1. The Organic Chemistry of Drug Design and Drug Action
Chapter 6
DNA-Interactive Agents

2. 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. 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

5. 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.

6. 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.

7. 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.

8. Hydrogen Bonding of Complementary Base Pairs (Watson-Crick Base Pair)
2 H-bonds

9. Hydrogen Bonding
3 H-bonds

10. Figure 6. 1 DNA structure. Reproduced with permission from Alberts, B
Figure 6.1 DNA structure. Reproduced with permission from Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D. (1989). Molecular Biology of the Cell, 2nd ed., p. 99. Garland Publishing, New York. Copyright 1989 Garland Publishing.

11. The 2 glycosidic bonds that connect the base to its sugar are not directly opposite each other, giving different spacings along helix.
Figure 6.2 Characteristic of DNA base pairs that causes formation of major and minor grooves

12. Duplex (double-stranded) DNA
(all inside)
Figure 6.3 Major and minor grooves of DNA. With permission from Kornberg, A. (1980); From DNA Replication by Arthur Kornberg. Copyright ©1980 by W. H. Freeman and Company. Used with permission.

13. Base Tautomerism most stable tautomer
Figure 6.4 Hydrogen bonding sites of the DNA bases. D, hydrogen bond donor; A, hydrogen bond acceptor

14. mimics thymine mimics adenine
Figure 6.5 Nonpolar nucleoside isosteres (6.4 and 6.5) of thymidine and adenosine, respectively, that base pair by non-hydrogen-bond interactions
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.

15. 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.

16. Packing of DNA into the Nucleus
Figure 6.6 Stages in the formation of the entire metaphase chromosome starting from duplex DNA. With permission from Alberts, B., (1994). Copyright ©1994 from Molecular Biology of the Cell, 3rd ed. By Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts and James D. Watson. Reproduced by permission of Routledge, Inc., part of The Taylor & Francis Group.

17. Figure 6.7 Artist rendition of the conversion of duplex DNA into chromatin fiber

18. Supercoiled DNA - Packing of Bacterial DNA
Facilitates RNA polymerase reaction
Helps in chromatin packing
circular DNA (plasmid)
supercoiled DNA
Figure 6.8 Conversion of duplex DNA into supercoiled DNA
Enzymes that interconvert supercoiled and relaxed DNA are called DNA topoisomerases.

19. DNA topoisomerases also resolve topological problems such as catenation and knotting.
catenanes
Figure 6.9 Catenane and knot catalog. Arrows indicate the orientation of the DNA primary sequence: a and b, singly linked catenanes; c and d, simplest knot, the trefoil; e–h, multiply interwound torus catenanes; i, right-handed torus knot with seven nodes; j, right-handed torus catenane with eight notes; k, right-handed twist knot with seven nodes; l, 6-noded knots composed of two trefoils. Adapted with permission from Wasserman, S. A. and Cozzarelli, N. R. Biochemical topology: applications to DNA recombination and replication. Science, 1986, 232, 952. Reprinted with permission from AAAS.

20. Figure 6. 10 Visualization of trefoil DNA by electron microscopy
Figure 6.10 Visualization of trefoil DNA by electron microscopy. Reproduced with permission from Griffith J.D., Nash, H.A., Proc. Natl. Acad. Sci. USA 1985, 82, 3124.

21. 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.

22. Table 6.1

23. Topoisomerase mechanisms
Figure 6.11 Mechanisms of DNA topoisomerase-catalyzed reactions. Drawings produced by Professor Alfonso Mondragón, Department of Molecular Biosciences, Northwestern University.

24. Topoisomerase mechanism
Scheme 6.1 DNA topoisomerase-catalyzed strand cleavage to cleavable complexes

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
Ready for another catalytic cycle
Relaxed DNA is released
Figure 6.12 Artist rendition of a possible mechanism for a topoisomerase I reaction. The colored sections are the topoisomerase, and the black lines are the double-stranded DNA. With permission from Champoux, J.J. (2010). With permission from the Annual Review of Biochemistry. Volume 70 ©2001 by Annual Reviews.

26. Mechanism for Topoisomerase I Decatenation (B)
Figure 6.13 Artist rendition of possible mechanisms of topoisomerase IA-catalyzed relaxation of (A) supercoiled DNA and (B) decatenation of a DNA catenane. From Li, Z.; Mondragon, A.; DiGate, R. J. The mechanism of IA topoisomerase-mediated DNA topological transformations. Mol. Cell 2001, 7, 301.

27. DNA Conformations Right-handed helices Left-handed helix
Figure 6.14 Computer graphics depictions of A-DNA, B-DNA, and Z-DNA. Reproduced with permission from the Jena Library of Biological Macromolecules, Institute of Molecular Biotechnology (IMB), Jena, Germany; Hühne R., Koch F. T., Sühnel, J. A comparative view at comprehensive information resources on three-dimensional structures of biological macromolecules. Brief Funct. Genomic Proteomic 2007, 6(3), 220–239.

28. A- and B-DNA glycosyl bonds are always 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).
(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 Schematic of how a drug could bind to DNA wrapped around histones in the nucleosome. Polach K. J. Mechanism of protein access to specific DNA sequences in chromatin: A dynamic equilibrium model for gene regulation. J. Mol Biol. 1995, 254, 130.

33. Reversible DNA Binders
Three ways small molecules can reversibly bind to duplex DNA.
Figure 6.17 Schematic of three types of reversible DNA binders. A, external electrostatic binder; B, groove binder; C, intercalator. In B and C, the pink bar represents the drug. Reproduced with permission from Blackburn G. M., Gait M. J., Eds. Nucleic Acids in Chemistry and Biology, 2nd ed., 1996; p By permission of Oxford University Press.

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. Netropsin is a minor groove binder
Figure 6.18 Model showing interaction of netropsin (colored ball model) with double helical DNA (colored stick model). The 2D structure of netropsin (6.8) is also shown. Image created by JanLipfert from crystallographic coordinates deposited in the Protein Data Bank, accession code 101D.

36. 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

37. Example of Intercalation: Ethidium bromide
Figure 6.19 Intercalation of ethidium bromide into B-DNA

38. 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.

39. Nalidixic acid binds to bacterial topoisomerase II

40. Other DNA intercalators

41. Selected Examples of DNA Intercalators
Acridines
Actinomycins
Anthracyclines

42. Amsacrine - acridine analog
Lead compound
Lead modification
antibacterial
anti-leukemia agent
stabilizes cleavable complex

43. Crystal Stucture of an Actinomycin Analog Bound to a DNA
dactinomycin - antitumor
from Streptomyces
Figure 6.20 X-ray structure of a 1:2 complex of dactinomycin with d(GC). Reprinted from Journal of Molecular Biology, Vol. 68, “Stereochemistry of actinomycin binding to DNA. II. Detailed molecular model of actinomycin DNA complex and its implications”, pp. 26–34. Copyright Academic Press, with permission from Elsevier.

44. Intercalation and topoisomerase II-induced damage
Anthracycline Analog
Complex stabilized by stacking energy and H-bonding
D ring (major groove)
Intercalation and topoisomerase II-induced damage
anti-leukemia agent
A ring (minor groove)
daunorubicin (daunomycin)
Figure 6.21 X-ray structure of daunorubicin intercalated into an oligonucleotide. Quigley, G. S.;Wang, A.; Ughetto, G.; Van der Marel, G.; Van Boom, J. H.; Rich, A. Molecular structure of an anticancer drug-DNA complex: Daunomycin plus d(CpGpTpApCpG). Proc. Natl. Acad. Sci. USA 1980, 77, p Reprinted with permission from Dr. C. J. Quigley.

45. Bis-intercalators do not always bind as tightly as expected
Figure 6.22 General structure of bis-quinoxaline intercalators

46. A bis-intercalator requires the correct linker

47. 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.
S mustard tried as antitumor agent, but too toxic.

48. Lead Modification Less toxic form of sulfur mustard sought.
first clinical trials of a nitrogen mustard
Marks beginning of modern cancer chemotherapy
(for advanced Hodgkin’s disease)

49. Chemistry of Alkylating Agents
Scheme 6.2 Nucleophilic substitution mechanisms
Reactivity of Nu- in general:
RS- > RNH2 > ROPO3= > RCOO-

50. For DNA:
N-7 of guanine > N-3 of adenine > N-7 of adenine > N-3 of guanine > N-1 of adenine > 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

51. If k1 > k2, SN2 If k2 > k1, SN1 anchimeric assistance
Scheme 6.3 Alkylations by nitrogen mustards
anchimeric assistance
If k1 > k2, SN2
If k2 > k1, SN1
Bifunctional alkylating agents
DNA undergoes intrastrand and interstand cross-linking
Compounds that cross-link DNA (bifunctional alkylating agents) are much more effective.

52. Interstrand Cross-linking of DNA by Mechlorethamine

53. Alkylation may change the preferred tautomer of the base

54. Hydrolysis of alkylated N-7 guanine leads to destruction of the purine nucleus.
Scheme 6.4 Depurination of N-7 alkylated guanines in DNA

55. Formation of cross-links in DNA
Scheme 6.5 Interstrand cross-links of abasic sites in duplex DNA by reaction with guanine

56. Slows rate of aziridinium formation
Mechlorethamine is quite unstable to hydrolysis (completely reacts within minutes of injection).
Therefore, a more stable analog is needed.
More stable
Slows rate of aziridinium formation
R = COOH too stable, but soluble
R = (CH2)3COOH chlorambucil

57. A naturally occurring mustard?
Scheme 6.6 Proposed mechanism for DNA alkylation by fasicularin

58. 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

59. Methanesulfonates Alkylates N-7 of guanine intrastrand cross-links
excellent leaving group
Alkylates N-7 of guanine
intrastrand cross-links

60. Cyclopropane-Containing Alkylators
From Streptomyces
All contain a 4-spirocyclopropylcyclohexadienone
Scheme 6.7 Reaction of nucleophiles with 4-spirocyclopropylcyclohexadienone

61. The nitrogen atom is conjugated with the cyclohexadienone which lowers the reactivity.
Scheme 6.8 Stabilization of the spirocyclopropylcyclohexadienone by nitrogen conjugation

62. 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 6.9 N-3 adenine alkylation by CC-1065 and related compounds

63. Metabolically-Activated Alkylating Agents
Stable compounds that require one or more enzymes or a reducing agent to convert them into the alkylating agent.

64. Can cross blood-brain barrier for brain tumors
Nitrosoureas
Lead compounds
6.38, where R = CH3 and R = H (modest antitumor activity)
(BCNU)
(CCNU)
Can cross blood-brain barrier for brain tumors

65. Mechanism of Action of Nitrosoureas
carbamoylating agent
alkylating agent
Scheme 6.10 Decomposition of N-methyl-N-nitrosourea

66. Evidence That Diazomethane (CH2=N+=N-) is Not the Active Alkylating Agent, But Methyl Diazonium Is
isolated
Scheme 6.11 Deuterium labeling experiment to determine mechanism of activation of nitrosoureas
If diazomethane was the actual alkylating agent, only 2 deuteriums would have been detected, but 3 deuteriums were found.

67. Evidence That the Alkylating Agent, Not the Carbamoylating Agent, is Responsible for Activity.
R = alkyl
N-nitrosoamides
N-nitrosourethanes
Cannot form carbamoylating agent; still anticancer agent
Also cannot form carbamoylating agent; still antitumor agent

68. However, nitrosoureas with no alkylating activity are inactive.
The carbamoylating agent (O=C=NR) acylates amines in proteins and inhibits DNA polymerase and repair enzymes.

69. Interstrand cross-link from carmustine (6.38, R = R = CH2CH2Cl)
1-[N3-deoxycytidyl]-2-[N-deoxyguanosinyl]ethane

70. 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 6.12 Mechanism proposed for cross-linking of DNA by (2-chloroethyl)nitrosoureas
Resistance is evidence for this intermediate
Resistance: O6-alkylguanine-DNA alkyltransferase - repair enzyme that excises O-6 guanine adducts

71. Fotemustine also causes cross links in DNA

72. Another Proposed Mechanism for Cross-Linking of DNA by (2-Chloroethyl) nitrosoureas
Scheme 6.13 Alternative mechanism for the cross-linking of DNA by (2-chloroethyl)nitrosoureas

73. Triazene Antitumor Drugs
Using [14C] dacarbazine (6.52), it was shown that formaldehyde is produced and DNA is methylated at N-7 of guanine.
Scheme 6.14 Mechanism for the methylation of DNA by dacarbazine

74. Mitomycin C
Scheme 6.15 Mechanism for the bioactivation of mitomycin C and alkylation of DNA

75. Scheme 6.16 Bioreductive monoalkylating agents

76. Bioreductive Bis-alkylators
Scheme 6.17 Bioreductive bis-alkylating agents

77. Leinamycin Requires thiol activation for antitumor activity
unusual functionality
Isolated from Streptomyces
Requires thiol activation for antitumor activity

78. Chemical Model Studies
Scheme 6.18 Model reaction for the mechanism of activation of leinamycin
these intermediates were proposed for activity

79. Mechanism Proposed for Leinamycin
Isolated, but does not directly alkylate DNA; in equilibrium with 6.64
Scheme 6.19 Mechanism for DNA alkylation by leinamycin
This reacts by an additional mechanism

80. Another Mechanism for How Leinamycin Damages DNA
Causes strand breakage
Scheme 6.20 Mechanism for hydrodisulfide activation of molecular oxygen to cause oxidative DNA damage

81. Strand Breakers Anthracycline Radical Formation
superoxide
Scheme 6.21 Electron transfer mechanism for DNA damage by anthracyclines
O2- and anthracycline semiquinone can generate HO
HO Cleaves DNA

82. Generation of HO from O2- and from 6.67
(ferric complex)
Fenton reaction
Scheme 6.22 Anthracycline semiquinone generation of hydroxyl radicals

83. 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

84. The cardiotoxicity of doxorubicin can be prevented by iron chelators
Scheme 6.23 Conversion of iron chelator prodrug 6.70 into iron chelator 6.71

85. Bleomycin From Streptomyces verticellus
Intercalates into DNA
Principal domains in bleomycin
Forms FeII complex with O2
Selective uptake by cancer cells

86. Ternary Complex of Bleomycin, Fe (II), and O2 Active Form

87. Activation of Bleomycin
Scheme 6.24 Cycle of events involved in DNA cleavage by ­bleomycin (BLM)
From another ternary complex or from NADPH-cytochrome P450 reductase

88. 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.

89. Proposed Mechanism for the Reaction of Activated BLM with DNA
DNA fragments
nucleic base propenals
3-phosphoglycolate
Scheme 6.25 Alternative mechanisms for base propenal formation and DNA strand scission by activated bleomycin: (A) Modified Criegee mechanism
(2 major products isolated)

90. Proposed Alternative Mechanism for the Reaction of Activated BLM with DNA
Scheme 6.25 Alternative mechanisms for base propenal formation and DNA strand scission by activated bleomycin: (B) Grob fragmentation mechanism

91. Tirapazamine Kills hypoxic cells in solid tumors
Damage to DNA backbone and bases
Scheme 6.26 Mechanism for formation of hydroxyl radicals by tirapazamine

92. Tirapazamine also reacts with DNA radicals under hypoxic conditions, acting as a surrogate O2.
Scheme 6.27 Mechanism for DNA-strand cleavage by tirapazamine

93. Enediyne Antitumor Antibiotics

94. Common Structural Features of Enediyne Antitumor Antibiotics
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

95. Mechanism for Esperamicins/Calicheamicins
Intercalates into DNA
Trisulfide reduction initiates the activation
Responsible for DNA strand scission
Scheme 6.28 Activation of esperamicins and calicheamicins

96. Dynemicin A Reductive Mechanism
Intercalates into DNA
Dynemicin A Reductive Mechanism
Causes DNA cleavage
Scheme 6.29 Reductive mechanism for activation of dynemicin A

97. Dynemicin A Nucleophilic Mechanism
Scheme 6.30 Nucleophilic mechanism for activation of dynemicin A

98. Zinostatin Activation Mechanism by thiols
Intercalates into DNA
Causes DNA cleavage
Scheme 6.31 Activation of zinostatin by thiols

99. Deactivation of zinostatin
Scheme 6.32 Polar addition reaction to deactivate zinostatin

100. 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
Strand scission
Strand scission
Scheme 6.33 DNA-strand scission by activated zinostatin and other members of the enediyne antibiotics. NCS, neocarzinostatin (Zinostatin)

101. Enediynes can also form quinones
Scheme 6.34 Catalytic antibody-catalyzed conversion of an enediyne into a quinone via oxygenation of the corresponding benzene biradical