Search

Hydroxymethyl analogs of 5-benzylacyclouridine (BAU) and 5-benzyloxybenzylacyclouridine (BBAU) were synthesized by the condensation of appropriately blocked 2-(chloromethyl)glycerols with substituted 2, 4-dimethoxypyrimidines. The HM derivatives were found to be potent inhibitors of the enzyme uridine phosphorylase and to potentiate significantly the growth-inhibiting action of FdUrd in cell culture.

8-Bromoguanosine cyclic 3',5'-monophosphate, 8-bromoguanosine 5'-monophosphate, and 8-bromoguanosine served as intermediates for the chemical synthesis of a series of 8-substituted seleno cyclic nucleotides, nucleotides, and their nucleosides. Selenourea was found to be a useful reagent in synthesizing these seleno-substituted nucleoside, nucleotide, and cyclic nucleotide. A nucleic acid analyzer was used to study the hydrolysis of these cyclic nucleotides by phosphodiesterase. It was found that all of the 8-substituted selenoguanosine cyclic 3',5'-phosphates synthesized, except 8-MeSe-cGMP, were resistant to hydrolyze by phosphodiesterase. These 8-substituted seleno cyclic GMP derivatives showed some antitumor activities against murine leukemic cells (L5178Y) in vitro and in vivo.

Amino analogs of BAU (5-benzylacyclouridine) and BBAU (5-benzyloxybenzylacyclouridine) and their 2′-hydroxymethyl derivatives were synthesized for evaluation as inhibitors of uridine phosphorylase and hence potential cancer chemotherapeutic agents. Both aminomethyl analogs were found to be potent inhibitors of this enzyme and good potentiators of the anti-tumor action of FUdR.

Racemic 2′-aminomethyl-5-benzyl-acyclouridine (AM-BAU, 5) and 2′-aminomethyl-5-benzyloxybenzyla-cyclouridine (AM-BBAU, 6) have been found to be very active inhibitors of uridine phosphorylase [1]. Their enantiomers were synthesized from chiral 2,2-dimethyl-1,3-dioxolane-4-methanol (7a,b). S-(—)-AM-BAU (5a) and S-(—)-AM-BBAU (6a) were prepared from the R-(—) isomer 7a, and R(+)-AM-BAU (5b) and R-(+)-AM-BBAU (6b) from the S-(+) isomer 7b. A different route from the S-(+) isomer 7b to S-(—)-AM-BBAU (6a) was also determined to be feasible.

Survival studies were performed in mice bearing Sarcoma 180 ascites tumor treated with 6-thio and 6-seleno analogs of guanine and guanosine. The selenium-containing analogs were somewhat superior to the sulfur-containing compounds in antitumor activity and therapeutic index. The formation of 6-SeGMP from 6-seleno-guanine (6-SeG) was demonstrated in Sarcoma 180 ascites cells. Guanine, 6-thioguanine (6-TG) and 6-SeG show comparable substrate activity whereas 8-azaguanine is a much poorer substrate for hypoxanthine-guanine phosphoribosyl transferase from Sarcoma 180 cells. Both 6-TG and 6-SeG are good substrates for purine nucleoside phosphorylase from Sarcoma 180 cells. Chemically and enzymatically synthesized 6-SeGMP behaved as a competitive inhibitor ( K i 1 × 10 −4 M) of erythrocytic and Sarcoma 180 guanylate kinases. Weak substrate activity was demonstrated in the presence of large amounts of erythrocytic guanylate kinase.

Die tierexperimentelle Untersuchung der Stoffwechselwirkungen von Brenzcatechinaminen und ihrer pharmakologischen Beeinflusbarkeit fuhrte zu folgenden Ergebnissen: 1. Adrenalin, Noradrenalin und Isoprenalin verursachen, i.m. injiziert, eine ausgepragte und langanhaltende Zunahme des Sauerstoffverbrauchs von narkotisierten Ratten ohne wesentliche Anderung des respiratorischen Quotienten. Isoprenalin war in dieser Hinsicht doppelt so wirksam wie Adrenalin und Noradrenalin. 2. Die stoffwechselsteigernde Wirkung von Adrenalin und Noradrenalin wird durch das „cocain-ahnlich“ wirkende Desipramin verstarkt. Dieser Effekt ist bei mit Reserpin vorbehandelten Tieren besonders ausgepragt. 3. Durch Vorbehandlung mit dem beta-Sympatholyticum Propranolol kann die calorigene Wirkung von Noradrenalin vollstandig verhindert werden. 4. Nach Ausschaltung der lipolytischen Wirkung von Noradrenalin mit Nicotinsaure wirkt dieses Amin noch immer stoffwechselsteigernd. 5. Adrenalin wirkt sehr stark, Noradrenalin deutlich und Isoprenalin kaum hyperglykamisch. Desipramin war ohne Einflus auf die blutzuckersteigernde Wirkung der Brenzcatechinamine. 6. Der lipolytische Effekt von Noradrenalin wird durch Desipramin nur bei mit Reserpin vorbehandelten Tieren deutlich verstarkt.

Messungen des Sauerstoffverbrauchs und der Kohlendioxidproduktion ergaben, das Amphetamin auch bei schilddrusenlosen Ratten calorigen wirkt, wenn der bei diesen Tieren bestehende Hormonmangel durch Zufuhr von Trijodthyronin ausgeglichen wird.

In attempts to increase the specificity of thiol agents and protect the genetic material of the cell, which is very sensitive to radiation damage, the synthesis of a group of compounds was undertaken in which cysteamine is attached through its amino group to a series of purine and pyrimidine components of nucleic acids. The 5-position of pyrimidines and the 8-position of purines seem well suited for the attachment of groups to be introduced into nucleic acids. Attempts were also made to introduce the bis( BETA -thioethyl) amino group into suitable carrier molecules to yield doublearmed cysteamines. While attempts to isolate bis-( BETA -mercaptoethyl)amine were unsuccessful, the oxidized form of the compound, 1,2-dithia-5-azepane was prepared in good yield. Thus, the preparation of N-substituted 1,2-dithia-5azepanes appeared feasible. A number of uracilyl compounds were prepared by reaction sequences which are shown. BETA -Benzylthioethylamine, prepared by the reaction of ethyleneimine with alpha - toluenethiol, was found to react with 5-bromouracil to yield 5-L BETA - benzylthioethylamino) uracil, This compound could also be obtained by the addition of alpha -toluenethiol to 5-( BETA -chloroethylamino)uracil (onearmed uracil mustard). Debenzylation with sodium in liquid ammonia resulted in the formation of 5-( BETA -mercaptoethylamino)uracil. Attempts to isolate themore » compound were unsuccessful; however, oxidation readily yielded its disulfide form. Bis( BETA -benzylthioethyl)amine reacted with 5bromouracil to form 5-bis( BETA - benzylthtoethylamino)uracil. An alternative synthesis of the compound involved the addition of the sodium salt of benzyl mercaptan to 5-bis( BETA chloroethylamino)uracil (uracil mustard). Reduction of the bis-benzylthio compound with sodium in liquid ammonia followed by air oxidation yielded N-(5- uracilyl)-1,2-dithta5-azepane. N,N'-bis(8-adeninyl)cystamine was prepared in annlogous fashion from 8-( BETA -benzylthioethylamino)adenine, which in turn was obtained by the reaction of 8bromoadenine with BETA -benzylthioethylamine. Difficulties were encountered in isolating mercaptans as such because of the rapidity of air oxidation. (P.C.H.)« less

The benzylacyclouridines, potent and specific inhibitors of mammalian uridine phosphorylase, were also found to be inhibitors of uridine phosphorylase but not thymidine phosphorylase from Escherichia coli. Competitive inhibition was observed in all cases and the most potent of these compounds was HM-BBAU (5-(3-benzyloxybenzyl)-1-[(2'-hydroxy-1'-hydroxymethyl)methyl]urac il) with a Ki value of 0.15 microM. The inhibitory potencies of these compounds parallel those obtained with enzymes from mammalian sources [Niedzwicki et al., Biochem. Pharmac. 31, 1857 (1982) and Naguib et al., manuscript in preparation] indicating that the structure of the active site of uridine phosphorylase from E. coli may resemble that of the mammalian enzyme.

The free-volume theory of transport is used to analyze the concentration dependence of the solvent self-diffusion coefficient for two polystyrene-solvent systems. The agreement between predictions of free-volume theory and the experimental data is good.

In view of the effectiveness of aminoethyl mercaptan (cysteamine) and of 2-aminoethylisothiouronium (AET) salts in protecting animals against ionizing radiation, efforts were made to prepare even more active analogs of the compounds. Sulfur is considered to be essential to the radioprotective action of cysteamine analogs; however, the selenium analog of cysteamine can shield mice against radiation. Since the atomic radii of sulfur and of selenium are similar, sulfur and selenium analogs may be assumed to have the same ability of fit receptor sites. The selenium analog of AET was prepared by the reaction of selenourea with 2-bromoethylamine hydrobromide. Treatment of the selenouronium salt with NaOH yielded 2-selenoethylguanidine, which was isolated as the flavianate salt. The reaction of 2-aminoethylisoselenouronium hydrobromide with boiling water resulted in the formation of 2-aminoselenazoline. Through the reaction of 2- methylaminoethyl. 2dimethylaminoethyl, and 2-diethylaminoethyl halides with selenourea the corresponding 2-alkylaminoethyl selenouronium salts were obtained. Similarly, 3-aminopropylisoselenouronium hydrobromide was synthesized.

A new class of nucleoside analogs, the pyridimine acyclonucleosides, are competitive inhibitors of uridine phosphorylase but have no effect on thymicline phosphorylase, uridine kinase or thymidine kinase. The most potent of the series is acyclothymidine [5-methyl-1-(2′-hydroxyethoxymethyl)uracil] with a Ki value of 3 μM. Ki values of less than 30 μM were estimated for other analogs substituted at the 5-position of the pyrimicline ring. Extracts of xenografts of six human tumors were assayed for tissue levels of uricline phosphorylase and thymicline phosphorylase and for inhibition of 5-fluoro-2′-deoxyuridine (FUdR) phosphorolytic activity by acyclouridine [1-(2′-hydroxyethoxymethyl) uracil]. FUdR cleavage was inhibited most in those tissues in which the ratio of thymidine phosphorylase to uricline phosphorylase was low. Potential usage of these uricline phosphorylase inhibitors with the chemotherapeutic agent FUdR is discussed.

Various pyrimidine acyclonucleosides (l-(2'-hydroxyethoxymethyl)uracils) are specific inhibitors of uridine phosphorylase [Niedzwicki et al., Biochem. Pharmac. 30 , 2097 (1981)]. 5-Benzyluracils have also been shown to inhibit this enzyme [Baker and Kelley. J. med. Chem. 13 , 461 (1970); Woodman et al., Biochem. Pharmac . 29 ,1059 (1980)]. We have synthesized the acyclonucleoside analogs of 5-benzyluracil (BU) and 5-benzyloxybenzyluracil (BBU). These compounds, 5-benzyl-1-(2'-hydroxy-ethoxymethyl) uracil (BAU) and 5-( m -benzyloxybenzyl)-1-(2'-hydroxyethoxymethyl)uracil (BBAU), are potent inhibitors of uridine phosphorylase. K i values of 98 and 32 nM were estimated for BAU and BBAU respectively. These compounds are better inhibitors of uridine phosphorylase than BU ( K i = 1575 nM), BBU ( K i = 270 nM), and all other compounds previously tested, and they have no effect on thymidine phosphorylase, uridine-cytidine kinase, or thymidine kinase. Potential chemotherapeutic applications of BAU and BBAU are discussed.

Amino analogs of BAU (5-benzylacyclouridine) and BBAU (5-benzyloxybenzylacyclouridine) and their 2′-hydroxymethyl derivatives were synthesized for evaluation as inhibitors of uridine phosphorylase and hence potential cancer chemotherapeutic agents. Both aminomethyl analogs were found to be potent inhibitors of this enzyme and good potentiators of the anti-tumor action of FUdR.

6-Chloro-9-(β-D-arabinofuranosyl)purine served as an intermediate for the chemical synthesis of a series of 6-substituted selenopurine arabinosides. In an in vitro test using murine leukemic cells (L-5178Y) these 6-substituted selenopurine arabinosides showed some cytotoxicity. Lengthening the side chain had no effect on their cytotoxicity. Selenourea was a useful reagent for synthesizing selenopurines, selenopurine nucleosides, and selenopurine arabinoside under mild conditions.

Racemic 2′-aminomethyl-5-benzyl-acyclouridine (AM-BAU, 5) and 2′-aminomethyl-5-benzyloxybenzyla-cyclouridine (AM-BBAU, 6) have been found to be very active inhibitors of uridine phosphorylase [1]. Their enantiomers were synthesized from chiral 2,2-dimethyl-1,3-dioxolane-4-methanol (7a,b). S-(—)-AM-BAU (5a) and S-(—)-AM-BBAU (6a) were prepared from the R-(—) isomer 7a, and R(+)-AM-BAU (5b) and R-(+)-AM-BBAU (6b) from the S-(+) isomer 7b. A different route from the S-(+) isomer 7b to S-(—)-AM-BBAU (6a) was also determined to be feasible.

Dimethyl DNS-chol (DDNS), a dansylated analog of acetylcholine, binds with specific sites in Schistosoma mansoni. This treatment provides fluorescent labeling of well-defined areas within the organism. It is believed that these regions of fluorescence reflect areas receiving cholinergic innervation; these regions might include both nervous and non-nervous (effector) tissue, and the DDNS binding sites might include both prejunctional amd postjunctional acetylcholine binding sites. Hycanthone (Hyc), an antischistosomal drug, blocks this labeling by DDNS. Atropine and other known antagonists of acetylcholine also prevent DDNS fluorescent labeling. Measurements of somatic motor activity demonstrate that Hyc and DDNS partially block the paralytic effects of the cholinomimetic drug carbamylcholine on S. mansoni. These data further support the hypothesis that Hyc is an anticholinergic agent, and show that DDNS may be useful as a histochemical label in studies involving structures receiving cholinergic innervation.

The toxicology and metabolism of 8-azaadenosine (8-azaAdo) were examined both as a single agent and in combination with the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF). The LD10 (mice) for 8-azaAdo alone on a once daily for 5 days (q.d. x 5) schedule was 30 mg . kg-1 . day-1. When the animals were pretreated with 0.1 mg . kg-1 . day-1 of dCF, the LD10 dose was reduced to 10 mg . kg-1 . day-1 x 5. The major organ toxicity seen was hepatic. Bone marrow cellularity was only slightly altered at the LD10 dose. 8-AzaAdo nucleotides were detected in the livers of treated mice as determined by high performance liquid chromatography. Further, after 2 hr of incubation, isolated rat hepatocytes accumulated 8-azaATP to levels of 2.2 mumoles/g of cells with 8-azaAdo (1 mM) alone and to 4.3 mumoles/g of cells when 8-azaAdo was used in combination with dCF (1 microgram/ml). ATP levels decreased to below the limits of detection after 2 hr in cells treated with the combination. The replacement of cellular ATP by 8-azaATP may provide an explanation for the hepatotoxicity observed in the murine toxicology studies.

Treatment of adenosine, 2-aminoadenosine, adenine arabinoside, formycin, 1-Me-AMP, cyclo-AMP, and 1-Me-cyclo-AMP with H2Se in aqueous pyridine gave the corresponding seleno compounds with good yield.

8-Bromoguanosine cyclic 3',5'-monophosphate, 8-bromoguanosine 5'-monophosphate, and 8-bromoguanosine served as intermediates for the chemical synthesis of a series of 8-substituted seleno cyclic nucleotides, nucleotides, and their nucleosides. Selenourea was found to be a useful reagent in synthesizing these seleno-substituted nucleoside, nucleotide, and cyclic nucleotide. A nucleic acid analyzer was used to study the hydrolysis of these cyclic nucleotides by phosphodiesterase. It was found that all of the 8-substituted selenoguanosine cyclic 3',5'-phosphates synthesized, except 8-MeSe-cGMP, were resistant to hydrolyze by phosphodiesterase. These 8-substituted seleno cyclic GMP derivatives showed some antitumor activities against murine leukemic cells (L5178Y) in vitro and in vivo.

Kinetic parameters for the phosphorolytic activity of uridine phosphorylase (UrdPase) from human and mouse livers have been determined. The values of these parameters are: K P i = 279.0 ± 66.0 μ M, K Urd = 242.0 ± 63.0 μ M and V max = 3940 ± 175 pmol/min/mg, and K P i = 76.0 ± 7.0 μ M, K Urd = 143.0 ± 9.0 μ M and V max = 293.0 ± 5.0 pmol/min/mg, for human and mouse livers respectively. Benzylacyclouridines, the specific inhibitors of UrdPase, and seventeen newly synthesized derivatives, modified at the pyrimidine ring, the benzyl moiety or the acycio tail, have been tested for their potency to inhibit UrdPase and thymidine phosphorylase (dThdPase) from both human and mouse livers. None inhibited dThdPase. In contrast, all of the compounds tested inhibited UrdPase. Competitive inhibition was observed in all cases. Several of the new compounds were superior in their inhibition of UrdPase to the parent compounds. The inhibitory potencies of these compounds with UrdPase from human liver roughly paralleled those obtained with UrdPase from mouse liver. The most potent of these compounds was AM-BBAU (aminomethyl-BBAU or 5-(3'-benzyloxybenzyl)-1-[(1'-aminomethyl-2'-hydroxy-ethoxy)methyl]uracil) with a K i value of 18 nM with UrdPase from mouse liver. Structure-activity relationships of the binding of these inhibitors of UrdPase are discussed.

6-Methylmercaptopurine ribonucleoside (MeMPR) is the most potent inhibitor known of PRPP amidotransferase, the first step in purine biosynthesis de novo. We have examined other 6-methylmercapto- and 6-methylselenopurine ribonucleosides for their ability to act as feedback inhibitors of this pathway as well as for their ability to form analog nucleotides. Methylmercaptopurine ribonucleoside (MeMPR), methylselenopurine ribonucleoside (MeSePR), and methylmercaptopyrazolo pyrimidine ribonucleoside (MeMPPR) were potent inhibitors (90 per cent), methylselenopyrazolo pyrimidine ribonucleoside (MeSePPR) was less potent (75 per cent), and methylselenoguanosine (MeSeGR) did not significantly inhibit this pathway. Large amounts of the corresponding 5'-monophosphate nucleosides were formed from MeMPR and MeMPPR, lesser amounts from MeSePPR and MeSePR and none from MeSeGR. The ability of 6-selenoguanine, its ribonucleoside and the α- and β-anomers of the deoxyribonucleoside were also examined as possible inhibitors of purine synthesis de novo. Further investigation is needed to determine if these compounds have significant activity as antineoplastic agents, but in any case they may lead to structural modifications which will produce active compounds.