version date: 1 December 2006 2 -Deoxyuridylate monophosphate 2 -De 2-Deoxy 5-fluorouridy late nt monophosphate thymidylate monophosphate 2-Deoxy 2-Deoxy - trifluoromethyl 5-trifluoromethyl nIalate phosphate Fig. 1 Superposition and comparisons of nucleotide-derived antineoplastic drugs capable of inhibiting TS, with the corresponding substrates <www.iupac.org/publications/cd/medicinalchemistry/> 6
6 Fig. 1 Superposition and comparisons of nucleotide-derived antineoplastic drugs capable of inhibiting TS, with the corresponding substrates. 2’-Deoxyuridylate monophosphate 2’-Deoxy- 5-fluorouridylate monophosphate 2’-Deoxy 5-fluorouridylate monophosphate 2’-Deoxy 5-trifluoromethyluridylate monophosphate 2’-Deoxy 5-trifluoromethyluridylate monophosphate 2’-Deoxythymidylate monophosphate <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 Dihydropholic acid Aminoe Trimetrexate Trimetrexate Piritrexim Piritrexim Fig 2 Superposition and comparisons of folate cofactor-derived antineoplastic drugs capable of inhibiting dhfr with the corresponding substrate Some physical similarities and differences among antineoplastic compounds (Table 1) belonging to groups I (TS)and VIl (DHFR)are illustrated, respectively, in Tables 2 and 3 Compared with substrates uridylate and thymidylate, both antimetabolites 5 fluorouracil and trifluridine show additional electronegative groups, which contribute to better nucleophilicity toward the thymidy late synthase target(Table 2). Fluorine is often considered an isostere of hydrogen even though it does not have the same valence as <www.iupac.org/publications/cd/medicinalchemistryl>
7 Fig. 2 Superposition and comparisons of folate cofactor-derived antineoplastic drugs capable of inhibiting DHFR, with the corresponding substrate. Some physical similarities and differences among antineoplastic compounds (Table 1) belonging to groups I (TS) and VII (DHFR) are illustrated, respectively, in Tables 2 and 3. Compared with substrates uridylate and thymidylate, both antimetabolites 5- fluorouracil and trifluridine show additional electronegative groups, which contribute to better nucleophilicity toward the thymidylate synthase target (Table 2). Fluorine is often considered an isostere of hydrogen even though it does not have the same valence as Dihydropholic acid Aminopterin Trimetrexate Methotrexate Piritrexim Piritrexim Methotrexate Trimetrexate Aminopterin <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 hydrogen. The atom is virtually the same size as hydrogen but more electronegative and thus can be used to vary the drug electronic properties without having any steric effect. Estimates of log P by using the fragment method show the greater fluorinated antimetabolites lipophilic character, as compared with the corresponding substrates(Table 2 ). Substituting fluorine for enzymically labile hydrogen can also disrupt the catalytic reaction since C-f bonds are not easily broken Table 2 Physicochemical similarities and differences of TS antimetabolites and substrate Molecular Dipol volume Compound Log P h bond h bond moment 2-Deoxyuridylate monophosphate 140.68 4.13 3.43 1.05 4.60 2-Deoxy-5-fluorouridylate monophosphate 140.33 -5.23 3.43 4.43 2-Deoxy-thymidylate monophosphate 147.58 1.053.05 2-Deoxy-5-trifluoromethyl-uridylate monophosphate(trifluridine MP) 16164 4.95 In contrast to the dihydrofolate substrate the methotrexate antagonist has an extra pteridine ring amino group, which improves the hydrogen bond interaction on the active site The replacement of the 4-oxo group of the substrate by the amino group will not appreciably change the size of the analog, but will have a marked effect on its polarity, electronic distribution, and bonding (Table 3). However, the N-methyl group containing methotrexate antagonist has a different shape and increased log P constant and liposolubility The methyl roup that generated steric hindrance may create constraints and impose particular favorable conformations for ligand and receptor interactions. Moreover, the N-methyl group inductive electron-donating effect disfavors ionization and gives rise to non-ionized forms, less soluble in water [71 <www.iupac.org/publications/cd/medicinalchemistry/> 8
8 hydrogen. The atom is virtually the same size as hydrogen but more electronegative and thus can be used to vary the drug electronic properties without having any steric effect. Estimates of log P by using the fragment method show the greater fluorinated antimetabolites lipophilic character, as compared with the corresponding substrates (Table 2). Substituting fluorine for enzymically labile hydrogen can also disrupt the catalytic reaction since C–F bonds are not easily broken [6]. Table 2 Physicochemical similarities and differences of TS antimetabolites and substrate. Compound Molecular volume (cm3 /mol) Log P (fragments) H bond acceptor H bond donor Dipole moment (debyes) 2'-Deoxyuridylate monophosphate 140.68 –4.13 3.43 1.05 4.60 2'-Deoxy-5-fluorouridylate monophosphate 140.33 –5.23 3.43 1.05 4.43 2'-Deoxy-thymidylate monophosphate 147.58 –3.48 3.44 1.05 3.05 2'-Deoxy-5-trifluoromethyl-uridylate monophosphate (trifluoridine MP) 161.64 –3.24 3.59 1.05 4.95 In contrast to the dihydrofolate substrate, the methotrexate antagonist has an extra pteridine ring amino group, which improves the hydrogen bond interaction on the active site. The replacement of the 4-oxo group of the substrate by the amino group will not appreciably change the size of the analog, but will have a marked effect on its polarity, electronic distribution, and bonding (Table 3). However, the N-methyl group containing methotrexate antagonist has a different shape and increased log P constant and liposolubility. The methyl group that generated steric hindrance may create constraints and impose particular favorable conformations for ligand and receptor interactions. Moreover, the N-methyl group inductive electron-donating effect disfavors ionization and gives rise to non-ionized forms, less soluble in water [7]. <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 Table 3 Physical similarities and differences of DHFR antimetabolites and substrate Molecular volume Log P H bond Name H bond moment Polar cm/mol)(fragments) acceptor donor (debye) surface area Dihydrofolic acid 224.90 3.60 2.12 224.86 Aminopterin 223.21 3.60 2.12 4.06 228.81 23442 3.3 3.3 3.61 20.02 Trimetrexate 1929 0.82 10441 Piritrexim 177.25 09.17 The development of resistance during acute leukemia therapy is probably due to the loss of the methotrexate cellular transport mechanism. Thus, investigation for a more lipophilic inhibitor led to trimetrexate and piritrexim, which are independent of the cell transport mechanism. They are analogs of methotrexate, in which one or two pteridine ring nitrogen atoms are replaced by carbon and a more lipophilic group replaces the benzoylglutamic acid chain. Table 3 lists log P values, showing the greater trimetrexate and piritrexim liposolubility and lower polar surface area, as compared to classical antimetabolites Identifying enzyme 3D pharmacophores Many drugs are effective by interacting with biological macromolecules such as enzymes, DNA, glycoproteins, or receptors. The target enzyme-substrate, -inhibitor or -cofactor (ligand) 3D complexes, can be downloaded from PDB onto a computer program and studied by molecular modeling. Ligand and target interactions may be due entirely to nonbonded forces, but occasionally a covalent interaction may be involved. Tight-binding ligands often have a high degree of target complementarity, which can be assessed and measured. A 3D pharmacophore specifies the group spatial relationships, corresponding to a set of features <www.iupac.org/publications/cd/medicinalchemistry 9
9 Table 3 Physical similarities and differences of DHFR antimetabolites and substrate. Name Molecular volume (cm3 /mol) Log P (fragments) H bond acceptor H bond donor Dipole moment (debye) Polar surface area Dihydrofolic acid 224.90 –3.60 3.52 2.12 5.24 224.86 Aminopterin 223.21 –4.00 3.60 2.12 4.06 228.81 Methotrexate 234.42 –3.32 3.35 1.87 3.61 220.02 Trimetrexate 192.97 –0.02 1.57 0.82 1.53 104.41 Piritrexim 177.25 0.90 1.76 1.07 4.72 109.17 The development of resistance during acute leukemia therapy is probably due to the loss of the methotrexate cellular transport mechanism. Thus, investigation for a more lipophilic inhibitor led to trimetrexate and piritrexim, which are independent of the cell transport mechanism. They are analogs of methotrexate, in which one or two pteridine ring nitrogen atoms are replaced by carbon and a more lipophilic group replaces the benzoylglutamic acid chain. Table 3 lists log P values, showing the greater trimetrexate and piritrexim liposolubility and lower polar surface area, as compared to classical antimetabolites. Identifying enzyme 3D pharmacophores Many drugs are effective by interacting with biological macromolecules such as enzymes, DNA, glycoproteins, or receptors. The target enzyme-substrate, -inhibitor or -cofactor (ligand) 3D complexes, can be downloaded from PDB onto a computer program and studied by molecular modeling. Ligand and target interactions may be due entirely to nonbonded forces, but occasionally a covalent interaction may be involved. Tight-binding ligands often have a high degree of target complementarity, which can be assessed and measured. A 3D pharmacophore specifies the group spatial relationships, corresponding to a set of features <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006
version date: 1 December 2006 common to active molecules, such as hydrogen bond donors and acceptors, positively or negatively charged groups, and hydrophobic groups of an appropriate size. The correlation of these structures with pharmacological action and complementary molecular interaction analyses between biological molecules and substrate/drug were possible by the use of a web accessible tool, Protein Explorer, a freeware option under PDb view Structure. The steps involved in the manipulation of Protein Explorer are described in the supplemental material The students are asked to render different format and color 3D enzymes from PDB and search the Display File list to access the catalytic site. Although some target enzyme active sites are not available from PDB files, it is possible to estimate how strongly a molecule will bind to a catalytic site by selecting the ligands surface contacts favorably interacting with specific functional groups of both ligand and macromolecule(see supplemental material). TS(group I)and dhfR (group VIl) are conveniently chosen enzymes to illustrate these tutorials due to their concomitant action in the cell de novo biosynthesis of thymidilate nucleotides Both enzymes have long been recognized as a drug target for inhibiting DNA synthesis in rapidly proliferating cells such as cancer cells or in bacterial or malarial infections. Traditional inhibitors clinically used as antineoplastic and antimicrobial agents, have been modeled on dUMP or the cofactor N, N-methylenetetrahydrofolate, and thus are structurally related to natural substrate and cofactor 3] Thymidylate synthase Backs ground Thymidylate synthase catalyzes the reductive methylation of 2 -deoxyuridine monophosphate (dUMP)to 2 -deoxythymidylate monophosphate(dTMP), using N, NO. methylenetetrahydrofolate cofactor, which is concomitantly converted to 7, 8-dihydrofolate <www.iupac.org/publications/cd/medicinalchemistry/> 10
10 common to active molecules, such as hydrogen bond donors and acceptors, positively or negatively charged groups, and hydrophobic groups of an appropriate size. The correlation of these structures with pharmacological action and complementary molecular interaction analyses between biological molecules and substrate/drug were possible by the use of a web accessible tool, Protein Explorer, a freeware option under PDB View Structure. The steps involved in the manipulation of Protein Explorer are described in the supplemental material. The students are asked to render different format and color 3D enzymes from PDB and search the Display File list to access the catalytic site. Although some target enzyme active sites are not available from PDB files, it is possible to estimate how strongly a molecule will bind to a catalytic site by selecting the ligand’s surface contacts favorably interacting with specific functional groups of both ligand and macromolecule (see supplemental material). TS (group I) and DHFR (group VII) are conveniently chosen enzymes to illustrate these tutorials due to their concomitant action in the cell de novo biosynthesis of thymidilate nucleotides. Both enzymes have long been recognized as a drug target for inhibiting DNA synthesis in rapidly proliferating cells such as cancer cells or in bacterial or malarial infections. Traditional inhibitors clinically used as antineoplastic and antimicrobial agents, have been modeled on dUMP or the cofactor N5 ,N10-methylenetetrahydrofolate, and thus are structurally related to natural substrate and cofactor [3]. Thymidylate synthase Background Thymidylate synthase catalyzes the reductive methylation of 2'-deoxyuridine monophosphate (dUMP) to 2'-deoxythymidylate monophosphate (dTMP), using N5 ,N10- methylenetetrahydrofolate cofactor, which is concomitantly converted to 7,8-dihydrofolate <www.iupac.org/publications/cd/medicinal_chemistry/> version date: 1 December 2006