Chapter 2 Drug Discovery,Design,and Development 21 Drug Discovery…… 4,8 A.Drug Discovery without a Lead 1.Penicillins 9 2.Librium.......1 B.Lead Disc0very… 11 1.Random Screening.......... 13 2.Nonrandom (or Targeted or Focused)Screening...............14 3.Drug Metabolism Studies............... …14 4.Clinical Observations................. .15 5.Rational Approaches to Lead Discovery、...… 16 2.2 Lead Modification:Drug Design and Development............... 17 A.Identification of the Active Part:The Pharmacophore...........17 B.Functional Group Modification............................................... 21 C.Structure-Activity Relationships.............................................. 21 D.Privileged Structures and Drug-Like Molecules............... 24 E.Structure Modifications to Increase Potency and the Therapeutic Index 25 1.Homologation..................................... 26 2.Chain Branching.................................................. 26 3.Ring-Chain Transformations....................................... 28 4. Bi0iS0 sterism,,…… 29 5.Combinatorial Chemistry........................................ a.General Aspects................ 34 b.Split Synthesis:Peptide Libraries............................ 36 c.Encoding Combinatorial Libraries.................... 3 d.Nonpeptide Libraries........................... 41 6.SAR by NMR/SAR by MS...................... 7.Peptidomimetics........... 47 F.Structure Modifications to Increase Oral Bioavailability........................... 51 1.Electronic Effects:The Hammett Equation.................................. 51 2.Lipophilicity Effects............................................ 53
8 Chapter 2 Drug Discovery,Design,and Development a.Importance of Lipophilicity................................... 53 b.Measurement of Lipophilicities. 55 c.Computerization of Log P Values........................................61 d.Membrane Lipophilicity.……4. 62 3.Effects of lonization on Lipophilicity and Oral Bioavailability 62 4.Other Properties that Influence Oral Bioavailability and Ability to Cross the Blood-Brain Barrier..........................65 G.Quantitative Structure-Activity Relationships.......................... 66 1.Historical......... 66 2.Steric Effects:The Taft Equation and Other Equations..................67 3.Methods Used to Correlate Physicochemical Parameters with Biological Activity......................... …,68 a.Hansch Analysis:A Linear Multiple Regression Analysis 68 b.Free and Wilson or de novo Method..........................70 c.Enhancement Factor............................. .71 d.Manual Stepwise Methods:Topliss Operational Schemes and Others.....71 e.Batch Selection Methods:Batchwise Topliss Operational Scheme, Cluster Analysis,and Others..73 4.Computer-Based Methods of QSAR Related to Receptor Binding: 3D-QSAR................... 75 H.Molecular Graphics-Based Drug Design.......................................... 78 l.Epilogue…444… 86 2.3 General References 87 2.4 Problems.................. 100 2.5 References............... 105 2.1 Drug Discovery Drug discovery is a very time-consuming and expensive process.Estimates of the average time required to bring a drug to the market range from 12-15 years at an average cost of about $800million.For approximately every 10.000 compounds that are evaluated in animal studies, 10 will make it to human clinical trials in order to get I compound on the market.The clinical trials consist of three phases prior to drug approval:phase I(generally a few months to a year and a half)evaluates the safety,tolerability (dosage levels and side effects),pharmacokinetic properties,and pharmacological effects in 20-100 healthy volunteers:phase II(about 1-3 years)assesses the effectiveness of the drug,determines side effects and other safety aspects, and clarifies the dosing regimen in a few hundred diseased patients;and phase III(about 2-6 years)is a larger trial with several thousand patients in clinics and hospitals that establishes the efficacy of the drug and monitors adverse reactions from long-term use.Once the new drug application(NDA)is submitted to the Food and Drug Administration(FDA).it can be several months to several years before it is approved for commercial use.Phase IV studies are considered to be the results found with a drug that has already been allowed onto the drug market and is in general use.Drug candidates(or new chemical entiries.NCE.as they are
Section 2.1 Drug Discovery 9 often called)that fail late in this process result in huge,unrecovered financial losses for the company.This is why the cost to purchase a drug is so high.It is not that it costs that much to manufacture that one drug.but that the profits are needed to pay for all of the drugs that fail to make it to market after large sums of research funds have already been expended. In general,drugs are not discovered.What is more likely discovered is known as a lead compound.The lead is a prototype compound that has a number of attractive characteristics, such as the desired biological or pharmacological activity,but may have other undesirable characteristics.for example,high toxicity,other biological activities,absorption difficulties. insolubility,or metabolism problems.The structure of the lead compound is modified by syn- thesis to amplify the desired activity and to minimize or eliminate the unwanted properties to a point where adrug candidate,a compound worthy of extensive biological,pharmacological. and animal studies,is identified:then a clinical drug,a compound ready for clinical trials,is developed.Prior to an elaboration of approaches to lead discovery and lead modification,two common drugs discovered without a lead are discussed. 2.1.A Drug Discovery without a Lead A.1 Penicillins In 1928 Alexander Fleming noticed a green mold growing in a culture of Staphylococcus aureus,and where the two had converged,the bacteria were lysed.This led to the discovery of penicillin,which was produced by the mold.Actually,Fleming was not the first to make this observation;John Burdon-Sanderson had done so in 1870,ironically also at St.Mary's Hospital in London,the same institution where Fleming made the rediscovery!2 Joseph Lister had treated a wounded patient with Penicillium,the organism later found to be the producer of penicillin(although the strains discovered earlier than Fleming's strain did not produce penicillin,but,rather,another antibiotic,mycophenolic acid).After Fleming observed this phenomenon,he tried many times to repeat it without success;it was his colleague. Dr.Ronald Hare,3.41 who was able to reproduce the observation.It only occurred the first time because a combination of unlikely events all took place simultaneously.Hare found that very special conditions were required to produce the phenomenon initially observed by Fleming.The culture dish inoculated by Fleming must have become accidentally and simultaneously contaminated with the mold spore.Instead of placing the dish in the refrigerator or incubator when he went on vacation as is normally done,Fleming inadvertently left it on his lab bench.When he returned the following month,he noticed the lysed bacteria.Ordinarily. penicillin does not lyse these bacteria;it prevents them from developing,but it has no effect if added after the bacteria have developed.However,while Fleming was on vacation(July to August)the weather was unseasonably cold,and this provided the particular temperature required for the mold and the staphylococci to grow slowly and produce the lysis.Another extraordinary circumstance was that the particular strain of the mold on Fleming's culture was a relatively good penicillin producer,although most strains of that mold(Penicillium)produce no penicillin at all.The mold presumably came from the laboratory just below Fleming's where research on molds was going on at that time. Although Fleming suggested that penicillin could be useful as a topical antiseptic,he was not successful in producing penicillin in a form suitable to treat infections.Nothing more was done until Sir Howard Florey at Oxford University reinvestigated the possibility of producing penicillin in a useful form.In 1940 he succeeded in producing penicillin that could be administered topically and systemically sI but the full extent of the value of penicillin
10 Chapter 2 Drug Discovery,Design,and Development was not revealed until the late 1940s.161 Two reasons for the delay in the universal utilization of penicillin were the emergence of the sulfonamide antibacterials (sulfa drugs,2.1:see Chapter 5.Section 5.4.B.2,p.254)in 1935 and the outbreak of World War II. H:N SO-NHR sulfa drugs 2.1 No studies related to the pharmacology,production,and clinical application of penicillin were permitted until after the war to prevent the Germans from having access to this wonder drug.Allied scientists who were interrogating German scientists involved in chemotherapeutic research were told that the Germans thought the initial report of penicillin was made just for commercial reasons to compete with the sulfa drugs.They did not take the report seriously. The original mold was Penicillium notatum,a strain that gave a relatively low yield of penicillin.It was replaced by Penicillium chysogenum,which had been cultured from a mold growing on a grapefruit in a market in Peoria,Illinois! For many years debate raged regarding the actual structure of penicillin(2.2),8 but the correct structure was elucidated in 1944 with an X-ray crystal structure by Dorothy Crowfoot Hodgkin(Oxford):the crystal structure was not actually published until 1949Several differ- ent penicillin analogs(R group varied)were isolated early on;only two of these early analogs (2.2.R =PhOCH2,penicillin V:and 2.2.R=CH2Ph,penicillin G)are still in use today. H日 CH CH; COOH H penicillin V(R=PhOCH2) penicillin G(R CH2Ph) 2.2 A.2 Librium The first benzodiazepine tranquilizer drug,chlordiazepoxide HCI [7-chloro-2-(methylamino)- 5-phenyl-3H-1.4-benzodiazepine 4-oxide:2.3:Librium],was discovered serendipitously.1 Dr.Leo Sternbach at Roche was involved in a program to synthesize a new class of tranquilizer drugs.He originally set out to prepare a series of benzheptoxdiazines(2.4),but when R was CHaNR2 and R2 was CHs,it was found that the actual structure was that of a quinazoline 3-oxide(2.5). NHCH HCI chlordiazepoxide HCI 2.3 2.5
Section 2.1 Drug Discovery 11 CH2CI H N NHCH3 CHNH2 2.6 CH NH2 CH2NHCH3 NHCH3 人 CH2-CI 0 2.3 OH Scheme 2.1 Mechanism for formation of Librium. However,none of these compounds gave any interesting pharmacological results.The program was abandoned in 1955 in order for Stembach to work on a different project.In 1957 during a general laboratory cleanup a vial containing what was thought to be 2.5(X=7-CL, R=CH2NHCH3,R2=C6Hs)was found and,as a last effort,was submitted for pharma- cological testing.Unlike all of the other compounds submitted,this one gave very promising results in six different tests used for preliminary screening of tranquilizers.Further inves- tigation revealed that this compound was not a quinazoline 3-oxide,but was instead the benzodiazepine 4-oxide,2.3,presumably produced in an unexpected reaction of the corre- sponding chloromethyl quinazoline 3-oxide(2.6)with methylamine (Scheme 2.1).If this compound had not been found in the laboratory cleanup,all of the negative pharmacolog- ical results would have been reported for the quinazoline 3-oxide class of compounds,and benzodiazepine 4-oxides may not have been discovered for many years to come. The examples of drug discovery without a lead are relatively few in number.The typical occurrence is that a lead compound is identified,and its structure is modified to give,eventually, the compound that goes to the clinic. 2.1.B Lead Discovery Penicillin V and Librium are,indeed,two important drugs that were discovered without a lead. However,once they were identified,they then became lead compounds for second-generation analogs.A myriad of penicillin-derived antibacterials have been synthesized as a result of the structure elucidation of the earliest penicillins.Diazepam(2.7,Valium)was synthesized at Roche even before Librium was introduced on to the market:this drug was derived from the lead compound Librium and is almost 10 times more potent than the lead
Chapter 2 Drug Discovery,Design,and Development 12 CH diazepam 2.7 The initial difficulty arises in the discovery of the lead compound.Several approaches can be taken to identify a lead.The first requirement for all of the approaches is to have a means to assay compounds for a particular biological activity,so that researchers can tell when a compound is active.A bioassay (or screen)is a means of determining in a biological system,relative to a control compound,if a compound has the desired activity. and,if so.what the relative potency of the compound is.Note the distinction between the terms activiry and porency.Activiry is the particular biological or pharmacological effect (for example,antibacterial activity or anticonvulsant activity):potency is the strength of that effect. Some screens are in vitro tests,for example,the inhibition of an enzyme or antagonism of a receptor;others are in vivo tests,for example,the ability of the compound to prevent an induced seizure in a mouse.In general,the in vitro tests are quicker and less expen- sive.Currently,high-throughput screens(HTS)very rapid and sensitive in vitro screens initially developed about 1989-1991,that now can be carried out robotically in 1536-or 3456-well titer plates on small(submicrogram)amounts of compound(dissolved in submi- croliter volumes)are becoming universally used.With these ultra-high-throughput screening approaches,it is possible to screen 100,000 compounds in a day!As we will see below, combinatorial chemistry(see Section 2.2.E.5,p.34)can supply huge numbers of compounds in a short period of time,which,theoretically,should provide an increased number of hits, i.e.,compounds that elicit a predetermined level of activity in the bioassay and.therefore, provide more leads.According to Drews,2 the number of compounds assayed in a large pharmaceutical company in the early 1990s was about 200,000 a year:that number rose to 5-6 million during the mid-1990s,and by the end of the 1990s it was>50 million!How- ever,the increase in the assay rate did not result in a commensurate increase in research productivity,as measured by new compounds entering the market.Of course,it can take 12-15 years for a drug to reach the market,so productivity in the early part of the 21st century should provide a more accurate ruler for success of drug discovery changes made at the end of the 20th century.Currently.HTS appears to have resulted in an increase in the number of hits,but this may be because more lipophilic compounds,which may have more drug-like properties (see Section 2.2.F.2,p.53),can be tested by dissolving them in dimethylsulfoxide DMSO)rather than in water.Nonetheless,it is not yet clear if this increase in hit rate is translating into a much greater number of leads and development compounds.[13] An exciting approach for screening compounds that might interact with an enzyme in a metabolic pathway was demonstrated by Wong.Pompliano,and coworkers for the discov- ery of lead compounds that block bacterial cell wall biosynthesis(as potential antibacterial agents)Conditions were found to reconstitute all six enzymes in the cell wall biosynthetic
Section 2.1 Drug Discovery 13 pathway so that incubation with the substrate for the first enzyme leads to the formation of the product of the last enzyme in the pathway.Then by screening compounds and looking for the buildup of an intermediate,it is possible to identify not only compounds that block the pathway(and prevent the formation of the bacterial cell wall),but to determine which enzyme is blocked(the buildup of an intermediate means that the enzyme that acted on that intermediate was blocked). Compound screening also can be carried out by electrospray ionization mass spectro- metryl15](the technique for which John Fenn received the Nobel prize in 2002)and by NMR spectrometry.16 Tightly bound noncovalent complexes of compounds with a macromolecule (such as a receptor or enzyme)can be observed in the mass spectrum.The affinity of the ligand (a small molecule that binds to a receptor)can be measured by varying the collision energy and determining at what energy the complex dissociates.This method also can be used to screen mixtures(a library)of compounds,provided they have different molecular masses and/or charges,so the m/z for each complex with the biomolecule can be separated in the mass spectrometer.By varying the collision energy,it is possible to determine which test molecules bind to the biomolecule best.TheHNMR method exploits changes in either relaxation rates or diffusion rates of small molecules when they bind to a macromolecule. This method also can be used to screen mixtures of compounds to determine the ones that bind best. Once the screen is developed,a variety of approaches can be taken to obtain a lead.As we will see below,the typical lead compound for a receptor or enzyme is the natural ligand for the receptor or substrate for the enzyme.Another good source of lead compounds is marketed drugs.17 In this case the target will generally be wellestablished,and the lead structure will be known to bind well to the target and to have good absorption properties.The main stumbling block to the use of marketed drugs as leads may be patent issues for commercialization.If the target macromolecule is not known or if no new leads have come from a marketed drug, other approaches can taken. B.1 Random Screening In the absence of known drugs and other compounds with desired activity,a random screen is a valuable approach.Random screening involves no intellectualization:all compounds are tested in the bioassay without regard to their structures.Prior to 1935(the discovery of sulfa drugs),this was essentially the only approach;today this method is still an important approach to discover drugs or leads,particularly because it is now possible to screen such huge numbers of compounds rapidly with HTSs.This is the lead discovery method of choice when nothing is known about the receptor target. The two major classes of materials screened are synthetic chemicals and natural products (microbial,plant,and marine).An example of a random screen of synthetic and natural compounds was the"war on cancer"declared by Congress and the National Cancer Institute in the early 1970s.Any new compound submitted was screened in a mouse tumor bioassay. Few new anticancer drugs resulted from that screen,but many known anticancer drugs also did not show activity in the screen used,so a new set of screens was devised that gave more consistent results.In the 1940s and 1950s,a random screen of soil samples by various pharmaceutical companies in search of new antibiotics was undertaken.However,in this case, not only were numerous leads uncovered,but two important antibiotics,streptomyein and the
14 Chapter 2 Drug Discovery,Design,and Development tetracyclines,were found.Screening of microbial broths.particular strains of Streptomyces. was a common random screen methodology prior to 1980. B.2 Nonrandom(or Targeted or Focused)Screening Nonrandom screening,also called targeted or focused screening,is a more narrow approach than is random screening.In this case,compounds having a vague resemblance to weakly active compounds uncovered in a random screen.or compounds containing different functional groups than leads,may be tested selectively.By the late 1970s,the National Cancer Institute's random screen was modified to a nonrandom screen because of budgetary and man- power restrictions.Also,the single tumor screen was changed to a variety of tumor screens because it was realized that cancer is not just a single disease. B.3 Drug Metabolism Studies During drug metabolism studies(see Chapter 7)metabolires(drug degradation products gen- erated in vivo)that are isolated are screened to determine if the activity observed is derived from the drug candidate or from a metabolite.For example,the anti-inflammatory drug sulin- dac (2.8.Clinoril)is not the active agent:the metabolic reduction product,2.9.is responsible for the activity COOH COOH CH sulindac 2.8 2.9 The nonsedating antihistamine terfenadine hydrochloride(2.10,Seldane)was found to cause an abnormal heart rhythm in some users who also were taking certain antifungal agents, which were found to block the enzyme that metabolizes terfenadine.This caused a buildup of terfenadine,which led to the abnormal heart rhythms.However,a metabolite of terfenadine, fexofenadine hydrochloride(2.11.Allegra),was also found to be a nonsedating antihistamine. but it can be metabolized even in the presence of antifungal agents.This,then,is a safer drug. Metabolites can be screened for other activities as well. CHy CH Ph HCI CHy HO OH terfenadine HCI 2.10
Section 2.1 Drug Discovery 15 CH: CO0H CH Ph HCI HO- OH fexofenadine HCI 2.11 B.4 Clinical Observations Sometimes a drug candidate during clinical trials will exhibit more than one pharmacological activity;that is,it may produce a side effect.This compound,then,can be used as a lead (or. with luck,as a drug)for the secondary activity.In 1947 an antihistamine,dimenhydrinate (2.12.Dramamine)was tested at the allergy clinic at Johns Hopkins University and was found also to be effective in relieving a patient who suffered from car sickness;a further study proved its effectiveness in the treatment of seasickness and airsickness.2 It then became the most widely used drug for the treatment of all forms of motion sickness dimenhydrinate 2.12 There are other popular examples of drugs derived from clinical observations.Bupro- pion hydrochloride(2.13).an antidepressant drug (Wellbutrin),was found to help patients stop smoking and is now the first drug marketed as a smoking cessation aid(Zyban).The impotence drug sildenafil citrate(2.14:Viagra)was designed for the treatment of angina and hypertension by blocking the enzyme phosphodiesterase-5.which hydrolyzes cyclic guano- sine monophosphate(cGMP).a vasodilator that allows increased blood flow 1211 In 1991 sildenafil went into phase I elinical trials for angina.In phase II clinical trials,it was not as effective against angina as Pfizer had hoped,so it went back to phase Iclinical trials to see how high of a dose could be tolerated.It was during that clinical trial that the volunteers reported increased erectile function.Given the weak activity against angina,it was an easy decision to try to determine its effectiveness as the first treatment for erectile dysfunction.Sildenafil works by the mechanism for which it was designed as an antianginal drug,except it inhibits the phosphodiesterase in the penis(phosphodiesterase-5)instead of the heart(Figure 2.1). Sexual stimulation causes release of nitric oxide in the penis. HN HCI bupropion HCI sildenafil citrate 2.13 2.14
16 Chapter 2 Drug Discovery,Design,and Development L-Arg- NO Nitric oxide synthase stimulates Erection Guanylate cyclase GTP smooth increased cGMP muscle blood relaxation flow GMP vasoconstriction PDE5 inhibits Viagra Figure 2.1 Mechanism of action of sildenafil(Viagra) Nitric oxide is a second messenger molecule that stimulates the enzyme guanylate cyclase, which converts guanosine triphosphate to cGMP.The vasodilator cGMP relaxes the smooth muscle in the corpus cavernosum,allowing blood to flow into the penis,thereby producing an erection.However,phosphodiesterase-5(PDE 5)hydrolyzes the cGMP,which causes vaso- constriction and the outflow of blood from the penis.Sildenafil inhibits this phosphodiesterase, preventing the hydrolysis of cGMP and prolonging the vasodilation effect. B.5 Rational Approaches to Lead Discovery None of the above approaches to lead discovery involves a major rational component.The lead is just found by screening techniques,as a by-product of drug metabolism studies,or from clinical investigations.Is it possible to design a compound having a particular activity? Rational approaches to drug design now have become the major routes to lead discovery.The first step is to identify the cause for the disease state.Many diseases,or at least the symptoms of diseases,arise from an imbalance (either excess or deficiency)of particular chemicals in the body,from the invasion of a foreign organism,or from aberrant cell growth.As will be discussed in later chapters,the effects of the imbalance can be corrected by antagonism or agonism of a receptor(see Chapter 3)or by inhibition of a particular enzyme(see Chapter 5): foreign organism enzyme inhibition or interference with DNA biosynthesis or function are important approaches to treat diseases arising from microorganisms and aberrant cell growth (see Chapter 6). Once the relevant biochemical system is identified,initial lead compounds then become the natural receptor ligands or enzyme substrates.For example,lead compounds for the contraceptives(+)-norgestrel (2.15,Ovral)and 17a-ethynylestradiol (2.16,Activella)were the steroidal hormones progesterone (2.17)and 17B-estradiol (2.18).Whereas the steroid hormones 2.17 and 2.18 show weak and short-lasting effects.the oral contraceptives 2.15 and 2.16 exert strong progestational activity of long duration