was not a spin off from the development of nerve gases. Indeed the research apparently began prior to the nerve gas research when industrial chemists in Germany began a search for synthetic chemicals to replace the insecticides nicotine, pyrethrum (see below) and rotenone which had to be imported into Germany. Based on earlier published work, a large number of organophosphate chemicals were synthesized from which active acetylcholinesterase inhibitors were obtained. It was only later, after the toxicity of the organophosphates was realized, that research on nerve gas began in earnest. Histamine: At least two responses are attributed to this neurotransmitter derived from the amino acid histidine. It is part of the allergic response (Hj receptor) and, in the stomach, stimulates the release of gastric hydrochloric acid (H receptor). Mild allergic responses, such as hay fever, have been treated for years with antihistamines. Their development pretty much followed the classical approach where 2

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histamine was first realized to be a crucial component of the allergic response. There already were known compounds which had antihistamine activity, but they were too toxic. The first clinically useful antihistamines were reported in 1942 to be followed by hundreds of useful compounds.© Co-recipient of the 1988 Nobel Prize in Medicine was Sir James Black who observed that antihistamines did not work against ulcers and postulated that there must be a second histamine receptor, now called the H receptor. This led to a new class of histamine antagonists (e.g. cimetidine and rantidine) which has greatly altered the treatment of peptic ulcers. No longer are patients with peptic ulcers dependent on dosing themselves with antacids. What is interesting to note from the structure activity relationship (SAR) aspect is the traditional antihistamines ( H ; blockers) bear little structural resemblance to histamine whereas the H blockers do. 2

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Cbrtisone/Hvdrocortisone: A large number of steroid analogues have been made in order to separate the glucocorticoid (antiinflammatory) response from the mineralcorticoid (fluid retention) with a fair degree of success. The result has been a large number of synthetic corticosteroids used systemically and topically for their antiinflammatory activity. Phenoxvacetic adds: The development of the 2,4-dichloro- and 2,4,5trichlorophenoxyacetic acids (R = H and CI, respectively) was an outgrowth of work based on the plant hormone activity (auxin) of indole-3-acetic acid. Based on the earlier concepts of rigid receptors, there was little likelihood that the chlorinated phenoxyacetic acids would show auxin activity. When it was realized that the margin of safety between induction of healthy root growth and the induction of excessive root thickening was too small for this group to be used as growth simulators, their use as herbicides developed. (6) Of course, today it is realized that substituted benzene rings can be used as bioisoteric replacements for heterocyclic rings such as indole. The opportunities for developing new approaches for attacking medical and agricultural problems is limited only by the complexity of the biochemical milieu of interest, i.e. the greater the complexity, the greater the number of opportunities. An example of exciting new approaches are the chapters in this book discussing the adenosine receptor in the heart which has potential use in developing new cardiotonic and antiarrhythmic agents, a group of potent parathyroid hormone antagonists that may prove useful in the treatment of hypercalcemia, and the serotonin receptor, the first of which are just coming onto the market.

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TOXICITY.

The ideal bioactive molecule acts solely on the target organ, organism, or receptor. The antibacterial agents in use today are excellent examples of this approach in human medicine. In general the key is to identify a metabolic reaction unique to the microorganism or an enzyme used by the microorganism or agricultural pest that is so physically or chemically different that the drug will have no significant effect on the patient's metabolism. Professor Adrian Albert takes a broader view of the term selective toxicity.Q) He considers antagonists as being toxic in the sense that they occupy receptors preventing the binding of the normal ligand. Thus an antihistamine shows selectivity by combining mostly with the H ; receptor and ignoring the H receptor. This class of drugs is not completely selective as evidenced by their centrally acting depressant actions leading to sedation and the anticholinergic response. The latter can range from a nuisance in some individuals to potentially harmful in asthmatics who may have trouble expectorating fluids from their lungs. The more restricted approach to selective toxicity will be used in this chapter and in the following examples. 2

Trimethoprim/Methotreiate: Both drugs inhibit dihydrofolate reductase, but trimethoprim (developed by George Hitchings and Gertrude Elion, also 1988 co-recipients of the Nobel Prize in Medicine) is selective for the bacterial dihydrofolate reductase while methotrexate is an inhibitor of the mammalian enzyme and is used in cancer chemotherapy. The latter drug cannot distinguish between the enzyme in malignant and normal cells with the result that it is a very toxic drug. Many times the antidote, calcium leucovorin which is the calcium salt of one of the forms of tetrahydrofolic acid, is administered to the patient following a course of intense methotrexate therapy. Because the pteridine ring is already reduced, calcium leucovorin does not require conversion by active dihydrofolate reductase into an active form. The discovery of methotrexate again shows how alert scientists exploit what first appeared to be a puzzling observation. It was found that administration of folates to patients with acute leukemia hastened the progress of the disease. Positive results from crude folate antagonists that were available at the time led eventually to the synthesis of methotrexate.© Tetracyclines: This very successful class of antibacterials act selectively on the bacterial ribosome inhibiting protein synthesis. They do not bind with mammalian ribosomes in the cytoplasm and, therefore, do not have a direct effect on the patient's metabolism. Like any drug, they are not free of potentially harmful side effects. They complex calcium and can interfere with development of the permanent teeth prior to their erupting through the gums As with the sulfonamides, this class


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of drugs was discovered as the result of an antibacterial screen of bacterial extracts. Acyclovir: This agent is quite effective against Herpes Simplex II (genital herpes) if the patient follows the regimen carefully. This drug is selective for viral thymidine kinase which converts acyclovir to the nucleotide triphosphate whereas the host cell kinase does not. Thus the drug remains in tl>e inactive prodrug form in noninfected cells. The acyclovir triphosphate, which lacks a 3'-OH, now inhibits viral DNA polymerase preventing the synthesis of the new viral DNA needed for herpes virus reproduction. Azidothvinidine: This drug shows a reasonable degree of selectivity for the viral RNA dependent DNA polymerase (reverse transcriptase), an enzyme found only in retroviruses. Because the Human Immunodeficiency Virus-I (HIV-1), the cause of Acquired Immunodeficiency Syndrome (AIDS), requires this enzyme to reproduce itself, azidothymidine slows the progress of this tragic, largely preventable disease. But bone marrow depression is a common complication indicating that it is inhibiting cell division in the patient. Antifungal Agents: It has been very difficult to design agents effective against fungal infections whether they are in humans, livestock or plants. In theory it should be possible to control fungal infections/infestations with the appropriate chemical because they do have unique biochemistry which can be exploited. For example, the imidazole class of antifungal agents (miconazole, ketoconazole) are selective for the incorporation of acetate into ergosterol, a route not found in humans. In practice, fungal infections can be very difficult to control. The structure activity relationships of the agents used for the fungal caused diseases are very diverse. Besides the imidazoles, there is the antibiotic griseofulvin which is active against both plant and animal fungal infections and binds preferentially to fungal RNA and the polyene antibiotics (nystatin, amphotericin B) which bind preferentially to the ergosterol in fungal membranes relative to cholesterol in mammalian cell membranes. (S) For more information, please see the chapter in this book describing approaches for developing fungicides used in agriculture based on taking advantage of the metabolic differences between the fungus and its host. Ideally, selective toxicity is the one of the best approaches to use in the design of biologically active molecules. It is very expensive and requires a considerable investment of time and capital because the metabolism of the pathogen must be elucidated in order to locate the unique transformations,

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enzymes or structures that can be the target of chemical intervention. In practice, it has worked best for treatment of bacterial diseases as there is a reasonable chance that there is some key metabolic difference between the pathogen and the host. Bacteria have cell walls rather than membranes (selective site for penicillin) and unique ribosomes (selective site of the tetracyclines). Bacterial infections are not feared in developed economies due to the large armamentarium of antibiotics available to the medical and veterinary professions. Fungal infections have proved to be more of a problem in both human and plant diseases. Fortunately, many fungi are found on the surface of the skin or on the plant and can be treated topically. This permits the use of some fairly toxic agents which, as long as they are not absorbed, cause little harm to the host and can be washed off the plant. Parasites and insects can have such complex metabolism and life histories that use of chemical agents has had only limited success when the offending organism is a pathogen for humans or livestock. First, it has been difficult to find chemicals that are selective for only the offending species. Many go through various changes as they move from egg to larva to mature adult. The organism's susceptibility will change with the stage in its life. If the organism cannot be stopped outside of the animal or plant host, a bioactive chemical will have to be introduced into the patient or plant in the form of a systemic agent. These can be very toxic to humans and animals requiring repeated applications until the patient is free of the organism. Compliance in humans is a problem due to the harsh side effects of these drugs. Use of these chemicals is complicated further in livestock or plants because it is more difficult to remove the chemical prior to or during food processing. Obviously it is easier to wash off a chemical from the surface of the plant or a dipped animal. At the same time it must be realized that since plants do not have nervous systems, very toxic pesticides can be applied without harm to plants. In other words, the principle of selective toxicity works very well in terms of protecting the plants against a variety of insects. Viruses have proved to be a real dilemma. First, they can only reproduce inside the cell. This means that the chemical agent must enter the cell in order to reach the virus. Compare this with the previously described pathogens in which the bioactive agent intercepts the organism before it penetrates the patient's cells. The alternative is to take an antiviral drug prophylactically. To date there is only one agent in the U. S. market, amantadine (see below), which is a very effective prophylactic agent against the Type A influenza virus. At the same time, most patients prefer vaccination because it is simpler and much less expensive. Most antiviral agents are nucleotide antimetabolites similar in structure to the nucleotide antimetabolites used in cancer chemotherapy. The viruses that lend themselves to drug therapy are those with complicated genomes. The herpes simplex virus has over 120 genes making it likely that there are unique enzymes, such as the viral thymidine kinase, required for

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its reproduction. Similarly, the HIV-I, a retrovirus with the unique reverse transcriptase enzyme, is potentially vulnerable to rationally designed molecules. METABOLISM OF FOREIGN

MOLECULES

(XENOBIOTICS).

A drug should be considered a xenobiotic. All mammals have the capabilities to transform these molecules. The drug metabolizing enzymes are a misnomer because their natural substrates are part of normal metabolism. Among other things, these enzyme systems hydroxylate steroids, degrade the porphyrin rings from aged erythrocytes, and conjugate the bile pigment, bilirubin, with glucuronic acid. Fortunately, the substrate specificities do not appear to be very strict with this diverse group of enzymes. The net result is that most drugs can be administered to humans and livestock with the correct assumption that, in most cases, they will be transformed and excreted. In other words, the elimination of administered drugs from the tissues of commercial livestock is due to these diverse group of enzymes. Indeed, the measurement of the biological half-life and determination of the metabolic fate are part of any application requesting permission to market a drug. It is now realized that many drugs are converted to active metabolites. Indeed, the parent drug molecule may largely be inactive. This has led to a systematic approach called prodrug design. (2) While there have been elegant approaches published for getting drugs to specific organs by the prodrug approach, the rigors of obtaining approval of new chemical entities has restricted this technique largely to the use of simple esters. This can be illustrated with Vitamin E or a-tocopherol. The acetate ester produces an oil soluble vitamin while the hemisuccinate yields a water soluble derivative. The new hypotensive drug, enalapril (see below) is marketed as the ethyl ester because the free acid, enalaprilic acid, is poorly absorbed orally. All of these examples are hydrolyzed in the patient to the active form. Examine the label on a container of multivitamins and notice how many of the vitamins are in a chemically more stable precursor form: retinol acetate or palmitate (retinal), pyridoxine (pyridoxal), and pantothenol (pantothenic acid). A study of a drug's metabolism can lead to the design of better compounds. This is illustrated by the popular local anesthetic, lidocaine, which also is an excellent drug for the treatment of arrhythmias. It has one drawback. It is so rapidly N-dealkylated by the hepatic cytochrome P450 enzyme system followed by hydrolysis that it cannot be given orally. (10) The initial N-deethylated product, monoethylglycinexylide, has excellent antiarrhythmic activity. This information led to the development of tocainide, an orally active antiarrhythmic drug. It can be considered the or-methyl analog of glycinexylide.

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Progress in the Design of Bioactive Molecules - American Chemical

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