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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-((β- d -ribofuranosyl) pyrazolo[4,3- d ]pyrimidine (NBTF) and 5-amino-7-[(4-nitrobenzyl)thio]-3-(β- d -ribofuranosyl) pyrazolo[4,3- d ]pyrimidine (NBTGF) were inhibitors of nucleoside transport in human erythrocytes and HL-60 leukemia cells. The IC 50 values for nitrobenzylthioinosine, NBTF and NBTGF with 10% erythrocyte suspensions were 18, 18 and 40 nM respectively. Specific binding studies with [ 3 H]NBTF yielded a K d of 3.4 nM with erythrocytes, approximately 10-fold higher than values reported for nitrobenzylthioinosine. NBTF and nitrobenzylthioinosine bound to HL-60 cells with K d 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.

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.

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.

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.

The C-glycosyl isostere of 4-demethoxy-9-desacetyldaunorubicin and its 7,9-diepi isomer have been synthesized via Diels-Alder reaction of a protected daunosaminyl-2,4-pentadiene with quinizarin quinone.

1-Hydroxyphenazine 5,10-dioxide showed antitumor properties against mouse leukemia P388. It also participated in biochemical mechanisms of quinoid antitumor agents, as indicated by inhibition of radiolabeled DNA-RNA precursors in cultured leukemia L1210 cells and by stimulation of oxygen consumption in mammalian microsomes. This suggests that the isosteric di-N-oxide system may be a biologically active substitute for 1,4-quinone, and that di-N-oxides of tetrahydrobenzo[b]phenazines can be explored as anthracyclinone N-isosteres. As potential synthetic intermediates, 7,8,9,10-tetrahydro-6,11-dihydroxybenzo[b]-phenazines have been prepared by 1) Diels-Alder addition of phenazine-1,4-quinone and 1-methoxy-3-(trimethylsilyloxy)-1,3-butadiene to give isolable but labile adducts and 2) condensation of 6,7-diamino-1,2,3,4-tetrahydro-2-hydroxy-5,8-dimethoxy-2-naphthoic acid with 3-methoxy-1,2-quinone. Attempts at N-oxidation gave instead oxidation of the 6,11-hydroquinone ring to quinone, regardless of hydroxyl protection. Despite previous literature indications, we have been unable to synthesize the 1,4-dihydroxyphenazine 5,10-dioxide system. We conclude that this hydroxyl substitution pattern (1,4) in an adjacent ring must be avoided in the redesign of anthracyclinone isosteres that have di N-oxide in place of quinone.

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).

Stereospecific coupling of a pentadienyl chain to C.1 of daunosamine followed by Diels-Alder reactions with quinones gave a new synthesis of C-glycosides, including the first intermediates to anthracycline C-glycosyl isosteres.

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.

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.

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.

Series of pyrimidine nucleoside analogs were tested for inhibition of DNA and/or RNA synthesis at L1210 cells. The structure-activity relationship was studied at the analogs of cancerostatic agents 5-fluorouracil and arabinosylcytosine. Out of them the 5'-chloro derivatives give some promise. The inhibitory activity of cyclocytidine vs DNA and RNA synthesis approaches the activity of cancerostatic antibotics.

C -Glycosylation of a 2-deoxypyranose has been achieved for the first time by conversion of 4- O-p -nitrobenzoyl- N -trifluoroacetyldaunosamine in a Wittig reaction into the corresponding derivative of ethyl 2-(daunosaminyl)acetate. The product was predominantly (54%) in the desired α- l configuration (separated from the β- l anomer, 15%) required for further elaboration of C -daunosaminyl derivatives. Conversion into the corresponding derivatives of 2-(α- l -daunosaminyl)acetaldehyde and 2-(α- l -daunosaminyl)ethanol was also achieved.

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.

2,3,5-Tri- O -benzyl- D -arabinofuranosyl bromide ( 4 ) was converted into 2,5-anhydro-3,4,6-tri- O -benzyl- D -glucononitrile ( 5 ), mixed with 20% of the D - manno epimer 6 . The mixture was reduced to the amine 7 , which via the N -nitrosoacetamide 10 afforded the 1-deoxy-l-diazo sugar 11 . Dipolar addition to dimethyl acetylene-dicarboxylate afforded the C -nucleoside derivative, dimethyl 3-(2,3,5-tri- O -benzyl-α-β- D -arabinofuranosyl)pyrazole-4,5-dicarboxylate ( 20 ). Selective ammonolysis afforded the 4-ester-5-carboxamide 21 , which was separated chromatographically into the α-(minor) and β-(major) anomers. Hydrazinolysis and Curtius reaction of the pair of 4-acid hydrazides (α- 22 and β- 22 ) afforded the anomeric 3-glycosyl-1 H -pyrazolo-[4,3- d ]pyrimidine-5,7-diones (α- 24 and β- 24 ). Hydrogenolytic debenzylation yielded the β- D )- arabino epimer ( 1 ) of oxoformycin B , and the α- D - arabino form 2 . These anomeric C -nucleosides were distinguished by circular dichroism spectra that showed the same relationship as α- and β- D - arabino anomers of normal purine nucleosides.

1,4-Dimethyldibenzothiophene (VII) was synthesized and was converted with butyl dichloromethyl ether to the 2-aldehyde (VIII). The structure was confirmed by an independent synthesis of the 2-ester (XI) derived from it. The 2-aldehyde (VIII) with aminoacetaldehyde diethylacetal formed the Schiff′s base (IX), and Pomeranz-Fritsch cyclization with 105% superphosphoric acid then formed thia-ellipticine (II), an isostere of the antitumor alkaloid, ellipticine.

Condensation of bis-(2-chloroethyl)phosphoramidic dichloride with 3′-amino-3′-deoxy-N,N-dimethyladenosine afforded the 2′,3′-cyclicphosphorodiamidate (III). By an improved synthesis, methyl 3-amino-3-deoxy-β-D-ribofuranoside was obtained as a model compound for conversion to the analogous 2,3-cyclicphosphorodiamidate (XII). Existence of the latter as two diastereomers due to phosphorus asymmetry was shown by nmr analysis, using comparison with the 5-(O-p-nitrobenzoate) (XIII) as a basis for assignments.

Nitration of 2,5-dimethylbenzoic acid was only 29% at the meta2 position vs. 41% at the ortho, in contrast to sulfonation which was 85% meta vs. 15% para. Identity of the products (and of their derived amino esters and phenolic esters) was determined from the nuclear magnetic resonance spectra and confirmed by chemical means.

Oxaellipticine has been synthesized from 1,4-dimethyldibenzofuran, by converting it to the 2-aldehyde, forming the Schiff's base with aminoacetaldehyde diethyl acetal, and cyclizing this with 105%superphosphoric acid. Alternatively, tetrahydrodimethyldibenzofuran was formylated mainly at the 3-position, and the 3-(2-nitrovinyl) derivative of the 3-aldehyde, by hydride reduction, then Bischler-Napieralski cyclization of the 3-(2-formamidoethyl) derivative, afforded hexahydrooxaellipticine, which could be aromatized only in poor yield. Isooxaellipticine, the pyrido-A' positional isomer, was similarly prepared from the nitrovinyl derivative of 1,4-dimethyl-2-dibenzofurancarboxaldehyde, through cyclization of the formamidoethyl intermediate and aromatization of the dihydro product. Likewise, isothiaellipticine was obtained from 1,4-dimethyl-2-dibenzothiophenecarboxaldehyde.

Oxidation with perbenzoic acid derivatives in neutral organic solvents of 11-desmethylellipticine (at the pyrido-N) and oxa-ellipticine afforded the corresponding N-Oxides. Thia-ellipticine afforded the sulfone N-oxide, which underwent N-deoxygenation with triphenylphosphine to yield thia-ellipticine sulfone.