Potential new artificial sweetener from study of structure-taste relationships.

4-(Methoxymethyl)-1,4-cyclohexadiene-1-carboxaldehyde syn-oxime is a new sweetening agent developed by systematic synthesis and taste evaluation of 80 new oximes analogous to the little-used osime sweetener, perillartine.

Dissociation of antitumor potency from anthracycline cardiotoxicity in a doxorubicin analog.

The search for new congeners of the leading anticancer drug doxorubicin has led to an analog that is approximately 1000 times more potent, noncardiotoxic at therapeutic dose levels, and non-cross-resistant with doxorubicin. The new anthracycline, 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin (MRA-CN), is produced by incorporation of the 3' amino group of doxorubicin in a new cyanomorpholinyl ring. The marked increase in potency was observed against human ovarian and breast carcinomas in vitro; it was not accompanied by an increase in cardiotoxicity in fetal mouse heart cultures. Doxorubicin and MRA-CN both produced typical cardiac ultrastructural and biochemical changes, but at equimolar concentrations. In addition, MRA-CN was not cross-resistant with doxorubicin in a variant of the human sarcoma cell line MES-SA selected for resistance to doxorubicin. Thus antitumor efficacy was dissociated from both cardiotoxicity and cross-resistance by this modification of anthracycline structure.

Electrocardiographic and transmembrane potential effects of 5-iminodaunorubicin in the rat.

5-Iminodaunorubicin (5-ID) is a quinone-modified anthracycline that retains antitumor activity but lacks the usual redox-cycling effects of quinoid agents. As a test for decreased cardiotoxicity, we have compared the dose- and time-dependent effects of multiple doses of 5-ID and doxorubicin (DXR) on the rat electrocardiogram (ECG) using a signal-averaging process and have related the ECG changes induced by 5-ID to transmembrane potential alterations in myocardial preparations isolated from treated rats. 5-ID was studied at dose levels of 16, 4, and 1 mg/kg, while DXR was given at 4, 2, and 1 mg/kg. At the high- and medium-dose levels, both agents produced widening of the QRS complex, increased R- and S-wave voltage, and prolonged the Q alpha T interval. The QRS widening reversed in all surviving rats, whereas Q alpha T prolongation was reversible with 5-ID but irreversible with DXR. At the lowest dose, 5-ID had no effect on the ECG until the end of treatment. Microelectrode studies on single cells showed that QRS widening occurring with 5-ID treatment was related to a decrease in the maximum rate of depolarization (Vmax) and that Q alpha T prolongation resulted from an increase in the duration of the action potential. Electron microscopic examination showed that although these toxic changes could not be related to specific morphological alterations, in general, the more severe the electrophysiological change, the greater the ultrastructural change. The most consistent ECG change was Q alpha T prolongation. Using this parameter as a marker for cardiotoxicity, 5-ID was about 4 to 5 times less cardiotoxic than was DXR at high- and medium-dose levels and was noncardiotoxic (i.e., below a threshold for cardiotoxicity) compared with DXR at 1 mg/kg over 20 (DXR) to 35 (5-ID) treatments. The decrease in cardiotoxicity relative to DXR is consistent with previous findings that quinone redox cycling is suppressed in 5-ID. However, the ECG and transmembrane potential effects that we identified at elevated doses of 5-ID can be associated with toxic changes in cardiac cell membranes. Therefore, membrane changes other than those due to quinone redox cycling and, presumably, lipid peroxidation must underlie the electrophysiological changes and structural modifications observed with 5-ID in this study. We believe that 5-ID is a useful mechanistic probe in anthracycline cardiotoxicity studies as well as being of obvious interest for clinical trials.

Metabolic disposition of 5-iminodaunorubicin in the rat.

We determined the metabolic disposition of 5-iminodaunorubicin (IMD) in rats receiving acute doses of IMD (16 and 4 mg/kg) i.v., i.p., and p.o. Major compounds found in plasma, liver, heart, lung, and brain of the rats receiving the higher dose, i.v. or i.p., were IMD and 5-imino-13-dihydrodaunorubicin. The aglycones, 5-iminodaunorubicinone and 5-imino-13-dihydrodaunorubicinone, were minor metabolites. No deoxyaglycones of IMD were detected in any tissue. We could not detect daunorubicin or its metabolites indicating IMD was not a prodrug of daunorubicin. Highest levels of IMD and metabolites were found in lung, liver, and heart, and lowest levels were found in the plasma and brain. Plasma levels of IMD after the higher i.v. dose decayed in a biphasic manner, and we calculated alpha-phase and beta-phase halftimes of decay of 1.4 and 10 hr, respectively. Patterns of distribution of IMD and metabolites were very similar following i.v. and i.p. treatments, except for higher amounts of IMD and metabolites in the liver after the latter route. A p.o. dose of IMD (16 mg/kg) yielded plasma levels of IMD that were only about 20% of those found by the parenteral routes. Summations of all compounds in all tissues at all times after this treatment yielded less than 2% of the corresponding totals found by the other routes. Results obtained following IMD (4 mg/kg) by the three routes generally confirmed conclusions derived from the studies at the higher dose, and we found an approximate linear dose relationship between the results from the two studies. Our inability to detect the formation of deoxyaglycone metabolites of IMD in vivo is consistent with earlier in vitro findings that IMD does not participate in the oxygen-cycling phenomenon typical of daunorubicin and doxorubicin to form drug and oxygen radicals and deoxyaglycone metabolites.

Metabolic disposition of N,N-dibenzyldaunorubicin in the rat.

The more efficacious and less cardiotoxic analogue of daunorubicin, N,N-dibenzyldaunorubicin (B2D), was found to be metabolized in rats by stepwise debenzylation that was superimposed on the known anthracycline metabolism via 13-ketone reduction and deglycosidation. Using high-pressure liquid chromatography for resolution and fluorescence for detection, we observed a series of metabolites in plasma, liver, heart, muscle, and lungs of rats receiving 10 mg B2D per kg, i.v., i.p., and p.o. Rats receiving 40 mg B2D per kg, i.v., died immediately, but this dose given p.o. was not lethal during 24 hr. Patterns of B2D and metabolites varied quantitatively with tissue and route of administration. Rat liver perfusion studies indicated extensive metabolism of B2D compared with limited metabolism of doxorubicin. These observations were consistent with an observed major first-pass effect on B2D in intact rats given B2D p.o. The predominant metabolites of B2D were the glycosidic derivatives, N-benzyldaunorubicin, daunorubicin, and their 13-dihydro derivatives. These metabolites of B2D had exhibited activity against mouse leukemia P388 as did B2D and were active in in vitro tests in which B2D was essentially inactive. These results indicate that B2D acts as a prodrug for a series of active metabolites. Conversion of B2D to these metabolites was relatively more efficient after p.o. administration than following i.v. or i.p. treatments.

Effects of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin and doxorubicin on the survival, DNA integrity, and nucleolar morphology of human leukemia cells in vitro.

The potential mechanisms of the extremely potent anthracycline analogue 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin (MRA-CN) have been compared with those of doxorubicin (DOX) by examination of drug effects on colony formation, macromolecular synthesis, DNA integrity, and ultrastructure of human leukemia cells in vitro. Following a 1-h exposure, MRA-CN was found to be 1400-fold more cytocidal than DOX which correlated with the drugs' inhibitory effects on DNA and total RNA synthesis. Treatment with MRA-CN resulted in a dose-dependent production of DNA interstrand cross-links as quantified by alkaline elution. One-h treatments with DOX or 3'-deamino-3'-(4-morpholinyl) doxorubicin (the non-cyano-containing analogue of MRA-CN) produced no DNA-DNA cross-links; rather they produced protein-concealed DNA single-strand breaks. After removal of MRA-CN, the DNA of KBM-3 cells displayed time-dependent fragmentation as indicated by rapid DNA filter elution during the pH 10 lysis step which preceded pH 12 elution. Within 4 h of MRA-CN exposure (10 nM, 1 h), 50% of the cellular DNA was in the lysis fraction. By 24 h, all the cellular DNA was in this fraction. MRA-CN (10 nM), 3'-deamino-3'-(4-morpholinyl)doxorubicin (1 microM), and actinomycin D (1 microM), but not DOX (3 mircroM), each produced distinctive nucleolar macrosegregation, indicating an effect on rRNA synthesis. The alpha-CN substituent on the morpholinyl moiety of MRA-CN appears to be responsible for the unique antitumor potency of this anthracycline. Nucleolar macrosegregation is probably associated with the morpholinyl moiety and is independent of the alpha-CN substituent.

Antitumor septacidin analogues.

In the first approach by total synthesis to the structure of the antitumor antibiotic septacidin, analogues have been obtained which show similar inhibition of RNA-DNA synthesis in cultured leukemia L1210 cells and similar activity against transplanted leukemia P388 in mice. In these analogues, the natural aminoheptose moiety is replaced by 4-amino-4-deoxy-and 4-amino-4,6-dideoxy-L-glucose, to retain the natural configuration of the pyranose ring. Also retained is the lipophilic fatty acid-amino acid side chain attached to the 4-amino group and glycosylation at the 6-NH2 of adenine. If the fatty acid chain was shortened from C16 to C6, if the fatty chain was shifted to the glycine unit, or if the glycine unit was omitted, activity was completely lost. However, activity was retained if the C16 chain was shortened only to C12 or if the glycine unit was extended to beta-alanine. Both active and inactive analogues were nonbinding to DNA and nonmutagenic to Salmonella strains. The synthetic approach was to start with a suitably protected sugar (L-fucose and L-galactose), construct the adenine moiety at C-1 introduce a 4-amino group, and finally attach the preformed side chain.

5-Iminodaunorubicin. Reduced cardiotoxic properties in an antitumor anthracycline.

Treatment of daunorubicin with methanolic ammonia affords 5-iminodaunorubicin, the first quinone-modified analogue of either daunorubicin or adriamycin. This product retains antileukemic activity in mice, is less cardiotoxic by electrocardiographic measurements in rats, and is nonmutagenic in Salmonella typhimurium (Ames test).

Intensely potent morpholinyl anthracyclines.

3'-Deamino-3'-(3-cyano-4-morpholinyl)doxorubicin is a new analogue that is 100 to 1000 times more potent than doxorubicin against tumors in cell culture or in mice, that is active by intraperitoneal, intravenous, or oral dosing, and that does not produce chronic myocardial lesions in mice. This analogue was encountered in studies on the reductive alkylation of doxorubicin and daunorubicin with 2,2' - oxybis [acetaldehyde], which constructs a morpholino ring incorporating the amino N. The morpholinyl nitrile byproducts are separated by virtue of their nonbasicity from the expected morpholino derivatives. The 13-dihydro and 5-imino derivatives are also described in this important new class of anthracyclines.

7-(Aminoethyl) ether and thioether of daunomycinone.

One-step treatment of daunomycinone with excess 2-aminoethanethiol and 2-aminoethanol in trifluoroacetic acid afforded at C-7 the thioether (77% yield) and ether (30% after recycling), respectively. Stereoselectivity for the natural 7S over the 7R configuration was greater for the ether (97:3) than for the thioether (2.5:1). Esterification of daunomycin at C-7 with beta-alanine was accomplished through the mixed anhydride of Z(OMe)-beta-alanine. Preliminary biological tests suggests that the antitumor and DNA interactive properties of the anthracyclines can be retained in such structures.

Enhanced antitumor properties of 3'-(4-morpholinyl) and 3'-(4-methoxy-1-piperidinyl) derivatives of 3'-deaminodaunorubicin.

Reductive N,N-dialkylation of daunorubicin with 2,2'-oxydiacetaldehyde and NaBH3CN occurred in two steps without interruption and with cyclization to form 3'-(4-morpholinyl)-3'-deaminodaunorubicin. This derivative retained the antitumor efficacy of doxorubicin against mouse leukemia P388 but at one-fortieth the dose; hence, it is the most potent anthracycline analogue synthesized so far. The 4-methoxy-1-piperidinyl derivative, similarly prepared with 3-methoxyglutaraldehyde, showed improved efficacy against P388, though at normal doses. Results with a series of analogues indicate that incorporation of the N in the new ring and the presence of an ether O at the 4-position are critical for enhanced activity.

New cyanomorpholinyl byproduct of doxorubicin reductive alkylation.

Previously we reported that reductive alkylation of doxorubicin with 2,2'-oxybis[acetaldehyde] and NaBH3CN to form the 4''-morpholinyl derivative also gave the intensely potent 3''-cyano-4''-morpholinyl as a byproduct, by addition of CN- to an iminium intermediate in place of hydride. We now find that sugar 4'-OH is a third nucleophile that can add to the iminium intermediate in this reaction. Bridging of the 4'-OH to the morpholine ring at C.5'' formed a novel byproduct with an oxazolidino ring fused to the sugar and morpholine. The new product was minor at neutral pH but predominant at an acidic pH. When tested against tumors in mice it was 4-6 times more potent than doxorubicin. Hence, in comparison with the 3''-cyano-4''-morpholinyl, potency was reduced up to 100-fold by the O bridge. Analytical HPLC showed the presence of three of the four possible diastereoisomers, and two were isolated. The diastereoisomers appeared to differ in stability. In vitro tests suggested that biological potency varied inversely with stability.

N-(cyanomethyl)- and N-(2-methoxy-1-cyanoethyl)anthracyclines and related carboxyl derivatives.

Treatment of doxorubicin with formaldehyde and NaCN afforded the N-(cyanomethyl) derivative as a stable alpha-cyanoamine with but moderate antitumor activity in mice, although it was prototypal to the intensely potent alpha-cyanomorpholine derivative. 2-Methoxyacetaldehyde and NaCN afforded the N-(2-methoxy-1-cyanoethyl) derivative as an open-chain analogue of the cyanomorpholine. This analogue underwent rapid hydrolysis to doxorubicin and appeared to act as a prodrug, giving increased antitumor efficacy although with decreased potency. N-(Carboxymethyl)daunorubicin was a highly water-soluble but inactive analogue, synthesized by N-alkylation with ethyl iodoacetate and saponification. The similar N-alkylation of N-(cyanomethyl) daunorubicin demonstrated the combining of N-alkyl chains having different functional substituents.

Anticancer specificity of some ellipticinium salts against human brain tumors in vitro.

Novel structure-activity relationships (SAR) distinct from known SAR for ellipticines have been revealed for certain ellipticinium salts. In particular, ellipticiniums such as the prototypical 9-methoxy-2-methylellipticinium (I- or OAc-) were found to be preferentially cytotoxic to the brain tumor cell line subpanel of the NCI 60 cell-line screening panel. Similar specificity also was apparent with 9-unsubstituted ellipticiniums, or others bearing 9-methyl or 9-chloro substituents. In contrast, 9-hydroxy-substituted ellipticiniums, as well as all nonquaternized ellipticines tested, were devoid of brain tumor specificity. Therefore, it did not appear that this unusual preference was correlated with the relative availability of redox cycling mechanisms, since redox cycling presumably is blocked in 9-methyl- and 9-chloroellipticiniums. Indeed, related investigations have indicated that the brain tumor specificity is mediated by preferential uptake and intracellular accumulation of the specific ellipticiniums. The present study further supports that the NCI in vitro "disease-oriented" primary screen can facilitate the discovery of novel, selectively cytotoxic leads for in vivo and mechanistic investigations.

Improved antitumor effects in 3'-branched homologues of 2'-deoxythioguanosine. Synthesis and evaluation of thioguanine nucleosides of 2,3-dideoxy-3-(hydroxymethyl)-D-erythro-pentofuranose.

The 3-(hydroxymethyl) branched homologue of 2-deoxyribofuranose was synthesized from the corresponding branched ribofuranose 2-O-(S-methyl dithiocarbonate) with tributyltin hydride in the first direct, one-step deoxygenation at C-2 of a ribofuranose. Nucleoside coupling afforded the corresponding 3'-branched 2'-deoxyribonucleosides of thioguanine. The alpha- and beta-nucleosides were equally inhibitory to growth of WI-L2 human lymphoblastoid cells, were phosphorylated and incorporated into the DNA of Mecca lymphosarcoma in mice to the same degree, and were more effective in these tests than the parent analogue alpha-2'-deoxythioguanosine. These results indicate that the hydroxy functions at the 3' and 5' positions of 2'-deoxynucleosides are interchangeable and that acceptance by the that the furanose ring oxygen and 2'-methylene are corresponding interchangeable, and that acceptance by the enzymes is improved if primary hydroxyls are provided at both the 3' and 5' positions.

N-(2-hydroxyethyl)doxorubicin from hydrolysis of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin.

The susceptibility of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin to hydrolysis at pH 7, 4, and 2 has been compared with that of the typically stable morpholine analogue. At pH 7, 74% of the cyanomorpholine was unchanged after 24 h at room temperature, but at pH 2 only 10% remained. Products identified were aglycon (8%) and N-(2-hydroxyethyl)doxorubicin (7%). Most of the losses were to unidentified polar products not eluted from HPLC. Authentic hydroxyethyl was synthesized from doxorubicin by reductive alkylation with glycolaldehyde. Antitumor potency was comparable to that of doxorubicin rather than of cyanomorpholine.

Further studies on the generation of reactive oxygen species from activated anthracyclines and the relationship to cytotoxic action and cardiotoxic effects.

The relative ease of generation of reactive oxygen species from a series of reductively activated aglycone and sugar modified anthracyclines was compared by the extents of single strand scission in supercoiled PM2-covalently closed circular (CCC)-DNA. The electrochemical properties of these agents were used as a quantitative measure of the ease of reduction and subsequent reoxidation of the reduced species. The relationship of these processes to various biological properties of the anthracyclines, in particular to the measured cardiotoxicity of the drugs, was examined. An attempt was made to identify those structural changes which ameliorate the cardiotoxic effects measured in other test systems while permitting the expression of antitumor properties.

Inhibition of nucleoside transport by nitrobenzylthioformycin analogs.

The formycin analogs of nitrobenzylthioinosine and nitrobenzylthioguanosine were synthesized and evaluated as nucleoside transport inhibitors. These analogs have a potential therapeutic advantage over their parent compounds in that their C-nucleosidic linkages prevent them from being degraded to the immunosuppressive agents, 6-mercaptopurine and 6-thioguanine. 7-[(4-Nitrobenzyl)-thio]-3-(beta-D-ribofuranosyl)pyrazolo[4,3- d]pyrimidine (NBTF) and 5-amino-7-[(4-nitrobenzyl)thio]-3-(beta-D- ribofuranosyl)pyrazolo[4,3-d]pyrimidine (NBTGF) were inhibitors of nucleoside transport in human erythrocytes and HL-60 leukemia cells. The IC50 value for nitrobenzylthioinosine, NBTF and NBTGF with 10% erythrocyte suspensions were 18, 18 and 40 nM respectively. Specific binding studies with [3H]NBTF yielded a Kd of 3.4 nM with erythrocytes, approximately 10-fold higher than values reported for nitrobenzylthioinosine. NBTF and nitrobenzylthioinosine bound to HL-60 cells with Kd values of 8.1 and 0.81 nM respectively. The octanol/water partition coefficients of nitrobenzylthioinosine, NBTF and NBTGF were 3.5, 3.2, and 2.8 respectively. NBTF could be expected to be equipotent with nitrobenzylthioinosine in whole blood where inhibitor concentrations of 10(-7) to 10(-6) M are required in order to saturate erythrocytic binding sites; hence, it may exhibit the advantages inherent in a C-nucleoside.

Tissue distribution of doxorubicin and doxorubicinol in rats receiving multiple doses of doxorubicin.

Plasma and tissue levels of doxorubicin (DXR) and doxorubicinol (DXR-OL) were measured fluorometrically after high-pressure liquid chromatography at 1, 3, and 24 h following one, nine, and 24 doses of 1.0 mg DXR/kg or one and eight doses of 4.0 mg DXR/kg, IP, to rats. Comparison of plasma levels of DXR found following single and multiple doses suggests significant build-up of DXR at 1 h with successive doses, but not at 3 h. Liver exhibited substantially higher levels of DXR (on a per gram of protein basis) than did plasma, and multiple doses did not produce higher levels than did a single dose. In contrast, the heart accumulated DXR slowly, attaining levels after multiple dosing in excess of those found in the liver. Skeletal muscle exhibited dose-related levels similar to those for heart but the absolute levels of DXR in muscle were only about one-tenth of those observed in heart. DXR-OL was at very low levels of less than or equal to 4% of the DXR levels in the tissues; it was, however, a major circulatory metabolite, attaining levels in the plasma as high as 85% of the concentration of DXR.

Effects of 5-iminodaunorubicin on nucleoli of rats.

We have confirmed that doxorubicin induces irreversible changes in the nucleolar ultrastructure of myocardial cells of rats. Similar changes were not caused by an equal dose of the synthetic analogue, 5-iminodaunomycin. These results combined with previous and current comparative tests with this analogue, doxorubicin, and daunomycin suggest that 5-iminodaunomycin may serve as a less cardiotoxic anthracycline derivative.