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Herein, we describe the structure-activity relationship study of a new 3-aryl-8H-thieno[2,3-b]pyrrolizin-8-one series of antitubulin agents. The pharmacological results from the National Cancer Institute in vitro human disease oriented tumor cell line screening allowed us to identify compound 1d (NSC 676693) as a very efficient antitumoral drug in all cancer cell lines tested. This prompted us to define the structural requirements essential for this antiproliferative activity. Among all analogues synthesized in this study, compound 1o was the most promising, being 10-fold more potent than compound 1d. Its activity over a panel of nine tumoral cell lines was in the nanomolar range for all of the histological types tested, and surprisingly, the resistant KB-A1 cell line was also sensitive to this compound. Moreover, a flow cytometric study showed that L1210 cells treated by the most potent compounds were arrested in the G(2)/M phases of the cell cycle with a significant percentage of cells having reinitiated a cycle of DNA synthesis without cell division. This interesting pharmacological profile, resulting from inhibition of tubulin polymerization, encouraged us to perform preliminary in vivo studies that led to a new prodrug chemical approach.

Synthesis of several new 5-amino-substituted derivatives of 5-amino-6H-imidazo[4,5,1-de]-acridin-6-one bearing in the benzene ring OH, OCH3, CH3, tert-butyl, or OCH2O groups is described. 8-OH-substituted compounds or double-substituted 7-OH-10-OCH3 compounds demonstrated potent in vivo activity against murine P388 leukemia. The highest activity was exhibited by 5-[[2-[[2-(diethylamino)ethyl]amino]ethyl]amino]-8-hydroxy-6H- imidazo[4,5,1-de]-acridin-6-one (4c).

Three general methods for the synthesis of derivatives of the bis-alkylating agent, myleran [1,4-bis(methane-sulfonoxy)butane (1)], are presented. These derivatives contain a lipophilic group attached to the 2 position of 1,4-bis(methanesulfonoxy)butane by means of a sulfonyl function. As examples of the scope of the methods, the synthesis of seven such derivatives (12a-g) is presented. All seven were inactive against L1210 and P388 leukemia in the mouse.

A series of benzimidazole-4,7-dione derivatives, bearing substituents at positions 1, 2, 5, and 6 of the benzimidazole ring, has been synthesized and tested for antitumor activity in vivo on P388 leukemia. Some of the synthesized compounds show significant antitumor activity, associated with high toxicity, however. Compounds 7, 18, and 27 show the highest antitumor activity in this series, whereas 17, 19, and 22 are scarcely active. Some hypothetical biological precursors of these quinones are devoid of antitumor activity. Some structure-activity relationships are discussed.

5,6-Dihydro-8(7H)-quinolinone was synthesized and converted into thiosemicarbazones which could be considered to be semirigid analogues of the 2-formylpyridine thiosemicarbazone class of antitumor agents. The Z and E isomers were separated and identified by 1H NMR and UV. Although the compounds showed essentially no inhibitory activity against the enzyme alkaline phosphatase, several of these agents had demonstrable anticancer activity in mice bearing the P388 leukemia. The E-configuration analogues in general were slightly more active than their corresponding Z isomers.

A select number of mitomycin C derivatives with a substituted hydrazine group at position 7 were synthesized and tested for antineoplastic activity by using an in vivo test with murine P388 leukemia. Several of the adducts displayed activity comparable to that of mitomycin C. The X-ray-determined crystal structure of one of the derivatives (3f) is reported.

Salts of 2,3,8,9-tetrasubstituted alkoxy-, hydroxy-, and acetoxybenzo(c)phenanthridines as well as the corresponding 6-methoxy-5,6-dihydrobenzo(c)phenanthridines were prepared from appropriate chalcones through the tetralone and the 4b,10b,11,12-tetrahydrobenzo(c)phenanthridine intermediates. Complete O-demethylation of the tetramethoxybenzophenanthridine was achieved by fusion with pyridine hydrochloride at elevated temperature. The title compounds are active against leukemias L1210 and P388 in mice and some are curative against Lewis lung carcinoma. The importance of the nature of the environment about the nitrogen atom of these compounds and the substituents is discussed. 3,4-Dimethoxy-3,4-methylenedioxychalcone possesses activity against leukemia P388.

Compounds containing an N*-N*-S* tridentate ligand were synthesized and tested for antitumor activity against P-388 lymphocytic leukemia in mice. Of these, only 2,2'-bipyridyl-6-carbothioamide (1a) showed antitumor activity at relatively high dosage levels. Compound 1a was also evaluated against L-1210 and S180 cells in culture and found to have significant activity.

On the basis of qualitative structure-activity relationships developed in the preceding article, a series of 32 new mitomycin A analogues were prepared and tested in antitumor screens. Seven of them gave greater prolongation of life (ILS) than mitomycin C in the mouse P388 leukemia assay. They included examples with 7-O substituents such as cyclic ethers and nitrogen heterocycles. A Hansch analysis was attempted with log P and MR as the independent variables, but no statistically significant correlation could be made. Seven compounds, chosen mainly for their good potency (MED), were tested in the subcutaneous B16 melanoma assay in mice and four of them showed greater ILS than mitomycin C.

6-Substituted 6-deoxy-L-galactose (L-fucose) derivatives were synthesized as potential antimetabolites of L-fucose. The cytotoxic effects of these compounds were evaluated on P388 leukemia cells in culture. The L-fucose analogues which showed the most potent growth inhibition were the sulfonyl ester, bromo, and iodo derivatives; since these compounds were all capable of alkylation, it is conceivable that their cytotoxic action is a consequence of this property. In agreement with this interpretation, none of the agents synthesized showed specific inhibition of the incorporation of L-[3H]fucose into glycoprotein.

A number of N,N:N',N':N",N"-tri-1,2- ethanediylphosphoric triamide (TEPA) and N,N:N',N':N",N"-tri-1,2- ethanediylphosphorothioic triamide (thio-TEPA) derivatives containing either two aziridine moieties (1a) or two (2-chloroethyl)amino functions (1b) and either a 2,2,6,6-tetramethylpiperidine, 1-oxy-2,2,6,6-tetramethylpiperidine or 1-hydroxy-2,2,6,6-tetramethylpiperidine component were synthesized and tested against lymphocytic leukemia P388 in mice. In a structure-activity comparison it was found that at optimum dose all compounds containing the nitroxyl radical were more active than the corresponding hydroxylamine derivatives. The open-chain compounds (1b) were less active than the corresponding aziridine ring compounds (1a). The replacement of the X = bridge in 1a with the X = N(CH3) group resulted in lowering of the anticancer activity.

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.

A series of 26 mitomycin A analogues including 23 new ones was prepared by a variety of methods. The most useful methods were alkoxide exchange on mitomycin A and treatment of 7-hydroxymitosane with 3-substituted 1-phenyltriazenes. Many of the new analogues were superior to mitomycin C in the P388 leukemia assay and the more stringent subcutaneous B16 melanoma assay both in mice. Four of them gave long-term survivors in the latter assay. Quantitative correlations between log P and antitumor activity were not possible, but some guidelines for future analogue development are proposed.

5-[[N-[(Ethoxycarbonyl)alkyl]amino]carbonyl] (6-9) and the corresponding aminothiocarbonyl (12-15) derivatives of 5,6,7,8-tetrahydrofolic acid were prepared as multisubstrate analogues of the substrate--cofactor adduct in the reactions catalyzed by the folate-mediated one-carbon transfer reactions. Evaluation in vitro showed that 7 (alkyl = hexyl) was cytotoxic to H.Ep.-2 cells (ED50, 4 microM) but noncytotoxic to proliferating L1210 cells. No activity was observed for 7 against the P388 leukemia in mice.

Treatment of tropolones with benzaldehyde diethyl acetals gave monotropolone (12) and bistropolone (13) derivatives at the benzylic position, whereas the related 1-ethoxyisochroman and the diethyl acetals of crotonaldehyde and cinnamaldehyde gave only the monotropolone derivatives (5, 10, or 11). The monotropolone derivatives (5, 10, 11, and 12) had poor potency against P388 leukemia in mice, but the bistropolone derivatives (13 and 14) showed significant potency and prolongation of life.

2-Haloethyl and ethyl (methylsulfonyl)methanesulfonates were prepared via sulfene intermediates. 2-Chloroethyl (methylsulfonyl)methanesulfonate is highly active against P388 leukemia in vivo; the majority of leukemic mice treated with this compound at 50 mg/kg per day, qd 1-5, survived more than 30 days and about 37% survived for more than 60 days. 2- Fluoroethyl (methylsulfonyl)methanesulfonate is also highly effective against P388 cells in vivo, but it is more toxic. Other (methylsulfonyl)methanesulfonate esters are more active than the analogous methanesulfonates and chloromethanesulfonates .

Because certain (2-chloroethyl)triazenes and (2-haloethyl)nitrosoureas have high antineoplastic activity, 2-chloroethyl and 2-fluoroethyl sulfonates were prepared to try to develop additional types of 2-haloethylating agents. In this initial study, it was demonstrated that antineoplastic activity much superior to that of the prototype, 2-chloroethyl methanesulfonate, could be found among 2-chloroethyl sulfonates. Among a variety of 2-chloroethyl alkane- and arenesulfonates, several substituted methanesulfonates displayed significant activity against P388 leukemia in mice; the chloromethanesulfonate showed high activity (T/C = 218%). None of the arenesulfonates were active in this test.

The tetrahydroprotoberberine alkaloids 5 and 7 possessing a trans-quinolizidine conformation display in vitro KB cytotoxicities in contrast to their corresponding diastereomers 4 and 6 which exist in the cis-quinolizidine conformation and are much less toxic. The DNA-binding parameters of these compounds as well as the protoberberine salts 1, 8, and 9 have been examined by equilibrium dialysis. Only the quaternary salts bind to DNA. The alcohol 5 showed low in vivo activity against leukemia P388 systems, while the quaternary salts 8 and 9 proved to be toxic to the host.

Some 2,3-bis(substituted methyl)naphthazarins and related compounds were synthesized by the Diels-Alder reaction of benzoquinone and 2,3-dimethylbutadiene followed by oxidation and substitution reactions. These compounds were prepared as potential biological alkylating agents. Screening results indicated that 1,4-diacetyl-6,7-dimethyl-4a,5,8,8a-tetrahydronaphthalene and 5,8-bis(benzoyloxy)-2,3-dimethyl-1,4-naphthoquinone possessed borderline activity against leukemia P388 and that naphthazarin diacetate possessed confirmed cytotoxicity against the cell culture of human epidermoid carcinoma of the nasopharynx.

The effects of various structural modifications on the antineoplastic activity of (arylsulfonyl)hydrazones of 4-pyridinecarboxaldehyde were examined in mice bearing either Sarcoma 180 or P388 leukemia. The introduction of different functional groups into the phenyl ring of the benzenesulfonyl moiety did not alter tumor inhibitory activity appreciably, and the pyridine ring could be replaced by 4-nitrobenzene without loss of antineoplastic activity. However, the aldehyde proton and the hydrazone proton alpha to the sulfonyl group were essential, and their substitution resulted in inactive anticancer agents.

As part of a study on the antitumor activities of tropolone derivatives prepared from hinokitiol, which naturally occurs in the plants of Chamaecyparis species, effects of aromatic substituents of alpha,alpha-bis(7-hydroxy-5-isopropyltropon-2-yl)toluenes on the activity were examined. Several of the compounds showed high potency in the P388 leukemia assay. 4-Hydroxy analogue 4d showed the most potent activity (T/C = 195%) at a 5 mg/kg dose. The introduction of large-size substituents, of which the steric influence prevents coplanarity of the substituted aromatic function, resulted in a remarkable decrease in the potency. X-ray structural analysis of highly potent 4-methoxy analogue 4b was undertaken.

5-Amino-4-(diazoacetyl)-1-beta-D-ribofuranosylimidazole (15), 5-amino-4-(chloroacetyl)-1-beta-D-ribofuranosylimidazole (16), and a number of related imidazole ribonucleosides have been synthesized. Compounds 15 and 16 are cytotoxic to both H.Ep.-2 and L1210 leukemia cells in culture. The (diazoacetyl)imidazole 15 is also active against the P388 leukemia in mice.

Bisphosphines related to bis(diphenylphosphino)ethane (dppe) and their gold complexes are described that are active in a spectrum of transplantable tumor models. When administered ip on days 1-5 at its maximally tolerated dose (MTD) of 40 mumol/kg, dppe reproducibly gives 100% increase in life span (ILS) in mice bearing ip P388 leukemia. Coordination of chlorogold(I) to each phosphine in dppe gave a complex that had similar activity but at a much lower dose level than dppe; the MTD for the gold(I) complex was 7 mumol/kg. Among other metal complexes of dppe, the Au(III) complex was active (greater than 50% ILS) whereas Ag(I), Ni(II), Pt(II), Pd(II), and Rh(I) complexes were inactive. Among dppe analogues, replacement of phenyl groups with ethyl or benzyl groups resulted in inactivity for both ligands and the corresponding gold complexes whereas substitution with cyclohexyl or heterocyclic ring systems yielded ligands and/or gold complexes with antitumor activity. Among substituted-phenyl dppe and dppe(AuCl)2 analogues, 3-fluoro, 4-fluoro, perdeuterio, 4-methylthio, and 2-methylthio analogues were active; 4-methyl, 3-methyl, 4-methoxy, 4-dimethylamino, and 4-trifluoromethyl analogues were marginal or inactive. Analogues in which the ethane bridge of dppe or dppe(AuCl)2 was varied between one and six carbons, unsaturated or substituted, revealed that activity was maximal with ethane or cis-ethylene. Compounds with good P388 activity were also active in other animal tumor models.

In an attempt to find new anticancer agents, a series of pyrido[3',4':4,5]pyrrolo[3,2-c]pyridines were synthesized and evaluated in the standard NCI screening. Among these new compounds, which are structurally related to 9-azaellipticines but differ by deletion of a cycle, those that have a 4-methyl group and a NHCH2CH2CH2NR2 side chain at the 1-position show significant cytotoxicity on L1210 cultured cells and antitumor properties in the in vivo P388 leukemia system. The in vivo antineoplastic activity of the most potent compounds were confirmed on the L1210 leukemia model.

Hycanthone analogues (5 and 6) containing 7-substituted hydroxyl groups were prepared and evaluated as antitumor agents. These compounds were significantly more active than the corresponding unsubstituted derivatives. The 7-hydroxylated 4-(hydroxymethyl)-9H-xanthen-9-ones, 11 and 12, were also active antitumor agents. However, the 7-hydroxy-9H-xanthen-9-one counterparts of the 7-hydroxylucanthones were totally devoid of antitumor activity. Results obtained thus far are consistent with the hypothesis that 4-hydroxymethyl substituents in the 9H-xanthen-9-one and 9H-thioxanthen-9-one series are required for antitumor activity.

Quantitative structure-activity relationships (QSAR) have been formulated for the hydrolysis of aniline mustards and their antitumor activity against Walker 256 tumor and L1210 and P388 leukemia. In general, the antitumor activity parallels hydrolysis under the conditions defined by Ross; toxicity (LD50) parallels antitumor efficacy. Chlorambucil is an exception. A most important finding is that ideal lipophilicity for effectiveness against Walker tumor appears to be much higher than for the leukemias which suggests that solid tumors may, in general, require more lipophilic drugs than leukemias.

A series of (o- and p-nitrobenzyloxycarbonyl)-5-fluorouracil derivatives were synthesized by reacting o- or p-nitrobenzyl chloroformate with 5-fluorouracil in the presence of triethylamine in DMF or Me2SO. The reductive activation of these agents was hypothesized to generate a reactive methide and 5-fluorouracil, two components that are capable of synergistic interaction through complementary inhibition. Measurement of the surviving fractions of EMT6 tumor cells treated with these agents in culture under conditions of hypoxia and aerobiosis resulted in equal cell kill regardless of the state of oxygenation. One of the synthesized agents, 3-(p-nitrobenzyloxycarbonyl)-5-fluorouracil (4), appeared to be superior to 5-fluorouracil in prolonging the survival time of mice bearing intraperitoneal implants of the P388 leukemia and Sarcoma 180.

The synthesis of 4,5-dihydropyrrolo[2,1-a]isoquinolines is reported. A key intermediate in the synthesis of 8-methoxy-4,5-dihydropyrrolo[2,1-a]isoquinolines, 6-hydroxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (6), was prepared by using a regiospecific phenolic cyclization reaction. The P388 lymphocytic activity is reported for 1,2-bis(hydroxymethyl)-5,6-dihydro-8-methoxy-3-methylpyrrolo [2,1-a]isoquinoline bis(isopropylcarbamate) (11a), 1,2-bis(hydroxymethyl)-5,6-dihydro-8-methoxy-3-methylpyrrolo[2,1-a ]isoquinoline bis(cyclohexylcarbamate) (11b), 1,2-bis(hydroxymethyl)-5,6-dihydro-3-methylpyrrolo[2,1-a]isoqui nol ine bis(methylcarbamate) (13a), 1,2-bis(hydroxymethyl)-5,6-dihydro-3-methylpyrrolo [2,1-a]isoquinoline bis(ethylcarbamate) (13b), and 1,2-bis(hydroxymethyl)-5,6-dihydroxy-3-methylpyrrolo[2,1-a]isoq uin oline bis(cyclohexylcarbamate) (13c); all of the compounds were active. Compound 11a was tested in an expanded tumor panel and was shown to be active against B16 melanocarcinoma, CD8F1 mammary, L1210 lymphoid leukemia, colon 38, and MX-1 human tumor breast xenograft systems.

A select number of 1-formyl- and 1-thioformyl-2-methylaziridine derivatives and the corresponding 1a-substituted mitomycin C analogues were synthesized and tested for antineoplastic activity by using an in vivo test with murine P388 leukemia. Select compounds were also tested in vivo with murine melanoma B16. Several of the mitomycin C derivatives displayed activity and some of the mitomycin C analogues were comparable in activity to the parent compound.