The Organic Chemistry of Drug Design and Drug Action

1
The Organic Chemistry of Drug Design and Drug
Action

• Chapter 6
• DNA-Interactive Agents

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DNA-Interactive Agents
DNA - another receptor Carries genetic
information in cells Few differences between
normal DNA and DNA from other cells. Therefore,
these drugs are generally very toxic used for
life-threatening diseases, such as cancer and
viral infections.
3
Cancer Cells
Rapid, abnormal cell division Constant need for
DNA and precursors

• Selective toxicity
• rapid uptake of drug molecules by cancer cells
• repair mechanisms too slow
• activation of proteins such as p53 in normal
  cells in response to DNA damage - leads to
  increased DNA repair enzymes, cell cycle arrest
  (to allow time for DNA repair), and programmed
  cell death (apoptosis)

4
Structure-Based Design of Potential Drugs for
Prevention of Hair Loss During Chemotherapy
Inhibition of cyclin-dependent kinase 2 (CDK2)
arrests hair follicle cell cycle, rendering it
less susceptible to anticancer agents. Lead
Known inhibitors of tyrosine kinases
5
Lead Modification
Inhibits CDK2 with IC50 60 nM
6
Crystal Structure of 6.2 and 6.3 Bound to CDK2
Figure 6.1
Note that the inhibitor is near Lys-33 (need a H
bond acceptor) and Val-18 (need a hydrophobic
group) the SO2NH2 group can be substituted.
Note the N of the thiazole can H bond to Lys-33,
and the S of the thiazole is hydrophobic. The
pyridine does not interfere, but can increase
solubility.
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Combination Chemotherapy

• In the late 1950s combination chemotherapy was
  introduced.
• Effectiveness compared to single drug
• Able to fight acquired resistance
• Different mechanisms of action increase
  effectiveness
• Some covalent modifications can be reversed by
  repair enzymes, so inhibitors of DNA repair can
  be added

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Drug Interactions
Care must be given to which mechanisms of action
are involved in drug combinations. For example, a
renal (kidney) cytotoxic agent should not be used
with a drug that requires renal elimination for
excretion.
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Drug Resistance
1. Increased expression of membrane glycoproteins
- affects membrane permeability (blocks drug
transport) 2. Increased levels of thiols
(destroys electrophilic anticancer drugs) 3.
Increased levels of deactivating enzymes
(destroys anticancer drugs) 4. Decreased levels
of prodrug-activating enzymes (prevents
activation of prodrugs) 5. Increased DNA repair
enzymes (repairs DNA modification) All involve
gene alterations.
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DNA Structure and Properties
purine
adenine
pyrimidine
cytosine
purine
guanine
pyrimidine
thymine
In double-stranded DNA the ratio of A/T and G/C
is always 1.
11
Hydrogen Bonding of Complementary Base Pairs
(Watson-Crick Base Pair)
2 H-bonds
12
Hydrogen Bonding
3 H-bonds
13
Figure 6.2
14
The 2 glycosidic bonds that connect the base to
its sugar are not directly opposite each other,
giving different spacings along helix.
Figure 6.3
15
Duplex (double-stranded) DNA
Figure 6.4
(all inside)
16
Figure 6.5
most stable tautomer
17
Figure 6.6
mimics thymine
mimics adenine
These can substitute for T and A in DNA
polymerase reactions. Therefore H bonding is not
essential only need the groups to fit snugly in
the binding site of DNA polymerase.
18
DNA Shapes
Human somatic cells - each of the 46 chromosomes
consists of a single DNA duplex about 4 cm long.
Therefore a total of 46 ? 4 1.84 m long of DNA
packed into the nucleus. Nucleus is only 5 ?m in
diameter Done with aid of richly basic proteins
called histones. Folded compact form of DNA
called chromatin.
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Packing of DNA into the Nucleus
Figure 6.7
20
Supercoiled DNA - Packing of Bacterial DNA
Facilitates RNA polymerase reaction Helps in
chromatin packing
Figure 6.9
circular DNA (plasmid)
supercoiled DNA
Enzymes that interconvert supercoiled and relaxed
DNA are called DNA topoisomerases.
21
DNA topoisomerases also resolve topological
problems such as catenation and knotting.
Figure 6.10
catenanes
22
Figure 6.11
knots
23
Two Principal Types of Topoisomerases
DNA topoisomerases I catalyze transient breaks of
one strand of duplex DNA. DNA topoisomerases II
(in bacteria called DNA gyrase) catalyze cleavage
of both strands of duplex DNA.
24
Mechanisms of Topoisomerase IA and IB
Scheme 6.1
25
Possible Mechanism of Topoisomerase I Reaction
Conformational change to make a gap for strand to
pass through
Attack of Tyr at 5?-phosphate
Cleavable complex
Religation of the two ends
Relaxed DNA is released
Ready for another catalytic cycle
Figure 6.12
26
Mechanism for Topoisomerase I Decatenation (B)
Figure 6.13
27
DNA Conformations
Figure 6.14
Right-handed helices
Left-handed helix
28
A- and B-DNA glycosyl bonds are always anti.
anti
(base in the opposite direction as the
5?-phosphate)
29
Z-DNA glycosyl bond is anti at pyrimidines but
syn at purines (responsible for zigzag
appearance).
syn
(base in the same direction as the 5?-phosphate)
30
Classes of DNA-Interactive Drugs
Reversible binders - reversible interactions with
DNA Alkylators - react covalently with DNA
bases Strand breakers - generate reactive
radicals that cleave polynucleotide strands
31
How Do Drugs Interact with DNA Packed as
Chromatin?
Figure 6.15A
Figure 6.15B
The outer surface of the DNA is accessible to
small molecules.
32
Also, nucleosomes are in dynamic equilibrium with
uncoiled DNA, so drug can bind after uncoiling.
Figure 6.16
33
Reversible DNA Binders
Three ways small molecules can reversibly bind to
duplex DNA.
External electrostatic
Groove binder
Intercalation
Figure 6.17
34
External electrostatic binders - cations that
bind to anionic phosphates. Groove binders -
proteins prefer major groove binding small
molecules prefer minor groove binding. Minor
groove generally not as wide in A-T regions as in
G-C regions. Therefore, flat aromatic, often
crescent-shaped molecules (6.11) prefer A-T
regions.
35
DNA Intercalators
Flat, generally aromatic or heteroaromatic
molecules Insert (intercalate) and stack between
base pairs Noncovalent interactions Drug is
perpendicular to helix axis Sugar-phosphate
backbone is distorted Energetically favorable
process Does not disrupt H-bonding Destroys
regular helix unwinds DNA Therefore interferes
with the action of DNA topoisomerases and DNA
polymerases, which elongate DNA chain and correct
mistakes in the DNA
36
Example of Intercalation
Figure 6.18
37
Topotecan binds to the DNA-topoisomerase I complex
antitumor agent
Does not appear to be a correlation between DNA
intercalation and antitumor activity. It is not
sufficient to intercalate without stabilization
of the cleavable complex.
38
Selected Examples of DNA Intercalators
Acridines
Actinomycins
Anthracyclines
39
Amsacrine - acridine analog
Lead compound
Lead modification
Proflavine antibacterial
anti-leukemia agent stabilizes cleavable complex
40
Crystal Stucture of an Actinomycin Analog Bound
to a DNA
Figure 6.19
dactinomycin - antitumor from Streptomyces
Resistance - efflux pump (P170 glycoprotein) and
impaired drug uptake
41
Anthracycline Analog
Figure 6.20
D ring (major groove)
D ring (major groove)
Complex stabilized by stacking energy and
H-bonding
A ring (minor groove)
daunorubicin (daunomycin)
Intercalation and topoisomerase II-induced
damage anti-leukemia agent
42
DNA Alkylators
Nitrogen mustards
Lead discovery Autopsies of soldiers killed in
World War I by sulfur mustard (6.23) showed
leukopenia (low white blood cells), bone marrow
defects, dissolution of lymphoid tissue,
ulceration of GI tract. These are all rapidly
replicating cells.
sulfur mustard
Suggested this may show tumor cytotoxicity
too. 1931 - S mustard tried as antitumor agent,
but too toxic.
43
Lead Modification
Less toxic form of sulfur mustard sought. 1942 -
first clinical trials of a nitrogen mustard Marks
beginning of modern cancer chemotherapy
(for advanced Hodgkins disease)
44
Chemistry of Alkylating Agents
Scheme 6.2
Reactivity of Nu- in general RS- gt RNH2 gt ROPO3
gt RCOO-
45
For DNA N-7 of guanine gt N-3 of adenine gt N-7 of
adenine gt N-3 of guanine gt N-1 of adenine gt N-1
of cytosine N-3 of cytosine, the O-6 of guanine,
and phosphate groups also can be alkylated.
Purines A/G
Pyrimidines T/C
46
Scheme 6.3
anchimeric assistance
If k1 gt k2, SN2 If k2 gt k1, SN1

• Bifunctional alkylating agents
• DNA undergoes intrastrand and interstand
  cross-linking
• Compounds that cross-link DNA (bifunctional
  alkylating agents) are much more effective.

47
Interstrand Cross-linking of DNA by
Mechlorethamine
48
Hydrolysis of alkylated N-7 guanine leads to
destruction of the purine nucleus.
Scheme 6.4
49
Mechlorethamine is quite unstable to hydrolysis
(completely reacts within minutes of
injection). Therefore, a more stable analog is
needed.
6.29
More stable Slows rate of aziridinium formation
R COOH too stable, but soluble
R (CH2)3COOH chlorambucil
50
Ethylenimines
Lower pKa of the aziridine N so it is not
protonated at physiological pH - attach
e--withdrawing group
Need at least 2 aziridines per molecule for
antitumor activity
e--withdrawing group
51
Methanesulfonates
excellent leaving group
Alkylates N-7 of guanine intrastrand cross-links
52
Cyclopropane-Containing Alkylators
From Streptomyces
All contain a 4-spirocyclopropylcyclohexadienone
Scheme 6.5
53
The nitrogen atom is conjugated with the
cyclohexadienone which lowers the reactivity.
Scheme 6.6
54
Binding of these molecules to the A-T regions of
DNA twists the nitrogen out of conjugation,
making the cyclopropane much more reactive. N-3
of adenine reacts.
Scheme 6.7
55
Metabolically-Activated Alkylating Agents
Stable compounds that require one or more enzymes
or a reducing agent to convert them into the
alkylating agent.
56
Nitrosoureas
Lead compounds 6.38, where R CH3 and R? H
(modest antitumor activity)
(BCNU)
(CCNU)
Can cross blood-brain barrier for brain tumors
57
Mechanism of Action of Nitrosoureas
Scheme 6.8
carbamoylating agent
alkylating agent
58
Evidence That Diazomethane (CH2NN-) is Not the
Active Alkylating Agent, But Methyl Diazonium Is
Scheme 6.9
isolated
If diazomethane was the actual alkylating agent,
only 2 deuteriums would have been detected, but 3
deuteriums were found.
59
Evidence That the Alkylating Agent, Not the
Carbamoylating Agent, is Responsible for Activity.
R? alkyl
N-nitrosoamides
N-nitrosourethanes
Also cannot form carbamoylating agent still
antitumor agent
Cannot form carbamoylating agent still
anticancer agent
60
However, nitrosoureas with no alkylating activity
are inactive. The carbamoylating agent (OCNR)
acylates amines in proteins and inhibits DNA
polymerase and repair enzymes.
61
Interstrand cross-link from carmustine (6.38, R
R? CH2CH2Cl)
1-N3-deoxycytidyl-2-N?-deoxyguanosinylethane
62
Proposed Mechanism for Cross-Linking of DNA by
(2-Chloroethyl) nitrosoureas
The same product is obtained when R cyclohexyl,
so 2-chloroethyldiazonium was proposed as the
intermediate.
Detected by electrospray MS
Scheme 6.10
Resistance is evidence for this intermediate
Resistance O6-alkylguanine-DNA alkyltransferase
- repair enzyme that excises O-6 guanine adducts
63
Triazene Antitumor Drugs
Scheme 6.12
Using 14C dacarbazine (6.52), it was shown that
formaldehyde is produced and DNA is methylated at
N-7 of guanine.
64
Mitomycin C
Bioreductive alkylation - metabolic reduction to
an alkylating agent Leads to cross-linking of
DNA.
Scheme 6.13
65
Leinamycin
unusual functionality
Isolated from Streptomyces Requires thiol
activation for antitumor activity
66
Chemical Model Studies
Scheme 6.16
these intermediates were proposed for activity
67
Mechanism Proposed for Leinamycin
Isolated, but does not directly alkylate DNA in
equilibrium with 6.64
Scheme 6.17
This reacts by an additional mechanism
68
Another Mechanism for How Leinamycin Damages DNA
Scheme 6.18
Causes strand breakage
69
Strand Breakers
Anthracycline Radical Formation
Scheme 6.19
superoxide
O2-? and anthracycline semiquinone can generate
HO? HO? Cleaves DNA
70
Generation of HO? from O2-? and from 6.67
Scheme 6.20
6.68
(ferric complex)
Fenton reaction
71
Third Possible Mechanism of DNA Damage by
Anthracyclines
Ferric complex
This could react with O2-? to give O2 Fe(II)
Fenton reaction of Fe(II) with H2O2 gives HO?
72
BleomycinFrom a Streptomyces
Intercalates into DNA
Principal domains in bleomycin
Forms FeII complex with O2
Selective uptake by cancer cells
73
Ternary Complex of Bleomycin, Fe (II), and
O2Active Form
74
Activation of Bleomycin
From another ternary complex or from
NADPH-cytochrome P450 reductase
Scheme 6.22
75
Possible Mechanisms for Activation of Bleomycin
All three mechanisms involve generation of free
radicals that can abstract H? from DNA, leading
to DNA strand scission.
76
Proposed Mechanisms for the Reaction of Activated
BLM with DNA
Scheme 6.24
Requires O2
DNA fragments
nucleic base propenals
(2 major products isolated)
3?-phosphoglycolate
77
Proposed Mechanisms for the Reaction of Activated
BLM with DNA (contd)
Scheme 6.24
78
Mechanism to Account for Water Incorporation into
Both C-4? and C-1? Positions of the Alkali-Labile
Product
Scheme 6.27
79
Tirapazamine
Kills hypoxic cells in solid tumors
Scheme 6.28
Damage to DNA backbone and bases
80
Tirapazamine also reacts with DNA radicals under
hypoxic conditions, acting as a surrogate O2.
Scheme 6.29
81
Enediyne Antitumor Antibiotics
?,?-Unsaturated ketone
Trisulfide
?,?-Unsaturated ketone
zinostatin
Trisulfide
82
Enediyne Antitumor Antibiotics (contd)
Like anthracyclines
83
Common Structural Features of Enediyne Antitumor
Antibiotics
(ene)
Macrocyclic ring with at least one double bond
and two triple bonds.
(diyne)

• Common modes of action
• intercalation into minor groove
• reaction (activation) with either a thiol of
  NADPH - generates radical
• radical cleavage of DNA

84
Mechanism for Esperamicins/Calicheamicins
Intercalates into DNA
Trisulfide reduction initiates the activation
Responsible for DNA strand scission
Scheme 6.30
85
Dynemicin A Reductive Mechanism
Intercalates into DNA
Causes DNA cleavage
Scheme 6.31
86
Dynemicin A Nucleophilic Mechanism
Scheme 6.32
87
Zinostatin Mechanism
Intercalates into DNA
Scheme 6.33
Causes DNA cleavage
88
Two mechanisms for DNA cleavage by any of the
biradicals generated in the presence of O2 under
reducing conditions
Strand scission
No Criegee rearrangement because under reducing
conditions
Major
Scheme 6.35
Strand scission
Strand scission