© Department of Chemistry, The University of Western Ontario

Chem 2223b Intersession 2008: Pharmaceutical Drugs

• In this chapter, we will discuss the process of pharmaceutical drug discovery, from the bench to the market, and some of its business aspects. We will also examine two selected topics: sulfanilamide, and the applications of chromophores.

• Background material, from Chem 2213a or otherwise, that is important includes:

o Reactions of amines and carboxylic acids

o Mechanism of substitution reactions

o Factors affecting the strengths of organic acids

o Singlet and triplet states

• After studying this section, attempt:

o Practice problems: chromophores :10; pharmaceutical drugs: all questions

o 2006 midterm: 12 – 16

o 2006 intersession midterm: 17 – 21, 24

o 2007 term test #2: 1 – 6, 12

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A. Pharmaceutical Development 1. Economics of Pharmaceutical Development

• While the exact costs involved in the discovery, development, and the bringing of a new drug to the market are unknown, there is little doubt that this process is an extremely expensive and long adventure.

• The estimated average cost, to research, discover, test, and bring a new drug to market exceeds USD 800 million, and this takes 12-15 years.

• Why are these costs so high? Many potential drug candidates fail after years of investment. It is estimated that out of 10,000 originally synthesized compounds, only one will clear all the hurdles required for commercialization.

• Drug development is a risky business for both the company and the investors.

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2. Food and Drug Administration (FDA) Model (also used by Health Canada)

• The development of new drugs can be classified into three broad stages.

a. Drug discovery and preclinical evaluation (this is there chemists make the most substantial contribution) • Identification of biological target • Compounds are identified or synthesized in the laboratory • Toxicological and efficacy studies in vitro and in animals • Drug discovery and preclinical evaluation • Patenting of new drug (patent length approximately 20 years)

b. Clinical evaluations • Three clinical trials assess the safety and efficacy of a compound • Very expensive and can take 2-10 years

c. FDA approval, marketing, and post-market surveillance • Once a drug is approved, it is monitored for long-term effects. • Pharmaceuticals are the second most-regulated industry.

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a. Drug discovery and preclinical evaluation

• In this stage, scientists identify new molecules that have the potential to achieve a desired biological effect. Some of these effects may include:

o The inhibition or stimulation of an enzyme, to alter a metabolic pathway

o Structural changes to cellular structure

• This may require research on:

o Fundamental mechanisms of disease or biological processes

o Action of known, existing therapeutic agents

o Random selection of potential drugs and broad biological screening

• New compounds can be synthesized in the lab or extracted from natural products. These new candidates are called new chemical entities (NCE). Since some drugs, such as peptides and monoclonal antibodies, are “biologicals” and not “chemicals,” the boarder term new molecular entity (NME) is often favoured over NCE.

• There are four primary methods to find new candidates.

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• Method 1: Extraction from natural sources is important an method, both historically and currently, of discovering potential drugs.

o Traditional, herbal, and holistic medicine should not be overlooked

Morphine and opiates (opium poppy)

Penicillin (Penicillium chrysogenum)

Taxol (Pacific yew tree)

Origin of Opium War Birth of antibiotics…

infections were no longer a death sentence

Greatest achievement in anti-cancer therapy

in the last 20 years

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• Method 2: Structure-Activity Relationship Studies (SARS) are used to make better compounds from those identified serendipitously or by purposeful searching.

o SARS determine the effect of structural changes to the molecule on its activity, e.g. how does changing a substituent affect the drug’s biological properties?

Codeine(in Tylenol 3)

HO

HO

ONH

O

O

ONH

OO

MeO

HO

ONH

MeO

MeO

ONH

Thebaine

Morphine Diacetylmorphine( )

OxycontinDilaudidVicodin

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• Method 3: Structure-Guided Drug Design (SGDD) is an in silico technique, where computerized modelling is used to design a drug that binds to the specified target.

o This method works best when the 3-D structure of the target is known.

o Note the meaning of structure in SARS and SGDD. In SARS, the structure of the drug is being changed. In SGDD, the structure of the target is known.

Modifications to the drug structure are made to make it fit better in the target of interest. Shown here is the anti-cancer drug methotrexate docked in a space-filling model of the enzyme dihydrofolate reducatase.

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o Some of the best-known drugs were developed using SGDD. These include Relenza and Tamiflu, both of which inhibit the influenza enzyme neuraminidase. Tamiflu is being stockpiled in case of a bird flu pandemic.

o Various internet-based projects allow you to volunteer your computer’s idle CPU time to assist in drug-discovery and protein-folding experiments.

Inhibitor docked in the active site of neuraminidase, the inhibition of which prevents influenza from replicating. However, scientists are still uncertain whether Tamiflu will be effective against the H5N1 strain of the bird flu, and cases of resistance have been reported.

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• Method 4: High-Throughput Screening (HTS) relies on the automated, combinatorial synthesis (combichem) of millions of sometimes-random compounds. These compounds are screened in parallel against a multitude of possible targets.

o Automated, small-scale synthesis, tracking, and screening on microwell plates

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• Regardless of method used, once ideal drug candidates are identified (typically 1-5 out of every 10000), they are then patented and subjected to preclinical evaluation.

• Preclinical studies include in vitro testing (test tubes studies using bacteria, human cells, enzymes, etc.) and ADMET studies in animals (rats, rabbits, mice, etc.). FDA approval is not required to perform in vitro or animal studies.

o Absorption o Distribution o Metabolism o Excretion o Toxicity

• The ADMET set of studies alone does not evaluate the efficacy of the drug.

• If the preclinical data are NOT desirable (e.g. drug candidate does not work, high toxicity, poor bioavailability, etc.), then the investment to date is virtually all lost.

• If the preclinical data are desirable, then an IND (investigational new drug) application is filed with the FDA for that specific NME. If the FDA approves the application, clinical evaluation in humans can commence.

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b. Clinical evaluation

• Clinical evaluation in humans occurs in three phases. The evaluation is usually aborted if data from any one of the phases is negative (bad or not desirable). The overall process takes many years and is expensive. Frequently, these evaluations are contracted overseas, where costs are lower.

• Phase I clinical trials establish the safety, ADMET, side effects, and dosage range in 20-100 healthy volunteers.

• Phase II clinical trials verify the effectiveness and short-term side effects in 100-500 volunteers who have the targeted disease or symptom.

• Phase III clinical trials verify the effectiveness and longer-term side effects in a larger population of volunteers (thousands). These trials also determine the optimum dosage and form of administration (oral, intravenous, transdermal. etc.).

• Permission needs to be granted by the FDA prior to proceeding to the next phase. The odds of passing all three phases are about 10%.

• While clinical trials are very long, the FDA may fast track an IND should it target a serious or life-threatening condition for which no current treatments exist.

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c. FDA approval and post-market surveillance

• If the clinical trials are positive, then the company submits an NDA (new drug application) to the FDA for the NME and the therapeutic use specified. An NDA is like a long lab report, which includes all data and results: 100,000 pages.

• If the FDA is satisfied that the drug candidate has a relatively high benefit-versus-risk ratio, then the drug is approved for marketing for the use specified.

• Although drugs are approved for very specific uses, physicians can prescribe a drug off-label and use it for something else besides the approved therapeutic use.

o Cancer drugs are often used off-label, since drugs approved for one cancer sometimes works for other types of cancers.

o There are also legal implications. Several years ago, antidepressants for adults were prescribed to children, without clinical trials involving children.

o Drug insurance policies often only cover a drug when used for its intended therapeutic purpose, not off-label usage.

• Note that patents expire 20 years from the date of filing, not the date of approval.

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• After approval, the safety and efficacy of the drug is still monitored. This post-approval monitoring is often called Phase IV trials. A drug may be withdrawn or may have restrictions placed on its usage if the data is negative. Examples:

Year Drug Primary health risk 2008 Trasylol (anti-bleeding for heart surgery) Death (reason unknown) 2007 Infant cold and cough medications Various, caused by misuse 2006 Tequin (antimicrobial) Severe hypo- or hyperglycemia 2005 Adderall XR (ADHD) Sudden death and stroke 2004 COX-2 inhibitors (anti-inflammatory) Stroke / myocardial infraction 2003 Serzone (antidepressant) Hepatoxicity 2002 Lotronex (gastrointestinal) Ischemic colitis 2001 Baycol (anti-cholesterol) Fatal rhabdomyolysis 2000 Propulsid (gastrointestinal) Fatal arrhythmia 2000 Rezulin (anti-diabetic) Hepatoxicity 2000 Phenylpropanolamine (decongestant) Hemorrhagic stroke

Some drugs were withdrawn as quickly as a year after approval, but others took decades. By consuming an “approved” drug, you’re volunteering to be a test subject.

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B. Sulfanilamide: An Unexpected Antibiotic 1. History

• In 1932, Gerhard Domagk was working at the German conglomerate IG Farben when he discovered that protonsil rubum, a red leather dye, protected mice when they were injected with a lethal dosage of streptococci bacteria.

• Shortly thereafter, Domagk’s daughter pricked herself with a needle and developed a streptococci infection. Her lymph nodes were lanced fourteen times to discharge fluid. Doctors insisted on the amputation of her arm, but her father disagreed.

• After ingesting some of the leather dye, she fully recovered.

N

NH2

H2NN S

O

O

NH2

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• What was puzzling was that protonsil was ineffective in test-tube experiments against bacteria, but it worked when ingested. This mystery was later solved by the Pasteur Institute in France, where it was found that the liver reduced the dye into two halves. This discovery gave birth to the discipline of drug metabolism.

N

NH2

H2NN S

O

ONH2

NH2

NH2

H2N

H2N SO

ONH2

azo group

Prodrug

did not killbacteria

Sulfanilamidedrug

sulfonamide group

• One half did not have any bacterial activity, but the other half, a chemical known as sulfanilamide, was fully active. i.e. the dye was metabolized into an active form.

• After this discovery, Protonsil was then rapidly abandoned in favour of chemically simpler sulfanilamide, as it was an existing chemical that was easily prepared.

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2. Mechanism of Antibacterial Activity

• Mammals need to consume folic acid as a vitamin, but bacteria can synthesize it from p-aminobenzoate (PABA). Folic acid is essential for metabolism.

NH

O

NH

N

OH

N

NH2NN

O

OH

HOOC

Folic Acid(vitamin B9)

Glutamic acid

PABAPteridinederivative

• Bacteria cannot transport folic acid across their cell membrane, so they must synthesize it themselves, and sulfanilamide inhibits the biosynthesis. Mammals are not affected by sulfanilamide, because they don’t have this biosynthetic pathway.

• The biosynthesis of involves two coupling reactions to link the three units together. An amide bond and a secondary amine are made. (At biological pH, PABA carries a negative charge at the carboxylate).

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• The first step involves the linking of the pteridine derivative to PABA, where the amine from PABA replaces the OH group on the pteridine via an SN2 reaction.

• The OH first needs to be converted into a good leaving group. In this case, it is phosphorylated twice.

NOH

N

NH2NN

HO

NOH

N

NH2NN

OPO

OO

POO

O

NH2OOC2 ATP

2 ADP

OOC

NH

NOH

N

NH2NN

Step 1 –H+

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• The second step forms an amide bond between the amino group of glutamic acid and the carboxylate of the product formed in the first step. ATP is used to activate the carboxylate by forming a mixed anhydride (compare to protein synthesis).

OOC

NH

NOH

N

NH2NN

NH2O

OH

HOOC

ATP

ADP

NH

O

NHN

OH

N

NH2NN

O

OH

HOOC

Step 2

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• How does sulfanilamide inhibit this process? It is structurally similar to PABA in terms of size and chemical functionality.

• The sulfonamide functional group is also weakly acidic, with a pKa of 10.43, and so it can deprotonate to form an anion. The anion is resembles PABA.

H2N S

O

O

NH2 H2N S

O

O

NH

• Thus, the deprotonated sulfonilamide is a substrate analog that competes with the natural substrate PABA (anion) for the enzyme in Step 1. If the drug is bound to the enzyme’s active site, the enzyme can no longer bind PABA.

• Without the biosynthesis of folic acid, and without a mechanism to transport it inside the cell, the bacteria cannot grow.

6.9Å 6.7Å

2.4Å 2.3Å

6.9Å 6.7Å

2.4Å 2.3Å

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3. Importance of Sulfonamide pKa

• Sulfanilamide has a pKa of 10.43, so only ~1 molecule in 1000 is in the deprotonated form (the active form) at physiological pH of 7.4.

• Thus, it is reasonable to expect that compounds existing entirely in the ionized form would be the best drugs, e.g. sulfanilic acid, a strong acid.

• However, this is NOT the case.

o Lowering the pKa increases the amount of the drug in the active, anion form, so it increases enzyme inhibition.

o Yet, the anion cannot cross the lipid cell membrane of the bacteria. Usually, only neutral species can cross hydrophobic membranes.

NH2

SO3

NH2

SO3H

sulfanilic acid(strong acid)

sulfanilateanion

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• In other words, a balance between the two is required. The drug must be neutral to cross the membrane, but once inside the cell, it must be acidic enough to ionize.

Drug-H

Drug

Drug-H

Drug

Bacterialcell

H2N SO

ON

R

H

• Scientists went on and prepared derivatives of sulfanilamide with different pKa values by adding a substituent to the sulfonamide nitrogen. The substituents were well-tolerated (did not cause toxicity) and didn’t interfere with drug action.

• These substituents can modify the acidity of the NH by stabilizing the conjugate base, the anion. Recall that for most acids, the more stable the conjugate base, the stronger the acid, and hence a lower pKa value.

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• Structure-activity relationship studies showed that the optimal pKa was about 5-7.

Some R pKa

H

N

N

S

N

N

C CH3

O

10.43

8.43

7.12

6.48

5.38pKa

Ant

ibac

teria

lact

ivity

0 124 8

• Although sulfanilamide is an older drug, it demonstrates some of the challenges involved in drug discovery. The same principles are still important today.

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• Sulfanilamide also has an important historical connection with the FDA.

• The first US law on food and drugs was passed in 1906, but it only prohibited the sale of counterfeit goods. There were no standards regarding purity or content, and no proof that a drug was safe and effective was needed... until 32 years later.

• Sulfanilamide had been sold as tablets, but they were not ideal for children. In 1937, a company sold Elixir Sulfanilamide, which contained the drug dissolved in a great-tasting liquid.

HOOH

• Despite over a hundred deaths, the company did not break any laws, even though the solvent was known to be toxic. These deficiencies were amended in 1938 and laid the groundwork for future amendments: pesticides, food additives, and medical devices.

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C. Pharmaceutical Applications of Light and Chromophores

• The pharmaceutical use of light and chromophores, known as phototherapy, is a relatively modern technique and has proven to be invaluable in medical fields such as oncology, dermatology, and ophthalmology.

• While the technique can be divided into photochemotherapy (PUVA) and photodynamic therapy (PDT), they share common features.

o A drug, or its precursor, is administered to the patient. Depending on the use, it can be consumed, injected, or applied.

o The affected area is irradiated with light of the appropriate wavelength.

o The drug is excited, which eventually leads to tissue destruction (mechanism varies).

• PUVA and PDT differ in the type of chromophore used and the mechanism of destruction.

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1. Photochemotherapy (PUVA)

• Photochemotherapy usually involves psoralens, which are found in certain plants (celery, parsley, etc). They absorb light in the UV-A region, so this type of photochemotherapy is termed PUVA therapy.

• After excitation to S1, it intersystem crosses to the T1 state. This triplet state acts as a crosslinking agent and reacts with thymine by a [2+2] photocycloaddition. The absorption of a second photo causes a second reaction.

O OO

OCH3

HNN

HO

O R N NH

H

O

O

R'N

HN

O

OR

CH3

N

NH

O

OR'

H3C UV

• PUVA therapy is approved for a treatment of various dermatological conditions,

such as psoriasis, eczema, and atopic dermatitis.

O OO

OCH3

methoxypsoralen(Methoxysalen)

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2. Photodynamic Therapy (PDT)

• In PUVA, the excited drug molecule is the actual species that damages DNA. Whereas, in PDT, the species that is directly responsible for the damage is not the excited drug, but rather, a reactive oxygen species (commonly termed ROS).

o Examples of ROS include singlet oxygen, superoxide, hydroxyl radical, hydroperoxyl radical, hydrogen peroxide, and others. They are powerful oxidants and can damage virtually all biomolecules.

• The correct structure of ground-state oxygen is not the one determined by Lewis-structure rules, but rather by molecular orbital theory. It has a single bond and two lone electrons.

• The two radicals are in different orbitals, so they can have the same spin (ms). i.e. they are not spin-paired, so it is in the triplet state.

• Ground-state oxygen is abbreviated 3O2. The triplet nature of oxygen also explains why it is paramagnetic; it has a non-zero net magnetic moment.

O OO O

wrong correct

O O

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• In the presence of the proper energy, 3O2 can be excited to form singlet oxygen (1O2*), an extremely powerful oxidizing agent, and this is the basis of PDT.

• However, the energy used for the excitation of oxygen does not come from direct excitation with light, but rather, by a mechanism known as energy transfer.

• A photosensitizer (the PDT drug) is excited with a laser. Efficient ISC to the triplet state occurs. Triplet states react quickly with other triplet states, and the reaction of 3Ps with 3O2 results in 1O2. Note that the process is catalytic, regenerating the drug.

• Because of this energy transfer process, the word dynamic is found in PDT.

O O O Oenergy

triplet(ground)

singlet(excited)

Ps Ps1. Laser

2. ISC

3

3O21O2

Energy transfer

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• The first PDT drug was Photofrin, developed by QLT Phototherapeutics. Approved to treat various cancers.

• Photofrin is a mixture of hematoporphyrin and related monomers and dimers, one of which is shown on the right.

• Wavelength of max absorption = 630 nm

• Advantages of Photofrin and PDT:

o Generation of 1O2 is highly localized to the area of irradiation o The porphyrin is essentially a catalyst, so only a small amount is needed

• Disadvantages of Photofrin: o Light of 630 nm can only penetrate a few millimeters into tissue, so it cannot be

used to treat deeply buried tumors o Long elimination half-life (21 days). Patients need to protect the eyes and skin

from exposure to sunlight or bright indoor light for 1 – 3 months.

HN

NNH

N

HN

NNH

N

COOH

COOH

OH

O

COOHHOOC

OH

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• Intense research is currently taking place to develop photosensitizers that absorb efficiently at longer wavelengths.

• The best wavelength is generally in the 650 – 950 nm area. There are many chromophores in tissue that absorb below 650 nm, and above 950 nm, water starts to absorb near-IR light.

• However, it is difficult to design a chromophore that absorbs in this range and yet is still able to efficiently produce 1O2.

• Many of the greatest discoveries in medicine and medical therapies are made by chemists, biochemists, and other basic-research scientists. Collaboration between disciplines is also an important key to successful research.

• Students who are interested in the mechanism of drug action should consider taking Chem 3393b, Medicinal Chemistry.