Chimica Farmaceutica

Drug design Selective Optimization of Side Activities (SOSA)

Sometimes slight changes to a structure can result in significant changes in pharmacological activity. For example, minaprine (Fig. 2) is an antidepressant agent that acts as a serotonin agonist. Adding a phenolic substituent resulted in 4-hydroxyminaprine, which is a potent dopamine agonist, whereas adding a cyano substituent gave bazinaprine, which is a otent inhibitor of the enzyme monoamine oxidase-A. Minaprine also binds weakly to muscarine receptors (Ki = 17 pM) and modifications were successfully carried out to give structure I with nanomolar activity (Ki = 3 nM) for the muscarinic receptor and negligible activity for dopamine and serotonin receptors. Minaprine also has weak affinity for the cholinesterase enzyme (IC50 = 600 pM). Modifications led to structure II, which has strong affinity (IC50 = 10 nM) for this target.

Laurea in Biotecnologie Page 2 Starting from the natural ligand or modulator. Natural ligands for receptors

The natural ligand of a target receptor has sometimes been used as the lead compound. The natural neurotrans mitters adrenaline and noradrenaline were the starting points for the development of adrenergic p-agonists such as salbutamol. dobutamine and xamoterol, and 5-hydroxytryptamine (5-HT) was the starting point for the development of the 5-HT1 agonist sumatriptan. The natural ligand of a receptor can also be used as the lead compound in the design of an antagonist. For example, histamine was used as the original lead compound in the development of the H2 histamine antagonist cimetidine. Turning an agonist into an antagonist is frequently achieved by adding extra binding groups to the lead structure. Other exam ples include the development of the adrenergic antagonist pronethalol, the H2 antagonist burimamide, and the 5-HT3, antagonists ondansetron and granisetron.

Laurea in Biotecnologie Page 3 Starting from the natural ligand or modulator. Natural ligands for receptors

Sometimes the natural ligand for a receptor is not known (an orphan receptor) and the search for it can be a major project in itself. If the search is successful, however, it opens up a brand-new area of drug design (Box 12.5). For example, the identification of the opioid receptors for morphine led to a search for endogenous opioids (natural body painkillers) which eventually led to the discovery of endorphins and enkephalins as the natural ligands, and their use as lead compounds. Natural ligands as lead compounds The discovery of cannabinoid receptors in the early 1990s led to the discovery of two endogenous cannabinoid messengers arachidonylethanolamine (anandamide) and 2-arachidonyl glycerol. These have now been used as lead compounds for developing agents that will interact with cannabinoid receptors. Such agents may prove useful in suppressing nausea during chemotherapy, or in stimulating appetite in patients with AIDS.

Laurea in Biotecnologie Page 4 Starting from the natural ligand or modulator. Natural substrates for enzymes

The natural substrate for an enzyme can be used as the lead compound in the design of an enzyme inhibitor. For example, enkephalins have been used as lead compounds for the design of enkephalinase inhibitors. Enkephalinases are enzymes which metabolize enkephalins, and their inhibition should prolong the activity of enkephalins. The natural substrate for HIV-protease was used as the lead compound for the development of the first protease inhibitor used to treat HIV. Other examples of substrates being used as lead compounds for inhibitors include the substrates for farnesyl transferase and matrix metalloproteinase

Enzyme products as lead compounds It should be remembered that enzymes catalyse a reaction In both directions and so the product of an enzyme-catalysed reaction can also be used as a lead compound for an enzymeLaurea ininhibitor. Biotecnologie For example, the design of the carboxypeptidase inhibitorPage 5L- benzylsuccinic acid was based on the products arising from the carboxypeptidase- catalysed hydrolysis of peptides. Natural modulators as lead compounds Many receptors and enzymes are under allosteric control. The natural or endogenous chemicals that exert this control (modulators) could also serve as lead compounds. In some cases, a modulator for an enzyme or receptor is suspected but has not yet been found. For example, the benzodiazepines are synthetic compounds that modulate the receptor for γ-aminobutyric acid (GABA) by binding to an allosteric binding site. The natural modulators for this allosteric site were not known at the time benzodiazepines were synthesized, but endogenous peptides called endozepines have since been discovered which bind to the same allosteric binding site and which may serve as lead com pounds for novel drugs having the same activity as the benzodiazepines.

Laurea in Biotecnologie Page 6 Combinatorial and parallel synthesis The growing number of potentially new drug targets arising from genomic and proteomic projects has meant that there is an urgent need to find new lead compounds to interact with them. Unfortunately, the traditional sources of lead compounds have not managed to keep pace and in the last decade or so, research groups have invested greatly in combinatorial and parallel synthesis in order to tackle this problem. Combinatorial synthesis is an automated solid-phase procedure aimed at producing as many different structures as possible in as short a time as possible. The reactions are carried out on very small scale, often in a way that will produce mixtures of compounds in each reaction vial. In a sense, combinatorial synthesis aims to mimic what plants do, i.e. produce a pool of chemicals, one of which may prove toLaurea be in Biotecnologiea useful lead compound. Combinatorial synthesisPage 7 has developed so swiftly that it is almost a branch of chemistry in itself. Parallel synthesis involves the small scale synthesis of large numbers of compounds at the same time using specialist miniaturised equipment. The synthesis can be carried out in solution or solid phase, and each reaction vial contains a distinct product. Computer-aided design of lead compounds A detailed knowledge of a target binding site significantly aids in the design of novel lead compounds intended to bind with that target. In cases where enzymes or receptors can be crystallized, it is possible to determine the structure of the protein and its binding site by X-ray crystallography. Molecular modelling software programs can then be used to study the binding site, and to design molecules which will fit and bind to the site: de novo drug design . In some cases, the enzyme or receptor cannot be crystallized and so X-ray crystallography cannot be carried out. However, if the structure of an analogous protein has been determined, this can be used as the basis for generating a computer model of the protein. NMR spectroscopy has also been effective in determining the structure of proteins and can be applied to proteinsLaurea that in Biotecnologie cannot be studied by X-ray crystallography. Page 8 Serendipity and the prepared mind

Frequently, lead compounds are found as a result of serendipity (i.e. chance). However, it still needs someone with an inquisitive nature or a prepared mind to recognize the significance of chance discoveries and to take advantage of these events. The discovery of cisplatin and penicillin are two such examples, but there are many more. Sometimes, the research carried out to improve a drug can have unexpected and beneficial spin offs. For example, propranolol and its analogues are effective β-blocking drugs (antagonists of β-adrenergic receptors). However, they are also lipophilic, which means that they can enter the central nervous system and cause side effects. In an attempt to cut down entry into the central nervous system, it was decided to add a hydrophilic amide group to the molecule and so inhibit passage through the blood-brain barrier. One of the compounds made was practolol. As expected, this compound had fewer side effects acting on the central nervous system, but more importantly, it was found to be a selective antagnist for the β-receptors of the heart over β-receptors in other organs: a result that was highly desirable, but not the one that was being looked for at the time. Frequently, new lead compounds have arisen from research projects carried out in a totally different field of medicinal chemistry. This emphasizes the importance of keeping an open mind, especially when testing for biological activity.

Laurea in Biotecnologie Page 9 Examples of serendipity

During the Second World War, an American ship carrying mustard gas exploded in an Italian harbour. It was observed that many of the survivors who had inhaled the gas lost their natural defences against microbes. Further study showed that their white blood cells had been destroyed. It is perhaps hard to see how a drug that weakens the immune system could be useful. However, there is one disease where this is the case: leukemia. Leukemia is a form of cancer which results in the excess proliferation of white blood cells, so a drug that kills these cells is potentially useful. As a result, a series of mustard like drugs were developed based on the structure of the original mustard gas. Another example involved the explosives industry, where it was quite common for workers to suffer severe headaches. These headaches resulted from dilatation of blood vessels in the brain, caused by handling trinitrotoluene (TNT). Once again, it is hard to see how such drugs could be useful. Certainly, the dilatation of blood vessels in the brain may not be particularly beneficial, but dilating the blood vessels in the heart could be useful in cardiovascular medicine. As a result, drugs were developed which dilated coronary blood vessels and alleviated the pain of angina. Workers in the rubber industry found that they often acquired a distaste for alcohol! This was caused by an antioxidant used in the rubber manufacturing process which found its way into workers' bodies and prevented the normal oxida tion of alcohol in the liver.

Laurea in Biotecnologie Page 10 Examples of serendipity

As a result, there was a build up of acetaldehyde, which was so unpleasant that workers preferred not to drink. The antioxidant became the lead compound for the development of disulfiram (Antabuse) — used for the treat ment of chronic alcoholism. The following are further examples of lead compounds arising as a result of serendipity: Clonidine was originally designed to be a nasal vasoconstrictor to be used in nasal drops and shaving soaps. Clinical trials revealed that it caused a marked fall in blood pressure, and so it became an important antihypertensive instead, Imipramine was synthesized as an analogue of chlorpromazine, and was initially to be used as an antipsychotic. However, it was found to alleviate depression and this led to the development of a series of compounds classified as the tricyclic antidepressants.

Laurea in Biotecnologie Page 11 Examples of serendipity

Laurea in Biotecnologie Page 12 Computerized searching of structural databases

New lead compounds can be found by carrying out computerized searches of structural databases. In order to carry out such a search, it is necessary to know the desired pharmacophore. Alternatively, docking experiments can be carried out if the structure of the target binding site is known. This type of database searching is also known as database mining. Fragment-based lead discovery

So far we have described methods by which a lead com pound can be discovered from a natural or synthetic source, but all these methods rely on an active compound being present. Unfortunately, there is no guarantee that this will be the case. Recently, NMR spectroscopy has been used to design a lead compound rather than to dis cover one. In essence, the method sets out to find small molecules (epitopes) which will bind to specific but different regions of a protein's binding site. These molecules will have no activity in themselves since they only bind to one part of the binding site, but if a larger molecule is designed which links these epitopes together, then a lead compound may be created which is active and which binds to the whole of the binding site.

Laurea in Biotecnologie Page 13 Fragment-based lead discovery

Laurea in Biotecnologie Page 14 Fragment-based lead discovery Lead discovery by NMR is also known as SAR by NMR (SAR = structure-activity relationships) and can be applied to proteins of known structure which are labelled with 15N or 13C such that each amide bond in the protein has an identifiable peak. A range of low-molecular-weight compounds is screened to see whether any of them bind to a specific region of the binding site. Binding can be detected by observing a shift in any of the amide signals, which will not only show that binding is taking place, but will also reveal which part of the binding site is occupied. Once a compound (or ligand) has been found that binds to one region of the binding site, the process can be repeated to find another ligand that will bind to a different region. This is usually done in the presence of the first ligand to ensureLaurea that in Biotecnologie the second ligand does in fact bind to a distinctPage 15 region. Once two ligands (or epitopes) have been identified, the structure of each can be optimized to find the best ligand for each of the binding regions, then a molecule can be designed where the two ligands are linked together. Fragment-based lead discovery There are several advantages to this approach. Since the individual ligands are optimized for each region of the binding site, a lot of synthetic effort is spared. It is much easier to synthesize a series of small molecular weight compounds to optimize the interaction with specific parts of the binding site, than it is to synthesize a range of larger molecules to fit the overall binding site. A high level of diversity is also possible, as various combinations of fragments could be used. A further advantage is that it is more likely to find epitopes that will bind to a particular region of a binding site than to find a lead compound that will bind to the overall binding site. Moreover, fragments are more likely to be efficient binders, having a high bind ing energy per unit molecular mass. Finally, some studies have demonstrated a 'super-additivity' effect where the binding affinity Laurea of the in Biotecnologie two linked fragments is much greater thanPage one 16 might have expected from the binding affinities of the two independent fragments. The method described above involves the linking of fragments. Another strategy is to grow' a lead compound from a single fragment — a process called fragment evolution. This involves the identification of a single fragment that binds to part of the binding site, then finding larger and larger molecules that contain that fragment, but which bind to other parts of the binding site as well. Fragment-based lead discovery

A third strategy is known as fragment self-assembly and is a form of dynamic combinatorial chemistry. Fragments are chosen that can not only bind to different regions of the binding site, but can also react with each other to form a linked molecule in situ. This could be a reversible reaction. Alternatively, the two fragments can be designed to undergo an irreversible linking reaction when they bind to the binding site. This has been called 'click chemistry in situ'. NMR spectroscopy is not the only method of carrying out fragment-based lead discovery. It is also possible to identify fragments that bind to target proteins using the techniques of X-ray crystallography, in vitro bioassays and mass spectrometry. X-ray crystallography, like NMR, pro vides information about how the fragment binds to the binding site, and does so in far greater detail. However, it can be quite difficult obtaining crystals of protein-frag- ment complexes because of the low affinity of the fragments. Recently, a screening method called CrystalLEAD has been developed which can quickly screen large num bers of compounds, and detect ligands by monitoring changes in the electron density map of protein-fragment complexes relative to the unbound protein. Finally, it is possible to use fragment-based strategies as a method of optimizing lead compounds that may have been obtained by other means. The strategy is to identify distinct fragments within the lead compound and then to optimize these fragments by the procedures already described. Once the ideal fragments have been identified, the full structure is synthesized incorporat ing the optimized fragments. This can be a much quicker method of optimization than synthesizing analogues of the larger lead compound..

Laurea in Biotecnologie Page 17 Fragment-based lead discovery

Laurea in Biotecnologie Page 18 Click chemistry in situ

Laurea in Biotecnologie Page 19 Properties of lead compounds

Some of the lead compounds that have been isolated from natural sources have sufficient activity to be used directly in medicine without serious side effects; for example morphine, quinine and paclitaxel. However, most lead compounds have low activity and/or unacceptable side effects which means that a significant amount of struc tural modification is required. If the aim of the research is to develop an orally active compound, certain properties of the lead compound should be taken into account. Most orally active drugs obey the rules laid down in Lipinskis Rule of Five or Veber's parameters. A study of known orally active drugs and the lead compounds from which they were derived demonstrates that the equivalent rules for a lead compound should be more stringent. This is because the structure of the lead compound almost certainly has to be modified and increased, both in terms of size and hydrophobicity. The suggested properties for a lead compound are that it should have a molecular weight of 100-350 amu, and a cLogP value of 1-3. (cLogP is a measure of how hydrophobic a compound is). In general, there is an average increase in molecular weight of 80 amu, and an increase of 1 in cLogP when going from a lead compound to the final drug. Studies also show that a lead compound generally has fewer aromatic rings and hydrogen bond acceptors compared to the final drug. Such considerations can be taken into account when deciding which lead compound to use for a research project if several such structures are available.

Laurea in Biotecnologie Page 20 Properties of lead compounds

Another approach in making this decision is to calculate the binding or ligand 'efficiency' of each potential lead compound. This can be done by dividing the free energy of binding for each molecule by the number of non-hydrogen atoms present in the structure. The better the 'ligand efficiency' the lower the molecular weight of the final optimized structure is likely to be. For fragment-based lead discovery, a rule of three has been suggested for the fragments used:

• a molecular weight less than 300 • no more than 3 hydrogen bond donors • no more than 3 hydrogen bond acceptors • cLogP = 3 • no more than 3 rotatable bonds • a polar surface area = 60 Å^2

Laurea in Biotecnologie Page 21 Drug Design and Relationship of Functional Groups to Pharmacologic Activity

Medicinal chemistry is the discipline concerned with determining the influence of chemical structure on biological activity. As such, it is necessary for the medicinal chemist to understand not only the mechansm by which a drug exerts its effect but also the physicochemical properties of the molecule. The term “physicochemical properties” refers to the influence of the organic functional groups within a molecule on its acid-base properties, water solubility, partition coefficient, crystal structure, stereochemistry, and so on. All these properties influence the absorption, distribution, metabolism excretion, and toxicity of the molecule. To design better medicinal agents, the medicinal chemist needs to understand the relative contributions that each functional group makes to the overall physicochcmical properties of the molecule. Studies of this type involve modification of the molecule in a systematic fashion and determination of how these changes affect biological activity. Such studies are referred to as studies of structure-activity relationships (SAR) — That is, what structural features of the molecule contributes to, or takes away from, the desired biological activity of the molecule of interest.

Chimica Farmaceutica Page 22 Relationship Between Molecular Structure and Biological Activity

Early studies of the relationship between chemical struc ture and biological ac tivity were conducted by Cramp Brown and Fraser in 1869. They showed that manv compounds containing tertiary amine groups became muscle relaxants when converted to quaternary ammonium compounds. Compounds with widely differing pharmacologic properties, such as strychnine (a convulsant), morphine (an analgesic), nicotine (deterrent, insecticide), and atropine (anticholinergic), could be converted to muscle relaxants with properties similar to those of tubocurarinc when methylated. Crum-Biown and Fraser therefore concluded that muscle-relaxant activity required a quaternary ammonium group within the chemical structure. This initial hypothesis was later disproven by the discovery of the natural neuro transmitter and activator of muscle contraction, acetylcholine. Even though Crum-Brown and Fraser's initial hypothesis concerning chemical structure and muscle relaxation was incorrect, it demonstrated the concept that molecular structure influences the biological activity of chemical compounds.

Chimica Farmaceutica Page 23 Relationship Between Molecular Structure and Biological Activity

With the discovery by Crum-Brown and Fraser that quaternary ammonium groups could produce compounds with muscle-relaxant properties scientists began looking for other organic functional groups that would produce specific biological responses. The thinking during this time was that specific chemical groups or nuclei (rings), were responsible for specific biological effects. This lead to the postulate, which took some time to disprove, that "one chemical group gives one biological action". Even after the discovery of acetylcholine by Loewi and Navrati, which effectively dispensed with Crum-Brown and Fraser's concept of all quaternary ammonium compounds being muscle relaxants, this was still considered to be dogma and took a long time to replace.

Chimica Farmaceutica Page 24 Relationship Between Molecular Structure and Biological Activity

Chimica Farmaceutica Page 25 Selectivity of Drug Action and Drug Receptors

Although the structures of many drugs or xenobiotics, or at least the composition of functional groups, were known at the start of the twentieth century, how these compounds exerted their effects was still a mystery. Utilizing his observations regarding the staining behavior of microorganisms, Ehrlich developed the concept of drug receptors. He postulated that certain "side chains" on the surfaces of cells were "complementary" to the dyes (or drug), thereby allowing the two substanccs to combine. In the case of antimicrobial compounds, this combining of the chemical to the "side chains” produced a toxic effect. This concept effectively was the first description of what later became known as the receptor hypothesis for explaining the biological action of chemical compounds Ehrlich also discussed selectivity of drug action via the concept of a "magic bullet" for compounds that would eradicate disease states without producing undue harm to the organism being treated (i.e.. the patient). This concept was later modified by Albert and generally is referred to as "selective toxicity." Utilizing this concept, Erhrlich developed organic arsenicals that were toxic to trypanosomes as a result of their irreversible reaction with mercapto groups (-SH) on vital proteins within the organism. The formation of As-S bonds resulted in death to the taiget organism. It was soon learned, however. that these compounds were toxic not only to the target organism but also to the host once certain blood levels of arsenic were achieved.

Chimica Farmaceutica Page 26 Selectivity of Drug Action and Drug Receptors

The "paradox" that resulted after the discovery of acetylcholine — how one chemical group can produce two different biological effects (i.e.. muscle relaxation and muscle contraction) — was explained by Ing usiing the actions of acetylcholine and tubocurarine as his examples, Ing hypothesized that both acetylcholine and tubocu rarine act at the same receptor, but that one molecule fits to the receptor in a more complementary manner and "activates" it, causing muscle contraction, (Ing did not elaborate just how this activation occurred.) The blocking effect of the larger molecule, tubocurarine, could be explained by its occupation of part of the receptor, thereby preventing acetylcholine, the smaller molecule, from interacting with the receptor. With both molecules, the quaternary ammonium functional group is a common structural feature and interacts with the same region of the receptor. If one closely examines the structures of other compounds with opposing effects on the same pharmacologic system, this appears to be a common theme: Molecules that block the effects of natural neurotransmitters (antagonists) generally are larger in size than the native compound. Both agonists and antagonists share common structural features, however, thus providing support to the concept that the structure of a molecule, its composition and arrangement of chemical functional groups, deter mines the type of pharmacologic effect that it possesses (i.e., SAR). Thus, compounds that are muscle relaxants acting via the cholinergic nervous system will possess a quaternary ammonium or protonated tertiary ammonium group and will be larger than acetylcholine.

Chimica Farmaceutica Page 27 Selectivity of Drug Action and Drug Receptors

Structure-activity relationships are the underlying principle of medicinal chemistry. Similar molecules exert similar biological actions in a qualitative sense. A corollary to this is that structural elements (functional groups) within a molecule most often contribute in an additive manner to the physicochemical properties of a molecule and, therefore, to its biological action. One need only peruse the structures of drug molecules in a particular pharmacologic class to become convinced of this (e.g.. histamine H1 antagonists, histamine H2 antagonists, and B- adrenergic antagonists). The objective of medicinal chemists in their quest for better medicinal agents (drugs) is to discover what functional groups within a specific structure are important for its pharmacologic activity and how can these groups be modified to produce more potent, selective, and safer compounds. An example of how different functional groups can yield compounds with similar pliysicochemical properties is shown with sulfanilamide antibiotics. The structures of sulfanilamide and p-aminobenzoic acid (PABA) are shown. In 1910, Woods demonstrated that PABA was capable of reversing the antibacterial action of sulfanilamide (and other sulfonamides antibacterials) and that both PABA and sulfanilamide had similar steric and electronic properties. Both compounds contain acidic functional groups, with PABA containing an aromatic carboxylic acid and sulfanilamide an aromatic sulfonamide. When ionized at physiological pH, both compounds have a similar electronic configuration, and the distance between the ionized acid and the weakly basic amino group also is very similar. It should therefore be no surprise that sulfanilamide acts as an antagonist to PABA metabolism in bacteria.

Chimica Farmaceutica Page 28 Selectivity of Drug Action and Drug Receptors

Chimica Farmaceutica Page 29 PHYSICOCHEMICAL PROPERTIES OF DRUGS: Acid-Base Properties The human body is 70 to 75% water, which amounts to approximately 51 to 55 L of water for a 73 kg individual. For an average drug molecule with a molecular weight of 200 g/mol and a dose of 20 mg. this leads to a solution concentration of approximately 2 X 10-6 M. When considering the solution behavior of a drug within the body, we therefore are dealing with a dilute solution, for which the Bronsted-Lowry acid-base theory is most appropriate for explaining and predicting acid-base behavior. This is a very important concept in medicinal ichemistry because the acid-base properties of drug molecules directly affect absorption, excretion, and compatibility with other drugs in solution. From general principles, it is possible to predict if a molecule is going to be ionized or un- ionized at a given pH simply by knowing if the functional groups on the molecule are acidic or basic. To be able to quantitatively predict the degree of ionization of a molecule, however, one must know the pKa values of the acidic and basic functional groups that are present and the pH of the environment to which the compound will be exposed. The Henderson-Hassalbach equation can be used to calculate the percentage ionization of a compound at a given pH (this equation was used to calculate the major forms of ciprofloxacin.

Chimica Farmaceutica Page 30 PHYSICOCHEMICAL PROPERTIES OF DRUGS: Acid-Base Properties The key to understanding the use of the Henderson-Hasselbach equation for calculating percentage ionization is to realize that this equation relates a constant. pKa. to the ratio of acidic form to the basic form of the drug. Because pKa is a constant for any given molecule, the ratio of acid to base will determine the pH of the solution. Conversely, a given pH determines the ratio of acid to base. When dealing with a base, the student must recognize that the conjugate acid is the ionized form of the drug Thus, as one should expect, a base behaves in a manner opposites to that of an acid. It is very important to recognize that for a base, the pKa refers to the conjugate acid or ionized form of the compound.

Chimica Farmaceutica Page 31 PHYSICOCHEMICAL PROPERTIES OF DRUGS: Acid-Base Properties

Chimica Farmaceutica Page 32 PHYSICOCHEMICAL PROPERTIES OF DRUGS: Acid-Base Properties: Predicting the Degree of Ionization of a Molecule

Chimica Farmaceutica Page 33 PHYSICOCHEMICAL PROPERTIES OF DRUGS: Water Solubility of Drugs The solubility of a drug molecule in water greatly affects the routes of administration that are available as well as its absorption, distribution, and elimination. Two key concepts to keep in mind when considering the water (or fat) solubility of a molecule are the hydrogen bond- forming potential of the functional groups in the molecule and the ionization of functional groups.

Chimica Farmaceutica Page 34 PHYSICOCHEMICAL PROPERTIES OF DRUGS: Predicting Water Solubility: Empiric Approach Lemke has developed an empiric approach to predicting the water solubility of molecules based on the carbon-solubilizing potential of several organic functional groups. If the solubilizing potential of the functional groups exceeds the total number of carbon atoms present, then the molecule is considered to be water soluble. Otherwise, it is considered to be water insoluble. Functional groups that can interact either through intramolecular hydrogen or ion-ion interactions will decrease the solubilizing potential of each group. It is difficult to quantitate how much such interactions will take away from water solubility, but recognizing these interactions will allow one to explain anomalous results.

Chimica Farmaceutica Page 35 PHYSICOCHEMICAL PROPERTIES OF DRUGS: Predicting Water Solubility: Analytical Approach Another method for predicting water solubility involves calculating an approximate logP or log of the partition coefficient for a molecule. This approach is based on an approximation method developed by Cates and discussed in Lemke. In this approach, one sums the hydrophobic or hvdrophilic properties of each functional group present in the molecule. Before we can calculate logP values, we must first digress to a brief explanation of the concept of partition coefficient. In its simplest form, the partition coefficient, P, refers to the ratio of the concentrations of drug in octanol to that in water. Octanol is used to mimic the amphiphilic nature of lipid. because it has a polar head group (primary alcohol) and a long hydrocarbon chain, or tail, such as that of fatty acids, which make up part of a lipid membrane. Because P is logarithmically related to free energy, it generally is expressed as logP and therefore, is the sum of the hydrophobic and hydriophilic characteristics of the organic functional groups making up the structure of the molecule. Thus, logP is a measure of the solubility characteristics of the entire molecule. Because each organic functional group contained within the molecule contributes to the overall hydrophobic/hydrophilic nature of the molecule, a hydrophobic/hydrophilic value (the hydrophobic substituent constant. p) can be assigned to each organic functional group.

Chimica Farmaceutica Page 36 PHYSICOCHEMICAL PROPERTIES OF DRUGS: Predicting Water Solubility: Analytical Approach When calculating logP from hydrophobic substituent constants, the sum is usually referred to as logPcalc or ClogP to distinguish it from au experimentally deter mined value (logP or MlogP). Over the years, extensive tables of p values have been compiled for organic functional groups and molecular fragments.

Chimica Farmaceutica Page 37 PHYSICOCHEMICAL PROPERTIES OF DRUGS: Predicting Water Solubility: Analytical Approach

Chimica Farmaceutica Page 38 STEREOCHEMISTRY AND DRUG ACTION

Stereoisomers are compounds containing the same number and kinds of atoms, the same arrangement of bonds, but different three-dimensional structures: in other words. they only differ in the three-dimensional arrangements of atoms in space. Stereoisomers are subdivided into two types, enantiomers and diastereoisomers. Enantiomers are compounds for which the three-dimensional arrangement of atoms is such that they are nonsuperimposable mirror images. Diastereoisomers are all stereoisomer compounds that are not enantiomers. Thus, the term "diastereoisomer" includes compounds containing double bonds (geometric isomers) as well as ring systems. Unlike enantiomers diastereoisomers exhibit different physicochemical properties, including, but not limited to, melting point, boiling point, solubility, and chromatographic behavior. These differences in physicochemical properties allow the separation of diastereoisomers from mixtures utilizing standard chemical separation techniques, such as column chromatography or crystallization. Enantiomers cannot be separated using such techniques unless a chiral environment is provided or they are converted to diastereoisomers.

Chimica Farmaceutica Page 39 STEREOCHEMISTRY AND DRUG ACTION

Chimica Farmaceutica Page 40 STEREOCHEMISTRY AND DRUG ACTION

The phisicochemical properties of a drug molecule are dependent not only on what functional groups are present in the molecule but also on the spatial arrangement of these groups. This becomes an especially important factor when a molecule is subjected to an asymmetric environment, such as the human body. Because proteins and other biological macromolecules are asymmetric in nature, how a particular drug molecule interacts with these macromolecules is determined by the three-dimensional orientation of the organic functional groups that are present. If crucial functional groups are not occupying the proper spatial region surrounding the molecule, then productive bonding interactions with the biological macromolecule (or receptor) will not be possible, potentially negating the desired pharmacological effect. If however, these functional groups are in the proper three-dimensional orientation, the drug can produce a very strong interaction with its receptor. It therefore is very important for the medicinal chemist developing a new molecular entity for therapeutic use to understand not only what functional groups are responsible for the drug's activity but also what three-dimensional orientation of these groups is needed.

Chimica Farmaceutica Page 41 STEREOCHEMISTRY AND DRUG ACTION

Approximately one in every four drugs currently on the market can be considered to be an isomeric mixture, yet for many of these compounds, the biological activity may reside in only one isomer (or at least predominate in one isomer). The majority of these isomeric mixtures are what are referred to as "racemic mixtures" (or "race- mates"). These are compounds, usually synthetic, that contain equal amounts of both possible enantiomers, or optical isomers. Enantiomers also are referred to as chiral compounds, antipodes, or enantiomorphs. When introduced into an asymmetric, or chiral. environment, such as the human body, enantiomers will display different physicochcmical properties, producing significant differences in their pharmacokinetic and pharmacodynamic behavior. Such differences can result in adverse side effects or toxicity, because one or more of the isomers may exhibit significant differences in absorption (especially active transport), serum protein binding, and metabolism. With the latter, one isomer may be converted into a toxic substance or may influence the metabolism of another drug.

The basic concepts of stereochemistry will be not reviewed as previously studied in the organic chemistry course.

Chimica Farmaceutica Page 42 STEREOCHEMISTRY AND DRUG ACTION Stereochemistry and Biological Activity: Easson-Stedman Hypothesis

In 1886, Piutti reported different physiologic actions for the enantiomers of asparagine, with (+)-asparagine having a sweet taste and (-)-asparagine a bland one. This was one of the earliest olbservations that enantiomers can exhibit differences in biological action. In 1933. Easson and Stedman reasoned that differences in biological activity between enantiomers resulted from selective reactivity of one enantiomer with its receptor. They postulated that such interactions require a minimum of a three-point fit to the receptor.

Chimica Farmaceutica Page 43 STEREOCHEMISTRY AND DRUG ACTION Stereochemistry and Biological Activity: Easson-Stedman Hypothesis

The Easson-Stedman Hypothesis states that the more potent enantioimer must be involved in a minimum of three intermolecular interactions with the receptor surface and that the less potent enantiormer only interacts with two sites. This can be illustrated by looking at the differences in vasopressor activity of (R)-(-)-Epinephrine, (S)-(+)-epinephrine and the achiral N-methyldopamine. With (R(-(-)-Epinephrine, the three points of interaction with the receptor site are the substituted aromatic ring, B- hydroxyl group, and the protonated secondary ammonium group. All three functional groug interact with their complementary binding sites on the receptor surface, producing the necessary interactions that stimulate the receptor. With (S)- (+)-Epinephrine, only two interactions are possible (the protonatcd secondary ammonium and the substituted aromatic ring). The B-hydroxyl group occupies the wrong region of space and therefore, cannot interact properly with the receptor. N- methyldopamine can achieve the same interactions with the receptor as (S(-(+ )- Epinephrine: therefore it is not surprising that its vasopressor response is the same as that of (S)-(+)-Epinephrine and less than that of (R)-(-)-Epinephrine.

Chimica Farmaceutica Page 44 STEREOCHEMISTRY AND DRUG ACTION Stereochemistry and Biological Activity

Not all stereoselectivity seen with enantiomers can be attributed to differences in reactivity at the receptor site. Differences in biological activity also can result from differences in the ability of each enantiomer to reach the receptor site. Because the biological system encountered by the drug is asymmetric, each enantiomer may experience selective penetration of membranes, metabolism, and absorption at sites of loss (e.g., adipose tissue) or excretion. Not all of these processes may be encountered by a particular enantiomer, but such processes may provide enough of an influence to cause one enantiomer to produce a significantly better pharmacologic effect than the other. Conversely, such processes also may contribute to untoward effects of a particular enantiomer.

Chimica Farmaceutica Page 45 STEREOCHEMISTRY AND DRUG ACTION Stereochemistry and Biological Activity

Chimica Farmaceutica Page 46 STEREOCHEMISTRY AND DRUG ACTION Stereochemistry and Biological Activity

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Chimica Farmaceutica Page 49 STEREOCHEMISTRY AND DRUG ACTION Conformational Isomerism and Biological Activity

With conformational isomerism, we are dealing with a dynamic process — that is, isomerization takes place via rotation about one or more single bonds. Such bond rotation results in nonidentical spatial arrangement of atoms in a molecule. Changes in spatial orientation of atoms because of bond rotation results in different conformations (or rotamers), whereas conversion of one enamiomer into another (or diastereoisomer) requires the breaking of bonds, which has a much higher energy requirement than rotation around a single bond. The neurotransmitter acetylcholine can be used to demonstrate the concept of conformational isomers.

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Each single bond within the acetylcholine molecule is capable of undergoing rotation, and at room temperature, such rotations readily occur. Even though rotation around single bonds was shown by Kemp and Pitzer in 1936 not to be free but, rather, to have an energy barrier, this barrier is sufficiently low that at room temperature, acetylcholine exists in many interconvertible conformations. Close observation reveals that rotation around the central Ca-Cb bond produces the greatest spatial rearrangement of atoms compared to rotation around any other bond within the molecule. In fact, several rotatable bonds in acetylcholine produce redundant structures, because all of the atoms attached to one end of some bonds are identical, resulting in no change in spatial arrangement of atoms (methyls). When viewed along the Ca-Cb bond, acetylcholine can be depicted in the Newman projections. When the ester and trimethylammonium group are 180° apart, the molecule is said to be in the anti or staggered, conformation (or conformer or rotamer). This conformation allows maximum separation of the functional groups and, therefore, considered to be the most stable conformation energetically. Other conformations possibly are more stable if factors other than steric interactions come into play (e.g., intramolecular hydrogen bonds).

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Rotation of one end of the Ca-Cb bond by 120° or 240° results in the two gauche, or skew, conformations. These are considered to be less stable than the anticonformer, although some studies suggest that an electrostatic attraction between the electron-poor trimethylammonium and electron-rich ester oxygen stabilizes this conformation. Rotation by 60°, 180°, and 240° produce conformations in which all of the atoms overlap, or what are referred to as eclipsed conformations. These are the least stable conformers. An interesting observation can be made with the two gauche conformers. These conformers are not distinct molecules, and they only exist for a transient period of time at room temperature. If these could be "frozen" into the conformations shown, however, they would be nonsuperimposable mirror images or enamiomers. Thus, a compound that is achiral, such as acetylcholine, can exhibit prochirality if certain conformational isomers can be formed. It is quite possible that such a situation could exist when acetylcholine binds to one of its receptors. Studies have suggested that the gauche conformation is the form that binds to the nicotinic receptor, whereas the anti form, which is achiral, binds to the muscarinic receptor.

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Chimica Farmaceutica Page 53 REFINEMENT OF THE LEAD STRUCTURE Determination of the Pharmacophore Once a lead compound has been discovered for a particular therapeutic use, the next step is to determine the pharmacophore for this compound. The pharmacophore of a drug molecule is that portion of the molecule containing the essential organic functional groups that directly interact with the receptor active site and therefore, confers on the molecule the biological activity of interest. Because drug-receptor interactions can be very specific, the pharmacophore may constitute a small portion of the molecule. It has been found on several occasions that what seem to be very complex molecules can be reduced to simpler structures with retention of the desired biological action. An example of this is the narcotic analgesic morphine, which is a tetracyclic compound with five chiral centers. Not only would simplification of the structure possibly provide molecules with fewer side effects, a reduction in the number of chiral centers would greatly simplify the synthesis of morphine derivatives and, thereby, decrease cost. It can be readily seen from the figure that the pharmacophore of morphine must consist of a tertiary alkylamine that is at least four atoms away from an aromatic ring.

Chimica Farmaceutica Page 54 REFINEMENT OF THE LEAD STRUCTURE Determination of the Pharmacophore

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