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PubMed1986

DNA sequence selectivity of guanine-N7 alkylation by three antitumor chloroethylating agents.

The DNA sequence selectivities of guanine-N7 alkylation produced by three chloroethylating antitumor agents, 1-(2-chloroethyl)-3-(cis-2-hydroxy) cyclohexyl-1-nitrosourea (cis-2-OH CCNU), 2-chloroethyl (methylsulfonyl)methanesulfonate, and 8-carbamoyl-3-(2-chloroethyl)imidazo-[5,1-d]-1,2,3,5-tetrazin-4(3H )-one (mitozolomide), were examined using a modification of the Maxam and Gilbert sequencing technique. In a region of pBR322 DNA, 2-chloroethyl (methylsulfonyl)methanesulfonate produced approximately the same degree of alkylation at all guanines. cis-2-OH CCNU, however, preferentially alkylated the middle guanines in runs of three or more guanines; the intensity of the reaction increased with the number of adjacent guanines in the DNA sequence. Mitozolomide produced the same pattern of preferential alkylation but not as intensely as cis-2-OH CCNU. Three other nitrosoureas, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1-(2-fluorethyl)-3-cyclohexyl-1-nitrosourea, and 1-(2-chloroethyl)-1-nitrosourea gave similar patterns of alkylation to that of cis-2-OH CCNU at pH 7.2. The ratio of 7-hydroxyethylguanine to 7-chloroethylguanine was approximately the same following treatment of the synthetic polymers dGn X dCn and (dG X dC)n with cis-2-OH CCNU, indicating that 7-chloroethylation and 7-hydroxyethylation were enhanced similarly by the presence of adjacent guanines. Ethylnitrosourea produced relatively little alkylation preference. The results suggest that the alkylating intermediates, 2-chloroethyldiazohydroxide and 2-hydroxyethyldiazohydroxide, tend to react preferentially with those guanine-N7 positions the electronegativity of which is enhanced by the presence of neighboring guanines. This is consistent with the presence of cationic character in the alkylating centers of these intermediates. 2-Chloroethyl (methylsulfonyl)methanesulfonate and ethyldiazohydroxide would not be expected to have strong cationic character, in agreement with their lack of sequence selectivity.

Alkylating Agents↗
PubMed1982

Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites.

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L1210 leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonuclease micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [14C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest. 64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210:bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210:bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.

Alkylation↗
PubMed1986

O-alkylation in DNA does not correlate with the formation of chromosome breakage events in D. melanogaster.

Postmeiotic cell stages of repair-proficient ring-X (RX) males were treated with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), diethylnitrosamine (DEN) or ethylnitrosourea (ENU) and then mated to either repair-defective (mei-9L1) or to repair-competent females (mei-9+). Absence of the mei-9+ function resulted in a hypermutability effect to all alkylating agents (AAs) when they were assayed for their ability to induce chromosomal aberrations (chromosome loss; CL), irrespective of marked differences in distribution of DNA adducts brought about by these AAs. This picture is different from that described previously for the induction of point mutations (Vogel et al., 1985a). There, evidence was presented indicating that reduction in DNA excision repair does not affect point mutation induction (recessive lethals) by those AAs most efficient in ring-oxygen alkylation such as ENU, DEN, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and isopropyl methanesulfonate (iPMS): the order of hypermutability of AAs with mei-9L relative to mei-9+ was MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females were plotted against those determined for mei-9+ females, straight lines of following slopes were obtained: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4, and iPMS = ENU = DEN = ENNG = 1. Those findings, together with the recent observation that AAs do not split into two groups when assayed for their ability to cause CL, point to the involvement of different DNA alkylation products in ENU- and DEN-induced chromosome loss vs. that of point mutations. It is concluded that with ENU and DEN chromosomal loss results from N-alkylation products whereas point mutations (SLRL) are the consequence of interactions with oxygen-sites in DNA. Thus, as a consequence of a very dominating role of O-ethylguanine (and possibly O4-alkylation of thymine), N-alkylation in DNA does not contribute measurably to mutation induction in the case of ENU-type mutagens while O-alkylation, very clearly, does not show a positive correlation with the formation of chromosome breakage events in Drosophila. Conversely, it appeared that with MMS-type mutagens (MMS; dimethyl sulfate, DMS; trimethyl phosphate, TMP), alkylation products such as 7-methylguanine and 3-methyladenine, if unrepaired or misrepaired, are potentially mutagenic lesions causing both mutations and chromosomal aberrations.

Alkylating Agents↗
PubMed1988

Alkylating activity in food products--especially sauerkraut and sour fermented dairy products--after incubation with nitrite under quasi-gastric conditions.

N-Nitroso compounds may well rank high among the genotoxic carcinogens present in our environment. Small amounts of such compounds may be formed in the human stomach after consumption of high-nitrate vegetables. Volatile nitrosamines can be conveniently determined but reliable methods of analysis for non-volatile N-nitroso compounds are still lacking. In this study we have used the 4-(4-nitrobenzyl)pyridine test to look for the formation of alkylating compounds such as N-nitroso-N-methylurea in a wide range of food products after incubation with nitrite under simulated gastric conditions. Our results indicate that many food products do not form alkylating compounds in appreciable amounts, even though the nitrite concentration used was five to ten times that found in saliva after a high-nitrate meal. Comparatively strong alkylating activity, however, was detected after incubation of samples of sauerkraut, certain dairy products (yoghurt, biogarde, quark, buttermilk and milk), wine and smoked mackerel. Samples of sauerkraut incubated with simulated gastric fluid, but without (added) nitrite, also displayed appreciable alkylating activity. The formation of alkylating substances in non-fat yoghurt was markedly inhibited by addition of ascorbic acid. The identity of the alkylating agents remains unknown. The isolation procedure was optimized for N-nitroso-N-methylurea, but will certainly result in the isolation of other compounds, such as C-nitroso-, C-nitro- or perhaps even C-nitroso-C'-nitro-compounds as well. Biogenic amines, glucosinolates, indole derivatives or other compounds may be involved as precursors. If alkylating agents are formed in vivo after ingestion of high-nitrate vegetables or drinking water, this is likely to occur only when the food products mentioned above are ingested simultaneously with or shortly after the nitrate load and not appreciably (except perhaps in the case of sauerkraut) when they are ingested alone, without a nitrate source. The health implications of these findings cannot yet be established. Many alkylating agents, however, have strong carcinogenic properties and continued investigation of food products (and their interaction products with nitrite) is indicated.

Alkylating Agents↗
PubMedDate not supplied

Biological aromatization of delta4,6- and delta1,4,6-androgens and their 6-alkyl analogs, potent inhibitors of aromatase.

Enzymic aromatization of delta6- and delta1,6-derivatives of the natural substrate androstenedione with human placental aromatase was first studied using gas-chromatography-mass spectrometry. The two steroids were aromatized with apparent Km and Vmax values of 62 nM and 32 pmol/min/mg protein for the delta6-steroid and 167 nM and 10 pmol/min/mg protein for the delta1,6-steroid, respectively. We next explored the aromatization of a series of 6-alkyl (methyl, ethyl, n-propyl, and n-pentyl)-substituted delta6-androstenediones and their delta1,6-analogs, potent competitive inhibitors of aromatase, to gain insight into the relationships between the inhibitory activity of the 6-alkyl-C19 steroids and their ability to serve as a substrate of aromatase. In a series of the delta1,6-androstenediones, all the 6-alkyl steroids were more efficient substrates than the parent delta1,6-steroid in which the aromatization rates of the alkyl steroids were about 2-fold that of the parent steroid, in contrast, all of the 6-alkyl-substituted delta6-androstenediones were converted into the corresponding 6-alkyl-delta6-estrogens with the rates of less than about a half that of the parent steroid. These results indicate that the 6-alkyl function decreases the aromatization rate of the delta6-steroid but enhances that of the delta1,6-steroid. The relative apparent Km values for the C19 steroids obtained in this study are different from the relative Ki values obtained previously, indicating that a good inhibitor is not essentially a good substrate in the 6-alkyl-substituted delta6- and delta1,6-androstenedione series.

Alkylation↗
PubMed1995

Sequence specificity of alkylation for a series of nitrogen mustard-containing analogues of distamycin of increasing binding site size: evidence for increased cytotoxicity with enhanced sequence specificity.

The covalent sequence specificity of a series of nitrogen mustard-containing analogues of distamycin was determined using modified sequencing techniques. The analogues tether benzoic acid mustard (BAM) and possess either one, two, or three pyrrole-amide units. Previous characterization of the biological profile of the series revealed an increase in cytotoxicity for each corresponding increase in the number of pyrrole units, while showing poor cross-link formation in isolated and cellular DNA. Examination of the sequence specificity revealed that BAM produced guanine-N7 lesions in similar manner to other conventional nitrogen mustards. The monopyrrole BAM conjugate also produced guanine-N7 alkylation in a similar pattern to BAM. However, alkylation of adenines was also seen that was found to be minor groove adenine-N3 lesions. The dipyrrole and tripyrrole conjugates did not produce detectable guanine-N7 alkylation but only alkylated in AT tracts. In addition, the tripyrrole conjugate preferentially alkylated only a subset of those sites alkylated by the monopyrrole and dipyrrole conjugates. Two sites, 5'-TTTTGG and 5'-TTTTGA, confirmed as guanine-N3 and adenine-N3 lesions, respectively, were strongly alkylated by the tripyrrole conjugate in preference to other similar sites including three occurrences of 5'-TTTTAA. Footprinting studies comparing distamycin and the tripyrrole conjugate showed identical non-covalent recognition of AT-rich sites. Hence, the drug that possessed the most enhanced sequence specificity for alkylation was also the most cytotoxic of this series.

Antineoplastic Agents, Alkylating↗
PubMed1994

Mapping of DNA alkylation sites induced by adozelesin and bizelesin in human cells by ligation-mediated polymerase chain reaction.

In this study, we have mapped the intracellular alkylation sites of adozelesin and bizelesin, two potent analogs of CC-1065, in individual genes at the single-nucleotide level. Human colon carcinoma cells were treated with adozelesin and bizelesin, and the position of adducts were mapped within the PGK-1 and p53 genes by means of ligation-mediated polymerase chain reaction. The monofunctional alkylating agent adozelesin was found to alkylate genomic DNA predominantly within 5'-(A/T)(A/T)A* sequences. Additional sites of alkylation were observed within 5'-(A/T)(G/C)(A/T)A* sequences; however, these were considered to represent sites of medium to low preference. Bizelesin, a bifunctional analog capable of both DNA monofunctional alkylation and DNA interstrand cross-link formation, was also found to alkylate 5'-(A/T)(A/T)A* sequences. Putative bizelesin DNA interstrand cross-link sites indicated that AT-rich sequences are preferred in the intervening sequence between the two cross-linked adenines. Both six- and seven-nucleotide regions were identified as putative sites of DNA interstrand cross-link formation with 5'-TTTTTTA*, 5'-TTTATCA* and 5'-GTACTAA* sequences being preferred. Non-adenine bases are not observed as potential intracellular sites of either DNA interstrand cross-linking formation or monofunctional alkylation. Thus, the patterns of alkylation induced by adozelesin and bizelesin in genomic DNA are similar but not identical to that observed in purified cell-free DNA.

Alkylating Agents↗
PubMed2006

Alkylation of phenol: a mechanistic view.

The current work utilizes the ab initio density functional theory (DFT) to develop a molecular level of the mechanistic understanding on the phenol alkylation in the presence of a cation-exchange resin catalyst, Amberlyst-15. The catalyst is modeled with the benzene sulfonic acid, and the effect of this acid on olefins such as isopropene (i-Pr) and tributene (t-Bu) in a phenol solution mimics the experimental condition. A neutral-pathway mechanism is established to account for early-stage high concentration of the phenolic ether observed in experiments. The mechanism involves an exothermic reaction between olefin and the benzene sulfonic acid to form ester followed by three reaction pathways leading to direct O-alkylation, o-C-alkylation, and p-C-alkylation. Our calculations conclude that O-alkylation to form the phenolic ether is the most energetically favorable in the neutral condition. An ionic rearrangement mechanism describes intramolecular migrations of the alkyl group from the phenolic ether to form C-alkylphenols, while the positively charged protonation significantly lowers transition barriers for these migrations. The ionic rearrangement mechanism accounts for high yields of o-C-alkylphenol and p-C-alkylphenol. Competition between the H atom and the alkyl R group at the substitutive site of the protonated ortho configuration is found to be the determining factor to the ortho/para ratio of C-alkylation products.

Algorithms↗
PubMed2002

Physicochemical characterization and solubility analysis of thalidomide and its N-alkyl analogs.

PURPOSE: The present study was primarily aimed at exploring the feasibility of improving percutaneous delivery via chemical manipulation of the thalidomide molecule to form analogs with improved physicochemical properties. N-Alkyl analogs were synthesized with the belief that these would be suitably hydrophobic and far less crystalline than the reference compound. This article presents their physicochemical properties. METHODS: Thalidomide and three of its N-alkyl analogs were synthesized. Identification and levels of purity (>96%) were assured through element analysis, fast atom-bombardment mass spectrometry, nuclear magnetic resonance spectroscopy, and high-performance liquid chromatography. N-Octanol/water partition coefficients were determined at pH 6.4. Solubilities in water and a series of n-alkanols were obtained. Best-fit solubility parameters were determined from the solubilities of the respective compounds in London solvents and were also calculated from respective hexane solubilities. melting points and heats of fusion. RESULTS: Methylation of the thalidomide molecule at its acidic nitrogen led to an aqueous solubility about 6-fold higher than thalidomide but, because the alkyl chain length was further extended from methyl to pentyl. aqueous solubilities decreased essentially exponentially. The destabilization of the crystalline structure with increasing alkyl chain length led to an increased solubility in nonpolar media. The log partition coefficient increased linearly with increasing alkyl chain length and the solubility parameters declined systematically through this series. By adding a methyl group to the thalidomide structure, the melting point dropped by more than 100 degrees C. Adding to the alkyl chain length led to further, more modest decreases. Heats of fusion decreased dramatically upon thalidomide's alkylation as well. CONCLUSION: Alkylation of the thalidomide molecule resulted in compounds with physicochemical properties that appear to be markedly better suited for percutaneous delivery.

Alkylation↗
PubMed1975

Isolation and identification of products from alkylation of nucleic acids: ethyl- and isopropyl-purines.

Ethylation and isopropylation of guanine in alkaline solution, or of adenine in formic acid, by alkyl methanesulphonates gave the following products: 1-, N2-, 3-, O6-, 7- and 9-alkylguanines; 1-, 3-, 7- and 9-alkyladenines. The products were identified from their characteristic u.v-absorption spectra, by comparison with either known ethyladenines or with the corresponding known methyladenines, and were also characterized by mass spectrometry. Their chromatographic properties on paper, t.l.c. and various columns were determined. DNA was alkylated in neutral solution with 14C-labelled alkyl methanesulphonates and the ratios of the alkylpurines formed were obtained, and compared for alkylation by methyl, ethyl and isopropyl methanesulphonates and by N-methyl-N-nitrosourea. The extents of alkylation at O-6 of guanine relative to those at N-7 of guanine varied with the reactivity of the methylating agents according to the predictions of Swain & Scott (1953) relating nucleophilicity of the groups alkylated with the substrate constants of the alkylating agents. The relative extents of alkylation at N-3 of adenine did not follow this correlation.

1-Propanol↗
PubMed2003

Specific targeting of a DNA-alkylating reagent to mitochondria. Synthesis and characterization of [4-((11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-8-oxy)butyl]-triphenylphosphonium iodide.

The selective manipulation of the expression and replication of mitochondrial DNA (mtDNA) within mammalian cells has proven difficult. In progressing towards this goal we synthesized a novel mitochondria-targeted DNA-alkylating reagent. The active alkylating moiety [(11aS)-8-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (DC-81)], irreversibly alkylates guanine bases in DNA (with a preference for AGA triplets), preventing its expression and replication. To target this compound to mitochondria it was covalently coupled to the lipophilic triphenylphosphonium (TPP) cation to form a derivative referred to as mitoDC-81. Incorporation of this lipophilic cation led to the rapid uptake of mitoDC-81 by mitochondria, driven by the large membrane potential across the inner membrane. This compound efficiently alkylated isolated supercoiled, relaxed-circular or linear plasmid DNA and isolated mtDNA. However mitoDC-81 did not alkylate mtDNA within isolated mitochondria or cells, even though it accessed the mitochondrial matrix at concentrations up to 100-fold higher than those required to alkylate isolated DNA. This surprising finding suggests that mtDNA within intact mitochondria may not be accessible to this class of alkylating reagent. This inability to alkylate mtDNA in situ has significant implications for the design of therapies for mtDNA diseases and for studies on the packaging, expression and turnover of mtDNA in general.

Alkylating Agents↗
PubMed1997

Biological fate of sulphur mustard: in vitro alkylation of human haemoglobin by sulphur mustard.

1. Human blood was incubated in vitro with a 1:1 mixture of [35S,12C4]- and [13C4]-sulphur mustard. Alkylated globin, containing the 2-hydroxyethylthioethyl (HETE) moiety, was isolated from the blood incubate following lysis of the erythrocytes and acidification with HCl in isopropanol. 2. The alkylated globin was hydrolysed with Pronase E to give a digest containing alkylated amino acids and alkylated dipeptides. A number of these were partially purified by hplc and identified by gc-ms and lc-ms. 3. The alkylated globin was hydrolysed with trypsin to give a digest containing alkylated peptides. Ten of these were partially purified by hplc, tentatively identified by lc-electrospray mass spectrometry, and the sequences and sites of alkylation determined using lc-electrospray tandem mass spectrometry. 4. (2-Hydroxyethylthioethyl)glutathione was also shown to be present in the pronase and trypsin digests of alkylated globin. 5. N-terminal valine, on both the alpha and beta chains, and histidine residues were identified as the key sites of interaction for targeting as biological markers of sulphur mustard poisoning.

Alkylation↗
PubMed1999

Stability of alkyl-dihydroxyacetonephosphate synthase in human control and peroxisomal disorder fibroblasts.

Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a peroxisomal enzyme that plays a key role in ether phospholipid biosynthesis. To determine the turnover of alkyl-DHAP synthase in several peroxisomal disorders, pulse-chase experiments were performed. In control fibroblasts, mature alkyl-DHAP synthase displayed a half-life of 23 +/- 12 h. In Zellweger syndrome and rhizomelic chondrodysplasia punctata fibroblast cell lines, in which alkyl-DHAP synthase cannot be imported into peroxisomes, the enzyme was mainly detected in its precursor form. This precursor form showed a much shorter half-life, 5 +/- 2 h. In contrast, when the precursor protein accumulated inside the peroxisome of a particular neonatal adrenoleukodystrophy cell line in which processing does not take place, a half-life of 18 +/- 8 h, resembling that of the mature protein in controls, was observed. In a cell line from a patient with a single deficiency in the activity of alkyl-DHAP synthase, the mature form was detected and its radioactivity decreased with a half-life of 16 +/- 7 h. Collectively, these results provide an explanation for the instability of alkyl-DHAP synthase outside its target organelle. Additionally, they indicate that both the precursor and mature form of alkyl-DHAP synthase exhibit considerable intraperoxisomal turnover.

Alkyl and Aryl Transferases↗
PubMed1984

Detection of IgG-associated determinants in reduced and alkylated preparations of human IgG3 by monoclonal antibodies.

Using classical typing antisera, previous experiments have failed to demonstrate IgG3 in partially reduced and alkylated preparations of human IgG intended for intravenous application (IGIV). To establish that IgG3 is actually present in such preparations, we designed an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies as solid-phase reagents and protein A-purified IgG3 as antigen. Three different samples of reduced and alkylated antigen were used: (1) IgG3 isolated from a ready-for-infusion IGIV; (2) IgG3 which was purified from an intramuscular (Cohn fraction II) IgG solution before being subjected to a mild reduction and alkylation procedure, and (3) completely reduced and alkylated IgG3. The reduction and alkylation procedure did not affect the solubility of IgG3, indicating that IGIV prepared in this manner should contain normal quantities of IgG3. In the ELISA, solid-phase monoclonals which were cross-reactive with multiple IgG subclasses clearly reacted with reduced and alkylated IgG3. Furthermore, there was no substantial difference between the quantities of modified and native antigen required for 50% maximal ELISA signal. In contrast, solid-phase monoclonals with IgG3-restricted specificity did not recognize reduced and alkylated material. These results indicate that IGIV prepared by reduction and alkylation has a normal IgG3 content and confirm that some IgG3-specific determinants are altered by the modification procedure.

Alkylation↗
PubMed2000

Clinical applications of quinone-containing alkylating agents.

Quinone-containing alkylating agents are a class of chemical agents that have received considerable interest as anticancer drugs. These agents contain a quinone moiety that can be reduced and an alkylating group that can form covalent bonds with a variety of cellular components. The oxidation state of the quinone element can modulate the activity of the alkylating element, and reduction of the quinone is required for activation of the alkylating activity of many of these agents. The quinone element may also contribute to the cytotoxic activity of quinone-containing alkylating agents through the formation of reactive oxygen species during redox cycling. The natural product, mitomycin C, has been the most widely used quinone-containing alkylating agent in the clinic, but other quinone-containing alkylating agents like porfiromycin, diaziquone, carbazilquinone, triaziquone and EO9 have also been used in the clinic for the treatment of cancer. In addition, many other quinone-containing alkylating agents have been tested in preclinical studies and the development of new agents is being actively pursued. This chapter describes the current and past clinical uses of these agents in the treatment of cancer and discusses new agents that are currently in clinical trials.

Antibiotics, Antineoplastic↗
PubMed1976

The disappearance kinetics of soluble immune complexes prepared with reduced and alkylated antibodies and with intact antibodies in mice.

Soluble immune complexes prepared with reduced and alkylated antibodies persisted longer in the circulation than complexes prepared with intact antibodies, when these were administered intravenously to mice. The disappearance of complexes with reduced and alkylated antibodies was delayed in part because the initial phase of vascular permeability was considerably less than that seen following the administration of complexes with intact antibodies. In addition, large complexes with lattice structure of more than two antigen and two antibody molecules persisted longer in the circulation after administration of complexes with reduced and alkylated antibodies than after administration of complexes with intact antibodies. Thus, the concentration of large latticed complexes with reduced and alkylated antibodies was significantly greater than the concentrations of large latticed complexes with intact antobodies at all observed times through 96 hours. The persistence of large latticed complexes with reduced and alkylated antibodies was associated with significantly decreased hepatic localization of complexes with reduced and alkylated antibodies compared to the hepatic localization of complexes with intact antibodies at 1, 4, 12, and 24 hours. The observations indicated that the removal of large latticed complexes from the circulation by the hepatic mononuclear phagocyte system was decreased when reduced and alkylated antibodies were used for the preparation of immune complexes. The persistence of large latticed complexes with reduced and alkylated antibodies in the circulation was associated with enhanced and prolonged presence of glomerular deposits of immune complexes, as reported in the accompanying article (Haakenstad AO, Striker GE, Mannik M: Lab Invest 35:293, 1976.

Alkylation↗
PubMed1988

Structural design, biochemical properties, and evidence for improved therapeutic activity of 5-alkyl derivatives of 5-deazaaminopterin and 5-deazamethotrexate compared to methotrexate in murine tumor models.

Studies are described examining a new class of 4-aminofolate analogues modified by an N to C conversion and alkyl substitution at the N-5 position of aminopterin and methotrexate. All of these analogues were equivalent to aminopterin and methotrexate as inhibitors of tumor cell dihydrofolate reductase (Ki = 3.49-5.16 pM). N to C conversion at the N-5 position of aminopterin reduced its influx (inferred from the change in Ki) 3-fold, but the same modification increased influx of methotrexate 2-3-fold in Sarcoma 180 cells. Alkylation (methyl or ethyl) of this position on 5-deazaaminopterin increased influx 3-fold, while a similar alteration of 5-deazamethotrexate increased influx 4-5-fold. Influx of the methotrexate analogues was increased a total of 14-fold as a result of these modifications. Similar differences among these analogues were observed for inhibition of Sarcoma 180 cell growth in culture. Inhibitory potency was in the ascending order methotrexate less than 5-deazamethotrexate less than 5-deazaaminopterin less than aminopterin less than 5-alkyl (methyl or ethyl) analogues of 5-deazaaminopterin and 5-deazamethotrexate (the ethyl analogues were 2-fold more inhibitory than the methyl analogues). All of the analogues examined were equivalent in regard to efflux from Sarcoma 180 cells. Differences in transport alone did not account for all of the increased inhibitory potency (up to 33-fold) of the 5-alkyl-5-deaza analogues compared to the parent compounds. The extent of polyglutamylation of 5-deazaaminopterin and 5-deazamethotrexate and their 5-alkyl derivatives in Sarcoma 180 cells was substantially less compared to aminopterin and equivalent to methotrexate. Transport inward of 5-deazaaminopterin in isolated crypt cell epithelium from mouse small intestine was 2-fold lower than aminopterin (influx Km = 14.2 +/- 2 microM), while influx of 5-deazamethotrexate was 2-fold greater than methotrexate (influx Km = 98.6 +/- 23). However, transport inward of all of the 5-alkyl derivatives of these 5-deaza analogues was intermediate [influx Km = 44.4 +/- 11 (SEM) to 49.8 +/- 12 microM] between values for aminopterin and methotrexate. These differences accounted, to some extent, for the reduced toxicity of the 5-alkyl-5-deazaaminopterin analogues compared to aminopterin and the increased toxicity of 5-methyl-5-deazamethotrexate compared to methotrexate. All of the 5-alkyl derivatives of aminopterin and methotrexate were more active in vivo than methotrexate against four murine tumor models.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗
PubMed1995

Sequence-specific DNA interactions by novel alkylating anthracycline derivatives.

New alkylating anthracycline derivatives with promising antitumor activity have been synthesized. We selected two of these compounds, 4-demethoxy-N,N-bis(2 chloroethyl)-4'-methylsulfonyl-daunorubicin (FCE 27726) and 4-demethoxy-3'-deamino-3'aziridinyl-4'-methylsulfonyl daunorubicin (FCE 28729), comparing their interaction with DNA and that of the non-alkylating derivative 4-demethoxy-4'-methylsulfonyl-daunorubicin (FCE 27894). The two alkylating derivatives were more cytotoxic than idarubicin and presented low cross-resistance with doxorubicin. Both FCE 27726 and FCE 28729 were found to alkylate guanines at the N7 position in the major groove with roughly the same specificity, but at different concentrations. FCE 27726 was 10 times more potent than FCE 28729 in alkylating DNA. At higher concentrations, FCE 27726 was able to alkylate adenines, possibly at the N3 position contained in a sequence 5'-PyAA. FCE 27726, as expected, was able to form DNA interstrand cross-links either in vitro and in vivo in treated cells. FCE 28729 did not form DNA interstrand cross-links in vivo. In vitro, at high concentrations, some DNA interstrand cross-links were evident. The non-alkylating derivative FCE 27894 did not produce any alkylation or DNA interstrand cross-links either in vitro or in vivo.

Animals↗