The Organic Chemistry of Drug Design and Drug Action

1
The Organic Chemistry of Drug Design and Drug
Action

• Chapter 5
• Enzyme Inhibition and Inactivation

2
Enzyme Inhibition and Inactivation
Why inhibit an enzyme?

• Many diseases arise from
• deficiency or excess of one metabolite
• infestation of foreign organism
• aberrant cell growth

All can be corrected by specific enzyme
inhibition.
Inhibition of an enzyme blocks the degradation of
its substrate (increasing its concentration) and
blocks the formation of its product (decreasing
its concentration).
3
Enzyme inhibitor - slows or blocks enzyme
catalysis Enzyme inactivator - irreversible
inhibitor Use of drugs to combat foreign
organisms and aberrant cells - chemotherapy
About one-quarter of FDA-approved drugs are
enzyme inhibitors Robertson, J. G. Biochemistry
2005, 44, 5561-5571.
4

• Enzymes are the most promising protein targets
  for rational drug design because
• enzymes are purified more easily
• enzyme inhibitors look like substrates
• can use enzyme mechanism in design

5
Ideal inhibitor - totally specific for one target
enzyme rarely occurs, if at all

• Look for differences in metabolic pathways
  between a foreign organism and humans

• Look for substrate differences

• Tumor cells have the same enzymes as normal
  cells, but replicate faster. They will take up
  antimetabolites (compounds that look like
  metabolites, but inhibit enzymes), which results
  in selective toxicity.

6
Ideal enzyme target in foreign organism or
aberrant cell - one that is essential for growth,
but nonessential in humans (or not even present)
7
Categories of Enzyme Inhibitors
Reversible - inhibition of enzyme activity that
is reversible, typically noncovalent Irreversible
- inhibits enzyme for an extended period of time,
typically covalent
First, consider two important concerns in drug
design drug resistance and drug synergism.
8
Drug Resistance
When a formerly effective drug dose is no longer
effective. Arises mainly from natural selection -
replication of a naturally resistant strain after
the drug has killed all of the susceptible
strains. On average, 1 in 10 million organisms in
a colony has one or more mutations that makes it
resistant.
9
Resistance is different from tolerance - this is
when the body adapts to a particular drug and
requires more of the drug to attain the same
initial effect - lowers the therapeutic index.
It is also possible to develop tolerance to
undesirable effects of drugs, such as sedation by
phenobarbitol - raises the therapeutic index.
10
Mechanisms of Drug Resistance
1. Altered drug uptake - exclusion of drug from
site of action by blocking uptake of drug -
altered membrane with more or - charges 2.
Overproduction of the target enzyme - gene
expression 3. Altered target enzyme (mutation of
amino acid residues at the active site) - drug
binds poorly to altered form of the enzyme 4.
Production of a drug-destroying enzyme - a new
enzyme is formed that destroys the drug
11
Mechanisms of Drug Resistance (contd)
5. Deletion of a prodrug-activating enzyme - the
enzyme needed to activate a prodrug is missing 6.
Overproduction of the substrate for the target
enzyme - blocks inhibitor binding 7. New
metabolic pathway for formation of the product of
the target enzyme - bypass effect of inhibiting
the enzyme 8. Efflux pump - protein that
transports molecules out of the cell
12
Examples of Mutated Target Enzyme
M184Vand M184I mutants are produced by these drugs
If your drug has a structure similar to the
substrate, mutations will lower binding of the
substrate as well as the inhibitor.
13
Approaches When a Drug-Destroying Enzyme is
Produced
1. Make an analog that binds poorly to this new
enzyme
2. Alter structure of drug so it is not modified
by the new enzyme, such as tobramycin (5.14),
which lacks the OH group of kanamycins (5.12)
that is phosphorylated by resistant organisms.
no OH group
resistant organisms phosphorylate here
3. Inhibit the new enzyme
14
Drug Synergism
Arises when the therapeutic effect of two or more
drugs used in combination is greater than the sum
of the effect of the drugs individually.
15
Mechanisms of Drug Synergism
1. Inhibition of a drug-destroying enzyme
protects the drug from destruction
2. Sequential blocking - inhibition of two or
more consecutive steps in a metabolic pathway -
overcoming difficulty of getting 100 enzyme
inhibition
3. Inhibition of enzymes in different metabolic
pathways- block both biosynthetic routes to
the same metabolite
4. Efflux pump inhibitors can be made to prevent
efflux of the drug
5. Use of multiple drugs for same target - about
1 in 107 bacteria resistant to a drug if you use
two drugs, then only 1 in 1014 is resistant to
both
16
Reversible Enzyme Inhibitors
Most common compete with substrate for active
site binding - competitive reversible inhibitors
structures similar to substrates or products
Scheme 5.3
(equilibrium attained with diffusion control)
Dissociation constant for breakdown of E?I
complex The smaller the Ki, the tighter (more
effective) the binding.
17
Inhibitor also may be a substrate - may be
unfavorable in drug design if the product is
toxic Inhibitor can act by binding to a site
other than active site (an allosteric site) -
called a noncompetitive inhibitor E?I
concentration depends on I and S and their
dissociation constants (Ki and Km, respectively).
As I increases, E?I increases. Both S and I
compete for E, so as either I decreases (by
metabolism) or S increases, E?I decreases and
E?S increases.
Therefore, drugs are administered several times a
day to maintain the enzyme in E?I complex.
18
Receptors vs. Enzymes
19
Example of Simple Competitive Reversible
Inhibition
Captopril, enalapril, and lisinopril -
antihypertensive drugs
Scheme 5.4
20
Hypertensive Effects of ACE

• Produces angiotensin II, a vasoconstrictor
  (hormone action)

• Involved in conversion of angiotensin II to
  angiotensin III, which releases another hormone,
  aldosterone (5.23). This hormone regulates the
  electrolyte balance by retention of Na and water.

• Catalyzes hydrolysis of a potent natural
  vasodilator, bradykinin

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg bradykinin
21
Lead Discovery
1965 - mixture of peptides in the venom of a
South American pit viper shows inhibition of
bradykininase activity and inhibits conversion of
angiotensin I to angiotensin II 1971 - several
other peptides found at Squibb but not effective
orally Today, a peptide combinatorial library
approach would be taken.
22
Lead Modification
Pro found best at C-terminal position, Ala best
in penultimate position, and an aromatic residue
in the antepenultimate position. ACE purified to
homogeneity from rabbit lung - contains one
Zn/enzyme
23
Mechanism of Action
Function of Zn cofactor
Figure 5.5
May be similar to carboxypeptidase A, another
Zn-dependent peptidase.
24
Hypothetical Active Site of Carboxypeptidase A
Figure 5.6
potent inhibitor of CPA
25
Potency of (R)-2-benzylsuccinic acid derived from
its resemblance to the collected products of
substrate hydrolysis.
Figure 5.7
26
With (R)-2-benzylsuccinic acid as a model, a
series of carboxyalkanoylproline derivatives were
tested as inhibitors of ACE.
good Zn coordination
Peptidomimetic To avoid orally unstable
dipeptide, made isostere
All were only weak inhibitors of ACE.
27
To increase potency, changed COO- to SH.
28
Hypothetical Binding of Inhibitors to ACE
Figure 5.8
isosteric exchange
29
Best binding properties Ki 1.7 nM
Highly selective for ACE
30
Effect of Structural Modifications
Every part of captopril is important for binding.
Table 5.1
31
Captopril was the first ACE inhibitor on the drug
market - hypertension and congestive heart
failure.
32
Combination Therapy
diuretic (for water retention caused by
aldosterone)
This combination is effective for 90 of the
hypertensive population. Sometimes a ?-blocker is
used in triple therapy for vasodilation.
33
Two side effects rashes and loss of taste -
reversible when drug withdrawn Merck hypothesized
side effects from SH group.
Went back to COO- instead of SH
Increased potency by adding groups to interact
with more sites on enzyme (i.e. - to increase the
pharmacophore).
34
Made more peptide-like, but protonated too
hydrophilic
Added a lipophilic group (R) to counteract
hydrophilicity and also to bind to S1 subsite
R (S)-PhCH2CH2 R? H enalaprilat
35
Binding of Enalaprilat to ACE
Figure 5.9
additional binding interactions
enalaprilat
Poorly absorbed orally - remedied by using ethyl
ester (at arrow) (enalapril) which is hydrolyzed
by esterases to give enalaprilat (a prodrug).
36
Does not require prodrug activation.
37
Dual-Acting DrugsDual-Acting Enzyme Inhibition
When inhibition of two different enzymes gives a
synergistic effect, a single inhibitor for both
enzymes can be designed.
38
Advantages of making a dual-acting drug instead
of two separate drugs
1. With two drugs need to develop two separate
syntheses, two formulations, and two metabolism
studies.
2. Two drugs have different pharmacokinetic rates
and metabolic profiles.
3. The likelihood that two drugs will progress to
the clinic at the same rate is small.
4. Must do three sets of safety studies and three
sets of clinical trials (one for each drug plus
the combination).
5. The odds of one drug in clinical trials
getting to market is 1 in 10 the odds of two
drugs is 1 in 100.
39
Example of a Dual-Acting Enzyme Inhibitor
Neutral endopeptidase (NEP) is Zn-dependent and
degrades atrial natriuretic peptide (ANP). ANP
causes vasodilation (lowers blood pressure) and
inhibits aldosterone formation.

• Therefore, ANP and angiotensin II have opposite
  functions.
• inhibition of NEP increases ANP
• inhibition of ACE decreases angiotensin II
• both result in blood pressure lowering

40
Peptidomimetic Dual-Acting Enzyme Inhibitors
Bristol-Myers Squibb
Lead modification
Lead compound
IC50 30 nM vs ACE IC50 400 nM vs NEP
potent in vitro for both ACE and NEP poorly
active in vitro
41
Conformationally-restricted Peptidomimetics
potent in vitro and in vivo for both ACE and NEP
IC50 5 nM vs ACE IC50 17 nM vs NEP oral
activity greater than captopril in rats
42
The 7,6-fused bicyclic thiazepinone analogue was
advanced to clinical trials
IC50 5 nM vs ACE IC50 8 nM vs NEP
Problems in phase III clinical trials had to be
withdrawn.
43
Dual-acting drugs do not have to inhibit only
enzymes. Could be an inhibitor of an enzyme and a
receptor antagonist or an antagonist for two
receptors, or an agonist for two receptors (or
any combination thereof). The approach is to
identify molecules that interact with each
receptor or enzyme, then combine parts of each
molecule to identify what is a common
pharmacophore.
44
Example of a Dual-Acting Receptor Agonist
Agonist for both the D2-receptor and
?2-adrenoceptor would be useful for the treatment
of airway diseases, such as chronic obstructive
pulmonary disease (COPD) and asthma.
45
D2-receptor agonist reduces cough and
mucous ?2-adrenoceptor agonist is an
antibronchoconstrictor
a weak ?2-adrenoceptor agonist
a potent D2-receptor agonist
These two were hybridized to give
46
Finally modified to give the clinical candidate
47
Example of Competitive Reversible Inhibitor Drug
that Also Acts as a Substrate
Sulfonamide Antibacterial Agents (Sulfa Drugs)
Lead discovery - Bayer Co. tested azo dyes
against streptococci. Prontosil was very
effective in mice (1935).
prontosil
No activity in vitro unless a reducing agent is
added. In vivo protonsil is metabolized by
reduction to the active agent (a prodrug).
48
sulfanilamide The active agent - active in vitro
and in vivo
Bacteriostatic - inhibits further growth of
bacteria, but does not kill them.
49
Lead Modification
Prontosil - beginning of modern
chemotherapy Thousands of compounds synthesized
and tested - the first SAR studies First examples
where new leads for other diseases revealed from
side effects in clinical studies - antidiabetic
and diuretic agents

• Other developments from these studies
• a simple assay for these compounds in body
  fluids
• showed antibacterial effect is proportional to
  the concentration in blood, which varied from
  patient to patient at a given dose - beginning of
  monitoring blood drug levels during chemotherapy

50
Mechanism of Action
1939 Stamp showed microorganisms contained a
substance that blocked the antibacterial action
of sulfonamides 1940 Woods hypothesized that
sulfanilamide must have a structure similar to a
substrate for an essential enzyme He
deduced that the structure was
p-aminobenzoic acid (PABA)
Showed that PABA was competitive with
sulfanilamide for microbial growth
51
1940 - Fildes proposes the antimetabolite theory
- a rational approach to chemotherapy is the
design of an enzyme inhibitor for an important
metabolic pathway whose structure is similar to
that of an essential metabolite
Mid 1940s - Miller shows sulfanilamide inhibits
folic acid biosynthesis
1948 - Nimmo-Smith et al. show sulfonamides are
competitively reversed by PABA
52
1969 - Two enzymes purified by Richey and Brown
Scheme 5.5
PABA
dihydrofolate
53
Sulfonamides are Substrates for Dihydropteroate
Synthase
35Ssulfamethoxazole
Scheme 5.6
Cannot produce dihydrofolate or tetrahydrofolate
needed for purines and DNA biosynthesis
Bacteriostatic - inhibition of tetrahydrofolate
biosynthesis inhibits DNA synthesis and
replication does not kill existing bacteria
54
Inhibition of dihydropteroate synthase has no
effect on humans--
We do not have this enzyme.
Folic acid is a vitamin (must be eaten), which we
convert to dihydrofolate and tetrahydrofolate by
enzymatic reduction.
Bacteria do not have a transport system for folic
acid therefore, cannot utilize the folic acid we
eat.
55
Dihydropteroate synthase satisfies at least 3 of
the 4 criteria for a good antimicrobial drug
target 1. Target is essential to survival of
microorganism 2. Target is unique to the microbe
so humans are not harmed 3. Structure and
function of the target is highly conserved across
a variety of species of that microbe for broad
spectrum 4. Resistance to inhibitors of target
not easily acquired
It generally takes 1-4 years for resistance to
emerge resistance to sulfonamides took almost
seven years.
56
Drug Resistance

• overproduction of PABA
• formation of a dihydropteroate synthase that
  binds PABA normally, but binds sulfonamides
  thousands of times less tightly
• altered permeability of sulfonamides

57
Drug Synergism
Combination of sulfadoxine with pyrimethamine-
used in treatment of malaria
(sulfa drug)
Sequential blocking - sulfa drug inhibits
synthesis of dihydrofolate pyrimethamine
inhibits dihydrofolate reductase (synthesis of
tetrahydrofolate)
58
Transition State Analogs
Enzyme binds to substrate tightest at transition
state of reaction (conformational change
involved) Bernhard and Orgel - inhibitor
molecules resembling transition state structure
should be more tightly bound than substrate -
transition state analog inhibitor Mechanism of
the enzyme reaction must be understood When more
than one substrate is involved, a single stable
compound is made with a structure similar to the
two or more substrates at the transition state -
multisubstrate analog inhibitor.
59
- antineoplastic
from Streptomyces antibioticus
inhibitor of adenosine deaminase Ki 2.5 x
10-12 M (2.5 pM) (107 times lower than Km for
adenosine!)
60
Hypothetical Mechanism of Adenosine Deaminase
Scheme 5.8
pentostatin mimics this
2?-deoxyadenosine
2?-deoxyinosine
61
Multisubstrate Analog N-Phosphonoacetyl-L-Asp
(PALA)
Aspartate transcarbamylase - de novo biosynthesis
of pyrimidines
Scheme 5.9
carbamoyl phosphate
isostere - no longer a leaving group, mimics
phosphate
N-carbamoyl-L-Asp
PALA
Tumor resistance

• tumor cells acquired ability to utilize
  preformed circulating pyrimidine nucleosides
• increased carbamoyl phosphate
• increased aspartate transcarbamylase

62
Slow, Tight-Binding Inhibitors
Slow-binding inhibitors - equilibrium between
enzyme and inhibitor is reached slowly (kon
small) therefore, time-dependent
inhibition Tight-binding inhibitors - substantial
inhibition when E and I are comparable (koff
small) Slow, tight-binding inhibitors - both
properties t1/2 for E?I complex can be seconds
to months
Cause unknown, but most likely a conformational
change in the enzyme.
63
Examples of Slow, Tight-Binding
InhibitorsLovastatin and SimvastatinAntihypercho
lesterolemic drugs
About 1/2 of all deaths in the U.S. is attributed
to atherosclerosis - fatty deposit buildup on
inner walls of arteries. Major component of
plaque is cholesterol. About 1/2 of body
cholesterol is biosynthesized the rest needs to
be eaten. Some people biosynthesize much more
than half of what they need.
Rate-determining step in cholesterol biosynthesis
is conversion of 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) to mevalonic acid.
64
Rate-Determining Step in Cholesterol Biosynthesis
Scheme 5.10
HMG-CoA
mevalonate
Inhibition of HMG-CoA reductase should lower
plasma cholesterol levels.
65
HMG-CoA
CoA
66
Lead Discovery
Endo and coworkers (Sankyo Co. in Japan) tested
8000 strains of microorganisms for metabolites
that inhibited sterol biosynthesis in vitro - 3
found from fungus Penicillium citrinum - most
potent called mevastatin.
The same compound isolated from a different
strain of Penicillium by Brown and coworkers
(Beecham Pharmaceuticals in England) - called it
compactin.
A more potent compound was isolated by Endo from
a different fungus - called monacolin K.
This same compound isolated at Merck - called
mevinolin.
Now, mevinolin is called lovastatin.
67
Mechanism of Action
Potent competitive reversible inhibitors of
HMG-CoA reductase Ki for lovastatin 6.4 x 10-10
M (affinity for lovastatin is 16,700 times
greater than for HMG-CoA)
68
(No Transcript)
69
HMG-CoA Reductase
Scheme 5.10
70
The active form of lovastatin is the hydrolysis
product. This mimics intermediate 5.70 in Scheme
5.10. Two important binding pockets
when H is replaced by CH3, it is simvastatin (2.5
times more potent than lovastatin)
Active form of lovastatin
71
A beneficial side effect of the statins is an
enhancement of new bone formation in rodents,
associated with increased expression of the bone
morphogenetic protein-2 gene in bone cells.
72
Peptidyl Trifluoromethyl Ketones Inhibition of
Human Leukocyte Elastase and Cathepsin G
Human leukocyte elastase and cathepsin G - serine
proteases released by neutrophils in lungs to
digest dead lung tissue and to destroy invading
bacteria. Natural inhibitors of these enzymes
(?1-protease inhibitor and bronchial mucous
inhibitor) are released to prevent these enzymes
from destroying lung, elastin, connective tissue.
An imbalance in protease and inhibitor
concentrations may cause emphysema or bronchitis.
73
Therefore, need inhibitor of elastase and
cathepsin G.
Most potent analog
74
Mechanism of Peptidyl Trifluoromethyl Ketones
Scheme 5.11
various conformational changes may be involved
75
Case History of Rational Drug Design of an Enzyme
InhibitorRitonavir and other anti-AIDS drugs
Human immunodeficiency virus-1 encodes an
aspartate protease (HIV-1 protease) that
processes the gag and gag-pol gene products into
mature, functional proteins. If this processing
does not occur, the progeny virions are immature
and noninfectious.
HIV-1 protease inhibitors should be effective
anti-AIDS drugs.
76
General Approach
First identify molecules with good potency
(preferably in the nanomolar range), then use
these molecules as leads to solve pharmacokinetic
problems.
77
Lead Discovery
HIV-1 protease has an unusual structure - active
enzyme has C2 symmetry. This symmetry element was
used as the starting point in the lead design to
get selectivity for HIV protease. Also used a
transition state analog approach, but had to
delete one of the two OH groups for stability and
convert the proline to a phenylalanine side chain
to have symmetry.
78
Lead Design/Discovery
Product of H2O addition to the peptide bond
Figure 5.10
to increase pharmacophore
C2 symmetric
C2 symmetric
5.79 analogs are generally 10 times more potent
than 5.78 ones Stereochemistry of OH not
important IC50 values 20 - 150 nM therapeutic
indices 500 - 5000
79
Lead Modification
With pharmacodynamics good, focus on
pharmacokinetic problems - peptidomimetic
approach Aqueous solubility poor A crystal
structure with 5.79e bound showed Cbz groups were
not interacting with the protein therefore,
they could be modified without affecting the
potency.
80
Polar heterocyclic bioisosteres of the Cbz phenyl
groups were used. Compound 5.80 was the best of
the series, but it still did not have oral
bioavailability.
81
Compounds in the 5.78 series were less potent
than those in the 5.79 series, but were more oral
bioavailable. A composite of the two was made
(5.81).
These were more potent than 5.79, but poorly
bioavailable.
82
Consider Lipinskis Rule of Five
The MW of 5.80 is 794 (should be lt
500) Therefore, needed to decrease size, which
means eliminate the C2 symmetry component.
SAR observations incorporation of carbamate
linkage gives higher potency than N-alkylurea
N-methylurea linkage gives higher oral
bioavailability than carbamate aqueous
solubility did not correlate with oral
bioavailability
83
The best of the first unsymmetrical series was
5.82 - aqueous solubility is 3.2 ?g/mL, oral
bioavailability is 32.
The plasma half-life, though, is only 2.3
h. Cause shown to be formation of 3 metabolites
the N-oxide of each pyridine nitrogen and the
bis(pyridine N-oxide), all from cytochrome P450
oxygenation.
84
To decrease metabolic oxidation, used
heteroaromatic isosteres with lower oxidation
potentials.
Potency increased with small alkyl substitution
on the P3 heterocycle (the left-hand one), but
not on the P?2 heterocycle (the right-hand
one). A crystal structure showed that this group
has a hydrophobic interaction with Val-82.
85
The position of the OH group is also important
compounds with the OH distal to the valine (5.83)
were 10-fold more potent than with the OH
proximal to the valine (5.84).
interacts with Val-82
86
The final modifications gave ritonavir (5.85)
Solubility is 6.9 ?g/mL, oral bioavailability is
78, Ki is 15 pM, and Cmax/ED50 (ratio of in vivo
plasma levels to effective dose for half the
animals) is 105.
Ritonavir also inhibits cytochrome P450, which is
why its metabolism is low.
87
Resistance
Resistance to ritonavir because of mutation of
Val-82 to larger groups - interferes with the
isopropyl group interaction. Modifications Excise
d the 3-isopropylthiazole (5.86) - poor
inhibition
88
Increased the pharmacophore by a ring-chain
transformation (5.87)
Modified the other thiazole (5.88)
89
Lopinavir has a low plasma half life (no thiazole
to inhibit P450). Therefore, ritonavir and
lopinavir are used in combination ritonavir
inhibits susceptible HIV-1 protease and P450, and
lopinavir inhibits the enzyme from the resistant
organism.
90
Irreversible Enzyme Inhibitors

• Reversible inhibitor is effective as long as I
  is suitable to drive equilibrium to the E?I
  complex.
• Irreversible enzyme inhibitor (inactivator)
• Structure similar to substrate or product
• Generally forms covalent bond to active site.
• Therefore, not necessary to maintain inactivator
  concentration once enzyme has reacted - complex
  cannot dissociate.

Smaller and fewer drug doses should be possible.
91
Irreversible inhibition does not mean permanent
loss of the enzyme - gene encodes for other
copies (may take hours or days). Two general
types of irreversible inhibitors Affinity
labeling agents (reactive compounds) Mechanism-bas
ed enzyme inactivators (unreactive compounds)
92
Affinity Labeling Agents
Scheme 5.12
reversible complex
covalent complex
generally alkylation or acylation
When equilibrium for E?I complex is rapid and
rate of dissociation of E?I complex is fast, the
kinact is rate-determining step, and
time-dependent loss of enzyme activity occurs.
93

• Affinity labeling agents are reactive -
  therefore, potentially toxic.
• Still can be effective
• Once E?I complex forms, unimolecular - more
  rapid than nonspecific bimolecular reactions.
• If E?I complex forms with other enzymes, needs
  nucleophile present at active site near reactive
  group for reaction to occur.
• In case of antitumor agents, DNA precursors
  rapidly transported and concentrated at tumor
  target.

94
Design of Effective Affinity Labeling Agents

• Low Ki for target enzyme for selectivity
• Modulate reactivity
• Incorporate reactive group so it is near
  nucleophile in active site of target enzyme (from
  crystal structure data)

95
Modulated Reactivity Quiescent Affinity Labeling
Inactivator whose reactivity is too low for
nucleophiles in solution. Only nucleophiles in
the active site of enzymes that use covalent
catalysis are reactive enough. For example,
peptidyl acyloxymethyl ketones (5.89) have low
chemical reactivity but inactivate cathepsin B
(important in osteoclastic bone resorption).
Scheme 5.13
96
Affinity Labeling Agents
Penicillins
penicillin G
ampicillin (R? H) amoxicillin (R? OH)
97
Cephalosporins/ Cephamycins
cefoxitin
cefaclor
When X H, cephalosporins When X OCH3,
cephamycins
98
Every atom excluding the lactam N has been
replaced or modified. Ideal drugs - inactivate
an enzyme essential for growth of bacteria, but
does not exist in animals.
99
Peptidoglycan transpeptidase
Scheme 5.1
Without a cell wall, high internal pressure
causes bacteria to burst.
100
Figure 5.2
A
B
Normal bacterial cell division
Bacterium treated with an antibacterial agent
(fosfomycin)
101
Comparison of a Penicillin with D-Ala-D-Ala
Figure 5.11
reactive bond
scissle bond
102
Penicillin acylates active site serine.
Scheme 5.14
hydrolysis is blocked transamidation is blocked
(steric or conformational change)
Modulated reactivity and nontoxicity - ideal drugs
103
Penicillin Resistance
?-Lactamase production - hydrolyzes ?-lactam
ring Also, transpeptidase becomes less
susceptible to acylation. Membrane permeability
is modified.
104
Drug Synergism
Combination therapy penicillin ?-lactamase
inhibitor (5.92 or 5.93)
105
Aspirin
Hippocrates (460 - 377 B.C.) recommended willow
bark for pain. Sodium salicylate used for
rheumatic fever in 1875. Bayer Co. makes analogs
of sodium salicylate in 1899 introduces
acetylsalicylate for fevers, inflammation, and
pain.
First drug to be tested in clinical trials before
registration. First major drug sold in solid form
(tablets) because of water insolubility.
106
Mechanism of Aspirin
Inactivation of prostaglandin synthase Prostagland
ins are released when cells are damaged - cause
inflammation, pain, and fever.
Scheme 5.16
(cyclooxygenase)
107
Aspirin causes specific acetylation of active
site Ser-530.
Scheme 5.17
108
Selective Cyclooxygenase Inhibition
Two forms of cyclooxygenase COX-1 and
COX-2 COX-1 constitutive produces
prostaglandins important in maintaining tissues
in stomach lining COX-2 induced in inflammatory
cells produces prostaglandins during
inflammation Aspirin and other nonsteroidal
antiinflammatory drugs (NSAIDs) inhibit both
COX-1 and COX-2, resulting in stomach irritation.
109
Group at Searle (now defunct) made 2500 compounds.
IC50 60 nM 10-100 nM
40 nM
COX-2 gt 1700 103 -
104 375
selectivity
(in vivo selectivity gt1000)
110
Other Selective COX-2 Inhibitors
(Merck)
(Searle)
2,000-fold selective
28,000-fold selective
Inhibition of COX-2 blocks production of
prostacyclin, which is believed to be a hormone
that restrains oxidative stress and platelet
activation contributing to atherosclerosis. This
causes the heart problems with the COX-2
inhibitors. FitzGerald, G. A. and co-workers,
Science 2004, 306, 1954-1957.
111
X-ray crystal structures of COX-1 and COX-2 are
known - very little difference in active sites.
COX-1 has an active site Ile-523
COX-2 has an active site Val-523
COX-2 selective inhibitors found to fit in active
site, but have a repulsive interaction with the
slightly larger Ile-523 of COX-1. Size difference
between Ile and Val too small to have been able
to use structure-based drug design.
Note that the COX-2 selective inhibitors are
reversible inhibitors, not irreversible
inhibitors.
112
Mechanism-Based Enzyme Inactivators

• Unreactive
• Structure similar to substrate or product
• Target enzyme converts into a species that
  inactivates it
• Inactivation occurs prior to release of the
  activated species

• Differentiated from affinity labeling agents
• initially unreactive
• target enzyme activates it by catalysis

113
Mechanism-Based Inactivation Kinetics
Scheme 5.19
partition ratio k3/k4
of turnovers to product per inactivation event
Ideally, partition ratio 0
114
Potential Advantages in Drug Design Relative to
Affinity Labeling Agents

• Unreactivity - nonspecific alkylation and
  acylation of other biomolecules is not a problem
• High specificity - only the target enzyme
  chemistry should be capable of activation of the
  inactivator
• Low toxicity

115
Examples of Mechanism-Based Inactivation
Vigabatrin - anticonvulsant agent Epilepsy -
described gt4000 years ago in Babylonian and
Hebrew writings Broadly defined as any CNS
disease characterized by recurring convulsive
seizures 1-2 of the world population has
epilepsy Can arise from imbalance in two
neurotransmitters L-Glu - excitatory
neurotransmitter GABA - inhibitory
neurotransmitter
116
Metabolism of L-Glu
Scheme 5.20
?-aminobutyric acid aminotransferase
glutamic acid decarboxylase
GABA
L-Glu
When GABA diminishes, convulsions begin. Inject
GABA into brain, convulsions cease. GABA does not
cross blood-brain barrier.
Design compound that crosses blood-brain barrier,
then inactivates GABA aminotransferase.
117
Mechanism of Aminotransferases
Scheme 4.18
118
Mechanism of Inactivation of GABA-AT by Vigabatrin
Scheme 5.21
30
vigabatrin
70
Michael addition
electrophile
119
Effect of Vinyl Group on Permeability of
Blood-Brain Barrier
Scheme 5.22
lipophilic and electron withdrawing
lowers pKa
120
Eflornithine - antiprotozoal drug
Polyamines - spermidine and spermine and the
precursor putrescine are important regulators of
cell growth, division, differentiation Required
for DNA synthesis
spermidine
putrescine
spermine
121
Rate-determining step
putrescine
spermidine
spermine
Scheme 5.23
122
Inhibition of ornithine decarboxylase should lead
to antitumor/ antimicrobial agents.
Causes almost complete reduction in putrescine
and spermidine, but slight effect on spermine
- inactivation of ornithine decarboxylase induces
an increase in SAM decarboxylase.
?-difluoromethylornithine (DFMO)
This increases the S-adenosylhomocysteamine
produced, which drives the reaction to
spermine. Therefore, poor antitumor effects
observed. However, effective against protozoal
infections (treatment of African sleeping
sickness).
123
Another indication discovered for eflornithine is
as a topical cream for reduction of unwanted
facial hair (Vaniqa). Possibly interferes with
polyamine biosynthesis in hair growth.
124
Mechanism of Decarboxylases
Scheme 4.16
125
Mechanism of inactivation of ornithine
decarboxylase by eflornithine
Scheme 5.24
electrophile
126
Alternative Approach for Inactivation
PLP decarboxylases are reversible, except for the
step where CO2 is lost. Therefore, you can start
with the product (putrescine) and catalyze the
reaction back to the intermediate (up to where
CO2 is reattached). Principle of microscopic
reversibility - for a reversible reaction the
same mechanistic pathway is followed in the
forward and reverse directions.
A mechanism-based inactivator designed from the
product also should be effective.
127
Product-Derived Mechanism-Based Inactivator
?-difluoromethyl putrescine
Scheme 5.25
128
Tranylcypromine - Antidepressant Agent

• Modern era of antidepressant therapeutics
• late 1950s
• monoamine oxidase (MAO) inhibitors
• tricyclic antidepressants also developed

First MAO inhibitor (iproniazid) initially used
as antituberculosis drug.
129
Brain concentrations of norepinephrine and
serotonin depleted in chronically depressed
individuals - correlation between increase in
these neurotransmitters and antidepressant
effect. Cardiovascular side effect caused
patient death. All deaths from hypertensive
crisis and all had eaten foods containing high
tyramine content (aged cheese, wine, beer).
Tyramine triggers release of norepinephrine,
which raises blood pressure. Normally, the
norepinephrine is degraded by MAO, but not if
taking a MAO inhibitor drug. Therefore, blood
pressure keeps rising (called cheese effect).
130
MAO exists in two isozymic forms called MAO A
(degrades norepinephrine and serotonin) and MAO B
(degrades dopamine and phenethylamine). Selective
MAO A inhibitors raise serotonin levels -
antidepressant agents Selective MAO B inhibitors
raise dopamine levels - antiparkinsonian
agents Must inhibit brain MAO A, not MAO A in
gastrointestinal tract to avoid cheese effect.
131
Nonselective MAO A/B Inhibitor (gives cheese
effect)
132
Mechanism of MAO
Scheme 4.32
133
Proposed mechanism of inactivation of MAO by
tranylcypromine
Scheme 5.26
134
Selegiline (L-deprenyl)Antiparkinsonian Drug

• Parkinsons disease - degenerative neurological
  disease afflicting gt 500,000 people in the
  U.S.
• Chronic, progressive motor dysfunction
• Tremors, rigidity

• Symptoms arise from degeneration of dopaminergic
  neurons and reduction in the enzyme that makes
  dopamine (L-aromatic amino acid decarboxylase)
• Selective inhibition of MAO B increases dopamine
  concentration

135
Cause for Parkinsons Disease
1977 - 23 year old man with an advanced case of
Parkinsons disease in Bethesda, MD was taking a
designer drug. This drug was injected into rats
with no neurodegenerative effect.
1982 - Four young Californians develop advanced
case of Parkinsons - also were taking a designer
drug, a synthetic narcotic with a structure
designed to be a variation of existing illegal
narcotic but not listed as a controlled substance.
136
Designer Drugs
137
Basis for the designer drug taken by the
Californians
Drug dealers designed a reverse ester of
meperidine.
Unstable to heat and acid (ionization of
propionoxyl group).
138
Decomposition product of 5.130 ionization
(MPTP)
This was found in the synthetic street narcotic.
Shown to produce symptoms of Parkinsons in mice
and primates, but not rats (the animal model used
in 1977 in Bethesda).
Symptoms do not appear until 60-80 of
dopaminergic neurons are destroyed.
139
MPTP is not the neurotoxic agent - effects are
blocked by MAO B-selective inactivators.
MAO B metabolizes MPTP to MPDP
MPDP
MPTP
MAO B metabolizes MPDP to MPP
MPP
Actual neurotoxic agent is charged so it cannot
diffuse out of the brain
140
selective MAO B inhibitor
Protects the brain from oxidation of neurotoxic
precursors and from degradation of dopamine.
141
Proposed Mechanism of MAO B by Selegiline
selegiline
Scheme 5.27
142
2-Fluoro-2?-deoxyuridylate, Floxuridine,
5-Fluorouracil - Antitumor Agents
Cancer - abnormal and uncontrolled cell
division Antimetabolite design - structures
related to pyrimidines and purines interfere with
biosynthetic pathways of metabolites by enzyme
inhibition or by incorporation into proteins or
DNA.
143
5-fluorouracil antimetabolite of uracil
floxuridine antimetabolite of 2?-deoxyuridine
5-Fluoro-2?-deoxyuridylate antimetabolite of
2?-deoxyuridylate
144
minor
Scheme 5.28
major
5-Fluoro-2?-deoxyuridylate is not active, but is
metabolized to the 2?-deoxynucleotide.
145
Principal site of action is thymidylate synthase,
the last step in de novo biosynthesis of
thymidylate. Only source of thymidylate, an
essential constituent of DNA Inhibition of
thymidylate synthase produces thymine-less death
Normal cells also require thymidylate synthase.
146
Selective Toxicity

• 1. Most tumor cells replicate more rapidly than
  most normal cells.
• Higher requirement for DNA precursors
• Also, thymidylate synthase activity is elevated
  in tumor cells.

2. 5-Fluorouracil, as well as uracil, is taken
up into tumor cells more efficiently than into
most normal cells.
3. Enzymes that degrade uracil in normal cells
also degrade 5-fluorouracil degradation not in
tumor cells.
147
Side Effects
Generally from destruction of rapidly
proliferating normal cells in intestines, bone
marrow, and mucosa.
148
Mechanism of Thymidylate Synthase
Scheme 5.29
dihydrofolate reductase
tetrahydrofolate
149
Mechanism of Inactivation of Thymidylate Synthase
by 5-Fluoro-2?-deoxyuridylate
Scheme 5.30
150
Drug synergism Combination of inhibitors of
thymidylate synthase and dihydrofolate reductase
- sequential blocking.