Biomedical subjects
Publications and source records attributed to A D Broom.
This communication describes a convenient, facile, and high-yield synthesis of 3-(beta-D-ribofuranosyl)isoguanine and its 8-methyl derivative, as well as nucleoside analogues of pteridines, from a common precursor, 5,6-diaminocytidine. 5,6-Diamino-2',3', 5'-tri-O-benzoylcytidine was synthesized from 4, 6-diamino-2-oxopyrimidine in three steps.
Poly(1-methyl-6-thioinosinic acid), or PMTI, is a single-stranded polyribonucleotide and is the first homopolyribonucleotide devoid of Watson-Crick hydrogen bonding sites to show potent human immunodeficiency virus (HIV) inhibition. PMTI was found to be active when evaluated against a variety of low passage clinical HIV isolates in fresh human peripheral blood cells, including T cell-tropic and monocyte-macrophage-tropic viruses, syncytium-inducing and non-syncytium-inducing viruses and viruses representative of the various HIV-1 clades (A through F). The compound was active against HIV-2, all nucleoside and non-nucleoside reverse transcriptase (RT) inhibitor drug-resistant virus isolates tested and interacted with AZT or ddl to synergistically inhibit HIV infection. In biochemical inhibition assays, PMTI was determined to be a potent inhibitor of HIV-1 and HIV-2 RT, including RTs with mutations that engender resistance to nucleoside and non-nucleoside RT inhibitors. PMTI inhibited both the polymerase and RNase H activities of HIV RT. PMTI did not inhibit HIV-1 protease or integrase. Cell-based mechanism of action assays indicated that PMTI also interfered with early events in the entry of HIV into target cells. Furthermore, PMTI inhibited the fusion of gp120-expressing and CD4-expressing cells, but at concentrations approximately 1 log10 greater than those that inhibited virus entry. These results suggest that the homopolyribonucleotide PMTI blocks HIV replication in human cells at its earliest stages by multiple mechanisms, inhibition of virus entry and inhibition of RT.
Several amphipathic (hydrophobic base, hydrophilic backbone) polyribonucleotides have recently been shown to have potent antiviral activity against HIV and human cytomegalovirus (HCMV). The working hypothesis developed during these studies was that the ability to form an ordered, non-hydrogen-bonded array in solution was an important criterion for activity. To explore further the role of structure and molecular size on the inhibition of virus replication, one new polynucleotide and two 32-mer oligonucleotides based on the triazolo[2,3-a]purine ring system have now been prepared. High-molecular-weight polynucleotide 4a (PTPR) and sulfur-containing 32-mer 5b (TTPR) were moderately active against HIV but showed greater potency against HDMV than ganciclovir. Both 4a and 5b gave clear evidence of cooperative melting behavior, whereas inactive 32-mer 5a showed no such behavior.
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The synthesis and biological activity of 42 6-substituted-2,4-diaminopyrido[3,2-d]pyrimidines (2,4-diamino-8-deazafolate analogues) are reported. The compounds were synthesized in improved yields compared to previous classical analogues using modifications of procedures reported previously by us. Specifically, the S-phenyl-; mono-, di-, and trimethoxyphenyl-; and mono-, di-, and trichlorophenyl-substituted analogues with H or CH3 at the N10 position and methyl and trifluoromethyl phenyl ketone analogues with H, CH3, and CH2C identical to CH at the N10 position were synthesized. The S10 and N10 alpha- and beta-naphthyl analogues along with the N10 CH3 analogues were also synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (pc) and Toxoplasma gondii (tg); selectivity ratios were determined against rat liver (rl) DHFR as the mammalian reference enzyme. Against pcDHFR the IC50 values ranged from 0.038 x 10-6 M for 2,4-diamino-6-[(N-methyl-2'-naphthylamino)methyl]pyrido[3,2-d]pyrimidine (28) to 5.5 x 10(-6) M for 2,4-diamino-6[(2',4'-dimethoxyanilino)methyl]pyrido[3,2-d]pyrim idi ne (15). N10 methylation in all instances increased potency. None of the analogues were selective for pcDHFR. Against tgDHFR the most potent analogue was 2,4-diamino-6-[(N-methylanilino)methyl]pyrido[3,2-d]pyrimidine (5) (IC50 0.0084 x 10(-6) M) and the least potent was 2,4-diamino-6[(2'-naphthylamino)methyl]-pyrido[3,2-d]pyrimidine (37) (IC50 0.16 x 10-6 M). N10 methylation afforded an increase in potency up to 10-fold. In contrast to pcDHFR, several of the 8-deaza analogues were significantly selective for tgDHFR, most notably 2,4-diamino-6-[(2'-chloro-N-methylanilino)-methyl]pyrido[3,2-d] pyrimidine (13), 2,4-diamino-6-[(3',4',5'-trimethoxyanilino)methyl]pyrido[3,2-d]pyr pyrimidine (29), and 2,4-diamino-6-[(2',4',6'-trichloroanilino)methyl]pyrido[3,2-d] pyrimidine (32) which combined high potency at 10-8 M along with selectivities of 8.0, 5.0, and 12.4, respectively. The potency of these three analogues are comparable to the clinically used agent trimetrexate while their selectivities for tgDHFR are 17-43-fold better than trimetrexate.
Polyribonucleotides (PTMG and PMTI) containing 1-methyl-6-thioguanosine or 1-methyl-6-thioinosine, respectively, as the sole nucleoside component are shown to be potent inhibitors of various strains of HIV-1 and HIV-2 in a number of human lymphocyte and macrophage cell lines in tissue culture as well as in fresh human peripheral blood lymphocytes and macrophages. PMTI and PMTG exhibit potencies in the range of 10(-7)-10(-8) M in these systems. The polynucleotides are active against virus strains resistant to AZT and pyridinone derivatives. Both PMTI and PMTG are synergistic with AZT and with ddI, and both inhibit HIV reverse transcriptase at nanomolar concentrations. The polymers show little or no toxicity in human cell lines at the highest doses tested (100 micrograms/mL, or about 0.2-1 microM). This class of compounds represents a new lead in AIDS therapeutic drug discovery.
A new approach has been devised for the synthesis of oligonucleotides containing 2'-deoxy-6-thioguanosine [d(s6G)]. The synthesis of oligonucleotides containing d(s6G) requires special protection and deprotection strategies to prevent the thione functionality from undergoing oxidation and hydrolysis. Previous attempted syntheses have neglected to address this problem. By using the cyanoethyl protecting group for the thione and phenoxyacetyl for the exocyclic amino group, it was possible to deprotect oligonucleotides with a mixture of sodium hydroxide and sodium hydrogen sulfide without any significant conversion of d(s6G) to deoxyguanosine. Application of this strategy will allow investigation of the structural as well as biological activity of d(s6G)-containing oligonucleotides.
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In contrast to methotrexate (MTX) and aminopterin (AMT), the 8-deaza analogues of these antifolates are not substrates for rabbit liver aldehyde oxidase. Since they are not converted to 7-hydroxy derivatives, they have been investigated with regard to their cytotoxicity for CCRF-CEM cells, transport into these cells, and conversion to polyglutamate forms. For this purpose 3H-labeled analogues were synthesized. The drug concentrations of the analogues required to inhibit cell growth by 50% are significantly lower than for the parent compounds particularly for a short exposure of cells to the drug. Vmax and Km for unidirectional influx do not differ greatly among the four drugs, but amounts of uptake over 1 h are markedly different and increase in the order MTX less than 8-deazaMTX less than AMT less than 8-deazaAMT. During 1 h of uptake a much greater proportion of the 8-deaza analogues is converted to polyglutamate forms than in the case of parent drugs. Only 52% of MTX is converted to polyglutamates, whereas in the case of the other three compounds the conversion is greater than or equal to 90%. However, MTX is relatively efficient in adding two glutamate residues, whereas the other drugs predominantly accumulate as forms with only one additional glutamate (+Glu1). During 1 h of efflux the drugs without additional glutamates decrease to low concentrations and there is also a major loss of +Glu1 form, but there is also an increase in longer chain forms, especially in the case of MTX. The net result is a still greater disparity in total intracellular levels of the four drugs after the period of efflux. MTX has much lower toxicity in mice in vivo than the other three compounds, 8-deazaAMT being the most toxic. At the maximum tolerated dose MTX produced a considerably greater increase in life span for mice bearing P388 than any of the other drugs, and a somewhat greater increase for mice bearing L1210. Thus the 8-deaza analogues do not offer a therapeutic advantage over MTX against leukemias in the mouse, primarily due to their much greater toxicity.
The synthesis and characterization of two "flexible" bisubstrate analogues of the intermediate in the thymidylate synthase reaction are reported. Steric constraints are minimized and diasteromeric mixtures avoided by using a pyrimidine-based analogue as the folate portion of the inhibitor while retaining all known important binding sites. A preliminary assessment of certain conformational parameters by NMR is presented. The compounds are shown to be potent competitive inhibitors with respect to dUMP or 5,10-CH2-H4PteGlu but gave mixed kinetics with respect to 5,10-CH2-H4PteGlu5 for human thymidylate synthase.
Recent demonstrations that deazafolate analogues may act as potent inhibitors of thymidylate synthase (TS) provided a firm rationale for the synthesis of N10-propargyl derivatives of 8-deazafolate and 8-deazaaminopterin (4). A complete assignment of the 1H NMR spectra of these compounds was made possible through application of 2D (COSY) techniques at 200 MHz. Data describing the inhibition of TS derived from human leukemia (K562) cells are presented. IC50 values of 2.25 and 1.26 microM were determined for 8-deaza-10-propargylfolate (3) and 8-deaza-10-propargylaminopterin, respectively. Comparison of the data for various folate analogues reveals a striking dependence of TS inhibitory potency upon the number of nitrogens in the folate pyrazine ring.
The design and synthesis of multisubstrate analog enzyme inhibitors offer new opportunities in the creation of potent, highly specific drug molecules. Selected examples of inhibitors of 1-carbon transfer serve to illustrate the potential of this approach. Inhibitors of indole-N-methyltransferase, L-aspartyl transcarbamoylase, and thymidylate synthase are illustrative of many compounds that have demonstrated considerable specificity and potency. Several of these inhibitors and the rationale for their syntheses are described.
The synthesis of a high-molecular-weight, putatively all-syn DNA analogue, poly(8-bromo-2'-deoxyadenylic acid), is described. The syn----anti transition was shown to be both salt and temperature dependent. Conditions were found which favored 'normal' Watson-Crick pairing and duplex formation with poly(dT).
The synthesis of an 8-deazafolate analogue of the intermediate in the methylation of 2'-deoxyuridylate is described. Alkylation of diethyl 5,6,7,8-tetrahydro-8-deazafolate with 3'-O-acetyl-5-(bromomethyl)-2'-deoxyuridine 5'-[bis-(trichlorethyl) phosphate], followed by removal of the trichloroethyl groups with a Zn/Cu couple and mild saponification, gave the target inhibitor N-[4-[[[2-amino-3,4,5,6,7, 8-hexahydro-4-oxo-5-[(2'-deoxyuridin-5-yl)methyl]-pyrido[3,2-d] pyrimidin-6-yl]methyl]amino]benzoyl]-L-glutamic acid 5'-monophosphate. The free nucleoside and the 5'-(methyl phosphate) diester were similarly prepared. Each of these reactions yielded a pair of diastereoisomers about C-6 of the reduced deazafolate in approximately a 1:1 ratio. These diastereoisomeric mixtures were evaluated as inhibitors of thymidylate synthetase derived from human tumor (HeLa) cells. The 5'-monophosphate was a potent inhibitor, competitive with respect to both 2'-deoxyuridylate (Ki = 0.06 microM) and tetrahydrofolate (Ki = 0.25 microM). In contrast, the nucleoside and the nucleotide methyl ester were poorer inhibitors by more than 3 orders of magnitude, attesting to the importance of the anionic function at the nucleoside 5'-position in the affinity of an inhibitor for the enzyme active site.
Poly(5-fluoro-2'-deoxyuridylic acid) was synthesized and its properties were compared with those of poly(dT) and poly(dU). It readily complexed with poly(dA). The 1:1 complex melted at about 20 degrees C lower than poly(dA) . poly(dT). A triple-stranded helix, poly(dA) . 2 poly(dF5U) was formed only in high salt (2.0 M NaCl).
Poly(6-thioinosinic acid), poly(s6I), a synthetic polynucleotide shown to inhibit L1210 ascites growth in vivo, was compared with 6-mercaptopurine (6-MP) for their effects on cellular kinetics of L1210 cells in vitro. These kinetic perturbations were monitored by changes in growth rates, cell volumes and cell-cycle distributions. At equimolar exposure doses (based on molar concentration of purine bases), 0.1 mM poly(s6I) was more effective than 6-MP in reducing growth rate and total cell number. A 20-fold increase in the 6-MP exposure dose (to 2 mM) was required to reduce total cell number similar to 0.1 mM poly(s6I). Concomitant determination of Coulter volume distributions of treated cell populations indicated 0.1 mM poly(s6I) increased the mean population volume more than either concentration of 6-MP. Cell-cycle distributions obtained by flow cytometry showed that, simultaneously with the increase in mean population volume, poly(s6I) exposure caused a decrease of cells in G1 and S and a pronounced accumulation of cells in G2. Treatment with 6-MP caused similar cell-cycle redistributions but always less than for poly(s6I). Flow sorting of these redistributed populations indicated the increase in mean population volume during treatment with 0.1 mM poly(s6I) was almost equally due to an increased percentage of G2 cells and to an increased volume of cells with the G2 DNA content. However, with 0.1 mM 6-MP, increased volume of G2 cells was predominant. Although poly(s6I) was synthesized in an attempt to develop more effective delivery forms of biologically active compounds like 6-MP and it has close structural similarities to 6-MP, its mechanism of action is unknown.
The polymerization of 2-fluoroadenosine 5'-diphosphate by polynucleotide phosphorylase to give high molecular weight poly(2-fluoroadenylic acid), poly(fl2A), is described. Both the single-stranded and double-stranded (acid) forms of poly(fl2A) exhibit strikingly similar ultraviolet and circular dichroism spectra to those of poly(A), and the enzymatic polymerization rates and thermal hyperchromicities of the two polymers are also very similar. However, the pKa of poly(fl2A) for protonation at N-1 is 2.9 compared to 5.9 for poly(A) under similar conditions. Poly(fl2A) forms a triple-stranded helix with poly(U) which has ultraviolet and cd spectra very reminiscent of poly(A) . 2 poly(U), but no conditions could be found which permitted the formation of a double helix. In the Escherichia coli ribosome system poly(fl2A) codes for the synthesis of polylysine, as does poly(A), although the rate and extent of incorporation were less in the former case. The role of basicity of adenine N-1 in these interactions is discussed.
The synthesis and characterization of poly(1-methylinosinic acid) are described. Laser Raman spectra of poly (mII) were obtained as a function of temperature in D2O solution. Thermal melting profiles derived from the intensity variations of the 712, 795, 814, 986, 1333, 1509, 1550 and 1680 cm-1 bands indicate a cooperative melting temperature of 9 +/- 1 degree C. The low temperature form of poly(mII) exhibits a carbonyl frequency at 1710 cm-I which is decreased to 1680 cm-I upon melting. The Raman hypochromism in the bands reported are equal to or much larger than any reported for other nucleic acids. The data are consistent with the low temperature form of poly(mII) being an ordered single stranded unit with a high degree of basestacking. The melting profiles obtained from the uv and cd spectra are consistent with and support the Raman data. This single stranded RNA exhibits an uncharacteristic behavior in that it melts cooperatively.