Thomas J. Bardos

STRUCTURE-ACTIVITY RELATIONSHIPS OF ALKYLATING AGENTS IN CANCER CHEMOTHERAPY*

There seems to be a widespread feeling among clinical investigators that the usefulness of alkylating agents in cancer chemotherapy has some inherent limitations and that the selectivity and range of efficacy of these agents against human malignancies cannot be substantially improved over those of the presently used drugs. This pessimism is not shared by the medicinal chemists who view the alkylating agents as reactive chemical compounds that exert their tumor-growth inhibitory effects by undergoing typical organic reactions with certain nucleophilic groups of the functional macromolecules of the cell. This presents to the medicinal chemist a unique and fascinating challenge. In attempting to design more effective and selective agents, he can apply and extend his knowledge of the mechanism of organic chemical reactions as well as of the physicochemical interactions between small and large molecules and of the biochemical processes involved in drug metabolism and transport. By appropriate structural design, one can alter the chemical properties or biochemical behavior of the alkylating agents in many different ways, and it seems almost inconceivable that some of the more ingenious or fortunate alterations should not result in substantial (even fundamental) improvements in the efficacy and selectivity of their pharmacologic action. Rising to the challenge, and not to be discouraged by the skepticism of the clinicians (who, after all, have tested so far only a handful of alkylating agents in cancer patients), the medicinal chemists have continued to synthesize with relentless enthusiasm thousands of alkylating agents; moreover, some of the new agents appear to be based on excellent rationales. Unfortunately, many of these compounds have received only limited pharmacologic testing, using different animal tumor systems or assay techniques, which makes it difficult to compare them with the established alkylating agents on the basis of their published test results. The problem of evaluating the chemotherapeutic potentials of the many new alkylating agents in a meaningful manner in order to select a lucky few that are to be given the chance of a clinical trial has been puzzling many investigators. This problem is discussed by Dr. Goldin (A. Goldin & H. B. Wood, Jr. Preclinical Investigation of Alkylating Agents in Cancer Chemotherapy. This monograph.), who is himself responsible for much of the progress made in this field. However, I should like to make reference at this point to a major study that was conducted several years ago by Dr. Leon Schmidt and associates under the sponsorship of the Cancer Chemotherapy National Service Center, in which the pharmacologic effects of a total of 141 alkylating agents (nitrogen mustards, ethylenimines and

Synthesis and biological activities of 2-pyrimidinone nucleosides. 2. 5-Halo-2-pyrimidinone 2'-deoxyribonucleosides.

1-(2-Deoxy-beta-D-ribofuranosyl)-5-bromo-2-pyrimidinone (BrPdR) and 1-(2-deoxy-beta-D-ribofuranosyl)-5-iodo-2-pyrimidinone (IPdR) have been synthesized by condensation of the appropriate silylated bases 2a and 2b, respectively, with 3,5-bis-O-(p-chlorobenzoyl)-2-deoxy-alpha-D-ribofuranosyl chloride (8) in 1,2-dichloroethane, in the presence of SnCl4, followed by separation of the anomeric blocked nucleosides via column chromatography and subsequent deprotection with methanolic ammonia. Both BrPdR and IPdR exhibited significant antiherpes activities against various strains of HSV-1 and HSV-2, the latter compound (IPdR) showing the higher activity as well as the stronger binding to the virus-specific thymidine kinase.

5-Mercaptodeoxyuridine—Its enzymatic synthesis and mode of action in microbiological systems☆

5-Mercapto-2′-deoxyuriCline (MUdR) was synthesized by enzymatic transfer of the deoxyribosyl group of thymidine (TdR) to 5-mercaptouracil (MU), by using the trans-N-deoxyribosylase from Lactobacillus helveticus. The acceptor and donor activities of MU, MUdR and their respective disulfides were studied in this enzyme system; the disulfides were found inactive as substrates, but they showed the same growth inhibitory activities as the corresponding thiols in the microbiological assays. MUdR was nearly as active as 5-fluoro-2′-deoxyuridine (FUdR) in the Lactobacillus leichmannii assay, but was considerably less active than FUdR in the Lactobacillus arabinosus and Streptococcus faecalis systems. Inhibition analysis studies indicated that MUdR, like FUdR, acts via inhibition of thymidylate synthetase. The significant differences found in the spectra of activities of MU and MUdR, as compared to those of 5-fluorouracil (FU) and FUdR, are related to differences in the routes and efficiency of their metabolic activation in which the mercapto analogs, due to the size of their 5-S− group, are restricted to the pathways available for the metabolic transformations of thymine and TdR, while the fluoro analogs may be converted to the nucleotide via the uracil pathway. Some synergism and cross-resistance studies are also reported.

Inhibition of DNA-dependent RNA polymerase with partially thiolated polynucleotides.

Abstract Various modified polynucleotides, prepared from polycytidylic and polyuridylic acids as well as from RNA and DNA isolates by partial thiolation in the 5-position of their uracil and/or cytosine bases, strongly inhibited the DNA-dependent RNA polymerase of Micrococcus lysodeikticus . The inhibition was in the presence of mercaptoethanol partially reversible by the DNA template in a concentration-dependent manner, but was irreversible without the addition of mercaptoethanol. Incubation of the enzyme with the template prior to the addition of the inhibitor afforded some protection and decreased the inhibitory effects. The results suggest that the partially thiolated polynucleotides act by competing with the DNA template for the “template site” of the enzyme. In the absence of mercaptoethanol, covalent binding via mixed disulfide linkages may take place between the inhibitor and the enzyme.

Cyclophosphamide, 2,2-dimethylaziridines and other alkylating agents as inhibitors of serum cholinesterase☆

Abstract The effects of cyclophosphamide (CTX) and of alkylating agents containing aziridine or 2,2-dimethylaziridine moieties on the procaine esterase activity of horse serum cholinesterase were investigated. The results indicated that CTX is a competitive, reversible inhibitor of the enzyme, while all the other agents studied caused irreversible inhibition. However, there was no over-all correlation between the cholinesterase inhibitory activities of these agents and their alkylating reactivities toward the model nucleophile 4-( p -nitrobenzyl)pyridine (NBP). The kinetics of inhibition were consistent with the formation of a reversible enzyme alkylating agent complex prior to the irreversible inactivation of the enzyme. In the case of the ring-C-unsubstituted aziridines (TEM. TEPA and AB-100), the inactivation process could be described by the Main equation from which a dissociation constant ( K d ) and a reaction rate constant ( k 2 )wcre calculated. The 2,2-dimethylaziridines(AB-132. AB-163 and TEPA-132) readily hydrolyzed. with rapid loss of alkylating activity (vs NBP). Simultaneously, the cholinesterase inhibitory activities of AB-132 and AB-163 significantly increased, reached a maximum and then gradually decreased on further hydrolysis; in contrast, TEPA-132 showed progressive loss of inhibitory activity. These results indicate that both AB-132 and AB-163 (but not TEPA-132) hydrolyze with the formation of an unstable intermediate(s) having little or no alkylating activity but acting as a potent, irreversible cholinesterase inhibitor(s).

Synthesis of Potential Dual Antagonists III: Ring-Substituted Ethyl [Bis(1-aziridinyl)phosphinyl]carbamates

The syntheses of a series of ring-substituted ethyl [bis(1-aziridinyl)phosphinyl]-carbamates are reported. Several of these compounds showed significant tumor inhibitory activities in experimental animals, and one of them, ethyl [bis(2,2-dimethyl-1-aziridinyl)phosphinyl]carbamate (AB-132), is currently under clinical investigation as an experimental antineoplastic agent.

Photoaffinity Labeling of a Cell Surface Polyamine Binding Protein

Intracellular polyamine pools are partially maintained by an active transport apparatus that is specific for and regulated by polyamines. Although mammalian transport activity has been characterized by kinetic studies, the actual protein itself has yet to be identified, purified, or cloned. As one approach to this problem, we attempted photoaffinity labeling of plasma membrane proteins using two specifically designed and synthesized polyamine conjugates as photoprobes. The first is a spermidine conjugate bearing the photoreactive moiety 4-azidosalicylic acid at the N position via an alkyl linkage, and the second is a norspermine conjugate with 4-azidosalicylic acid at the N position via an acyl linkage. Labeling of murine L1210 lymphocytic leukemia cells was carried out at 4°C to promote selective alkylation of cell surface proteins. Separation of plasma membrane proteins from cells cross-linked with the N-spermidine conjugate by SDS-polyacrylamide gel electrophoresis revealed two heavily labeled proteins at 118 and 50 kDa (designated p118 and p50, respectively). Band p118 was more well defined and much more intensely labeled. Analogous proteins were also observed in human U937 lymphoma cells. Specificity of labeling was strongly suggested by competition with polyamines and analogs during labeling and further indicated by the nearly identical labeling of the same protein by the N-norspermine photoprobe but not by the unconjugated photoreagent. Neuraminidase pretreatment of L1210 cells increased mobility of the p118, suggesting that it was glycosylated and, thus, of plasma membrane origin. In transport-deficient L1210 cells, p118 and p50 were found to have a slightly higher molecular mass and were accompanied by a less distinct protein band (100 kDa). These findings indicate the presence of a polyamine binding protein at the surface of murine and human leukemia cells, which could be directly or indirectly related to the polyamine transport apparatus.

POLYNUCLEOTIDES CONTAINING 5‐MERCAPTO‐SUBSTITUTED PYRIMIDINES: INHIBITION OF VIRAL DNA POLYMERASES AND THE BIOLOGICAL IMPLICATION*

Partially thiolated polycytidylic acids MPC I-III, containing 1.7%, 3.5% and 8.6% 5-mercaptocytidylate units, respectively) inhibited the DNA polymerase of Friend leukemia virus (FLV) in the endogenic reaction as well as in the presence of poly(A)-(dT)14 or poly[d(a-T)] templates; the inhibitory activities were directly related to the percent of thiolation. Various partially thiolated RNA and DNA isolates from Ehrlich ascites cells (containing one 5-mercaptopyrimidine nucleotide/50-100 nucleotide units) also inhibited the DNA polymerases of FLV in the endogenic reaction, and also in the presence of the synthetic templates. The thiolated DNA was the most active, but the thiolated tRNA also showed substantial inhibitory effects, while the thiolated ribosomal RNA was less effective. In a bacterial DNA polymerase (E. coli-K12, using denatured DNA as template), MPC I-III showed no activity. By contrast, MPC III and several partially thiolated nucleic acid isolates significantly inhibited a regenerating rat liver DNA polymerase (I) system; among those tested, the thiolated DNA from Ehrlich ascites cells showed the highest activity. Kinetic analysis of the inhibitory action of this thiolated DNA in the rat liver enzyme system, using as template the corresponding unmodified DNA, demonstrated that the thiolated DNA acts as a competitive inhibitor of the template, with a Ki/Km ratio of 0.5.

Absence of cross-resistance to alkylating agents in cyclophosphamide-resistant L1210 leukemia

Abstract A cyclophosphamide resistant strain of L1210 leukemia was sensitive to treatment with any of eight other alkylating agents containing either β chloroethylamine or aziridine functional moieties including mechlorethamine (nitrogen mustard); Tris(aziridinyl)phosphine sulfide (Thio TEPA); alanine, 3-[p-[bis(2-chloroethyl) amino] phenyl]-monohydrochloride (melphalan); four Tris(aziridinyl)phosphine oxide (TEPA) analogs and 1,3,4,5-Tetra o-acetyl-2-(di-2-chloroethyl)amino-2-deoxy- d -glucopyranose. Cross resistance was virtually absent excepr in the case of o-[bis(1-aziridinyl)phosphinyl]-N-hydroxyurethane and melphalan where partial cross resistance was documented.

Inhibition of some DNA polymerase activities from cultured Burkitt cells by thiolated ribonucleic acids

Although unmodified poly C and unmodified ribosomal RNA showed little ( < 20%) or no inhibition of 6–7S cytoplasmic, 3–4S cytoplasmic and 3–4S chromatin-associated DNA-directed DNA polymerases and of RNase sensitive endogenous DNA polymerase and DNA-directed DNA polymerase activity associated with a particulate material (p = 1.16 − 1.18 g/ml) from Burkitt cells the thiolated poly C and thiolated RNA were strongly inhibitory (70–97%). Moreover, the thiolated nucleic acids were more inhibitory to 6–7S enzyme than to 3–4S enzyme. Thiolation of nucleic acids thus appears to be a potentially important procedure for the development of agents which may be selective against certain polymerases.