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The studies on the chemistry of mitomycins are described : i) identification and structural elucidation of mitomycins D, E, F, G, I, J, K, L, M, albomitomycin A, and isomitomycin A from the fermentation broth; ii) derivation of mitomycins structurally close to naturally occurring mitomycins; iii) mitomycin rearrangement, the interconversion between mitomycin A, albomitomycin A, and isomitomycin A;iv) chemical con-version of isomitomycin A from mitomycin A; v) 3α-alkoxymitomycins, unexpected by-products in decarbamoylation of mitomycins;vi) the configuration of mitiromycin.

A series of 6-demethylmitomycins and 6-demethyl-6-halomitomycins having various mitomycin skeletons were synthesized, taking into account the electronic effect toward the quinone moiety and the partition coefficients. Treatment of enones 15 and 16 with selenenamide or N-halosuccinimide-Et2NH afforded the 6-demethyl intermediates 17, 18, and 21-24 via the tandem Michael addition/retro-Mannich reaction sequence. Subsequent conversions into the mitomycin skeletons resulted in the formation of the desired derivatives 7a-c, 8a-c, 11a-c, and 12a,b. These mitomycin derivatives including 3a-c and 4a-c were evaluated for their anticellular activity against HeLa S3 cells and antitumor activity against Sarcoma 180 in mice. The anticellular activity of 1 and 3a-c depends on the substituent at the C-6 position and the order of increasing activity is H < CH3 < Br < Cl. A similar tendency was observed in their antitumor potency (ED50). The activities of 9 and 11a-c also follow a pattern similar to that of 1 and 3a-c. Compounds 4b,c, 8b,c, and 12b having both a halogen at the C-6 position and a methoxy group at the C-7 position did not show the activities because of the instability of the compounds. Interestingly, a correlation between the anticellular activity (IC50) and the partition coefficients (log kappa') determined by HPLC was observed within the compounds studied except the unstable compounds, while their antitumor activity (ED50 or T/C) did not correlate with the quinone reduction potential (E1/2). These results would indicate the importance of the C-6 substituents and the mitomycin skeletons for exhibiting both anticellular and antitumor activities.

A series of 4-hydroxy-3-quinolinecarboxylic acid derivatives (6) and 4-hydroxy-2-oxo-1,2-dihydro-3-quinolinecarboxylic acid derivatives (7) were designed and synthesized as 5-HT3 receptor antagonists. Molecular modeling studies suggested that the 3-carbonyl moiety in 6 was almost coplanar to the plane of an aromatic ring, but in 7 there was a 30 degrees deviation. 4-Hydroxy substitution in quinoline derivatives enhanced affinity for the 5-HT3 receptors, and endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-4-hydroxy-3- quinolinecarboxamide (6f) exhibited the most potent activity in the Bezold-Jarisch (B-J) reflex test (ED50 = 0.1 micrograms/kg, iv) among quinoline derivatives 6. Although 4-hydroxy-2-oxo-1,2-dihydro-3-quinolinecarboxamide derivatives (7a) exhibited higher affinity (e.g., 7d: Ki = 0.48 nM) for the 5-HT3 receptors than ondansetron (Ki = 7.6 nM) or granisetron (Ki = 2.1 nM), these amides showed less potent activity in the B-J reflex test than the reference compounds. Interestingly, the ester derivatives 7c, 7f, and 7h eliminated affinity for the 5-HT3 receptors. These unusual structure-activity relationships and the deviation of the 3-carbonyl moiety from the plane of an aromatic ring suggest that the active conformation of 7a might be different from the proposed one for the preceding 5-HT3 antagonists. Thus, 6f was chosen for further studies. No receptor binding for a variety of ligands was significantly antagonized by 6f. Comparing the ratios of the ED50 value in the B-J reflex test (rat, iv) with the LD50 value in acute lethal toxicity (mouse, iv), 6f was proved to have a 600-fold wider margin of safety than ondansetron. Compound 6f dose-dependently attenuated both the incidence and frequency of emetic episodes induced by cisplatin in the dog (ED50 = 14 micrograms/kg, iv) more potently than ondansetron (ED50 = 210 micrograms/kg, iv). Compound 6f (KF-20170) is now under further investigation as a drug for treating gastrointestinal disorder.

KW-2149, a new mitomycin C (MMC) derivative, inhibited the growth of murine P388 leukemia in vitro at 20-fold lower concentrations than those of MMC. KW-2149 was also effective in inhibiting the growth of MMC-resistant P388 (P388/MMC) cells. To elucidate these characteristics of KW-2149, its uptake and efflux were compared with those of MMC in MMC-sensitive and -resistant P388 cells. Both MMC and KW-2149 accumulated rapidly in P388 cells after incubation at the concentration of 0.47 and 0.024 microM, respectively, which were the IC50 values at 1-h exposure. Although this concentration of KW-2149 was 20 times lower than that of MMC, its intracellular concentration was little more than that of MMC, suggesting that KW-2149 accumulated in the cells quite efficiently. The accumulated KW-2149 in the cells after 1-h treatment remained for as long as 24 h after the incubation of the cells in drug-free medium, suggesting that most of the intracellular KW-2149 or MMC was bound to cellular components. The ratios of resistance of P388/MMC cells to MMC and KW-2149 were 34 and 8.8, respectively, at 1-h exposure, suggesting that P388/MMC cells were partially resistant to KW-2149 in vitro. P388/MMC cells also showed partial resistance to cisplatin, Adriamycin, m-AMSA, and etoposide. The accumulation of MMC in P388/MMC cells was lower than that in P388 cells, although the size of the former cells was almost equal to that of the latter. As a result, the amount of DNA-bound MMC was lower in P388/MMC cells than in P388 cells, suggesting its involvement in the mechanisms of MMC resistance in P388/MMC cells.

After inspection of the X-ray crystallographic analyses of mitomycin A (1a) and albomitomycin A (2), an efficient chemical conversion of 1a to isomitomycin A (3) via 2 was accomplished in 68% overall yield. First, 1a was converted to (8aS)-8a-bromoalbomitomycin A (4b) in 89% yield, which was reduced with tributyltin hydride and AIBN to give 2 in 85% yield. Second, the C4a-N4 bond of 2 was selectively cleaved under hydrogenolysis conditions, and subsequent air oxidation afforded 3 in 90% yield from 2. Treatment of 2 or 3 with NH 3 in methanol afforded mitomycin C (1b) in good yield

A series of 6-alkyl-6-demethylmitomycins (1-5) was synthesized and eva- luated for anticellular and antitumor activities. These novel compounds were prepared by Michael addition of various carbanion species to 6-de- methyl-7,7-(ethylenedioxy)-6,7-dihydro-6-methylidenemitosane (6 and 9) followed by treatment with NH 3 or MeOH/K 2 CO 3 . Alkylation at the C-6 position of 6-demethyl-6-selenide (7) was also useful for the alterna- tive synthesis of 6-alkyl-6-demethylmitomycins. The antitumor activity of these derivatives was evalulated, and 6-demethyl-6-ethylmitomycin A (2a) was found to exhibit excellent activity against S-180 solid tumor in mice. The structure-activity relationship is also discussed

A series of C-6-substituted methyl mitomycins was synthesized and evaluated for anticellular and antitumor activities. These novel compounds were prepared by Michael addition of various alcohols or thiols to 6-demethyl-7,7-(ethylenedioxy)-6,7-dihydro-6-methylidenemitosanes followed by treatment with NH3 or MeOH/K2CO3. Most compounds were potent against HeLa S3, and some of them showed superior activity to that of mitomycin C (MMC) against P388 leukemia and sarcoma 180 in mice. In addition, some compounds exhibited remarkable activity against MMC-resistant P388 in mice. FAB-MS spectra of these mitomycin derivatives showed the elimination of the C-6-methyl substituents from the mitomycin skeletons to form quinonemethides. Interestingly, treatment of 6-demethyl-6-[[(2-pyrimidinyl)thio]methyl ]mitomycin C (12v) with diethylamine afforded 6-demethyl-6-[(diethylamino)methyl]mitomycin C (31) in good yield. These results suggested that the C-6-substituted methyl mitomycins would have different biological character from that of MMC.

A series of mitomycin derivatives 1-3 having unique condensed-ring structures was synthesized and evaluated for their anticellular and antitumor activity. These compounds were synthesized by the Michael addition of 1, 3-dicarbonyl compounds to 6-demethyl-7, 7-(ethylenedioxy)-6, 7-dihydro-6-methylenemitosanes (4-6, and 14) and the subsequent cyclization. For the preparation of 1, the allyloxycarbonyl (Aloe) group was employable for the protection of the aziridine (1a-N-H), since the deprotection proceeded without decomposition of the substrates under the mild conditions with Pd(0) and HCO2H-NEt3. Among these structurally unique derivatives, compounds 1a, 1b, 1d and 1e were quite potent against HeLa S3 human tumor cells and sarcoma 180 solid tumor in mice.

A series of 6-demethylmitomycins and 6-demethyl-6-halomitomycins having various mitomycin skeletons were synthesized, taking into account the electronic effect toward the quinone moiety and the partition coefficients. Treatment of enones 15 and 16 with selenenamide or N-halosuccinimide-Et2NH afforded the 6-demethyl intermediates 17, 18, and 21-24 via the tandem Michael addition/retro-Mannich reaction sequence. Subsequent conversions into the mitomycin skeletons resulted in the formation of the desired derivatives 7a-c, 8a-c, 11a-c, and 12a,b. These mitomycin derivatives including 3a-c and 4a-c were evaluated for their anticellular activity against HeLa S3 cells and antitumor activity against Sarcoma 180 in mice. The anticellular activity of 1 and 3a-c depends on the substituent at the C-6 position and the order of increasing activity is H < CH3 < Br < Cl. A similar tendency was observed in their antitumor potency (ED50). The activities of 9 and 11a-c also follow a pattern similar to that of 1 and 3a-c. Compounds 4b,c, 8b,c, and 12b having both a halogen at the C-6 position and a methoxy group at the C-7 position did not show the activities because of the instability of the compounds. Interestingly, a correlation between the anticellular activity (IC50) and the partition coefficients (log kappa') determined by HPLC was observed within the compounds studied except the unstable compounds, while their antitumor activity (ED50 or T/C) did not correlate with the quinone reduction potential (E1/2). These results would indicate the importance of the C-6 substituents and the mitomycin skeletons for exhibiting both anticellular and antitumor activities.

A series of esters and amides of 1-alkyl-2-oxo-1,2-dihydroquinoline-4- carboxylic acid or 2-alkoxy-quinoline-4-carboxylic acid containing a basic azabicycloalkyl moiety has been synthesized and evaluated for affinity for the [3H]quipazine-labeled 5-HT3 receptors. Most of the esters exhibited 10-fold more potent activity than that of ondansetron (1; Ki = 7.6 nM). Lipophilic substituents at the 1- or 2-position of the quinoline ring enhanced affinity for the receptors. Compounds 21 and 37 showed the highest affinity (Ki = 0.32 and 0.31 nM, respectively) among them. On the other hand, most of the amides showed 100-fold lower affinity than that of the esters. Molecular modeling studies indicated that the carbonyl moiety in 19 (ester) or 31 (amide) was not coplanar to the plane of an aromatic ring (over 20 degrees deviation). Although some of the selected compounds exhibited potent activity in the Bezold-Jarisch (B-J) reflex test, good correlation was not observed between the affinity for the 5-HT3 receptors and the activity in the B-J reflex test (in vivo). From these data, it was suggested that our quinoline derivatives might interact with the 5-HT3 receptors in a different way from that of the reported 5-HT3 receptor antagonists presumably due to the presence of the heterogeneity of the 5-HT3 receptors between brain and heart.

The first example of C-3 alkoxylation in mitomycins has been achieved. 3 alpha-iso-Propoxy-10-O-decarbamoylmitomycin D (4) and 3 alpha-iso-propoxymitomycin D (5) were derived from mitomycin D (3) under decarbamoylation conditions with iso-propoxide. Under similar conditions 3 alpha-iso-propoxy-10-O-decarbamoylporfiromycin (8) and 3 alpha-methoxy-10-O-decarbamoylmitomycin B (11) were also derived from porfiromycin (6) and mitomycin B (9), respectively. The mechanism of generation of these novel analogs was based on the premise that the key intermediate of hydroquinone iminium salt (14) was led through the iminium salt (13), followed by alkoxide addition and oxidation.

The configuration of mitiromycin (2) was first determined by NMR experiments including the NOE technique. The absolute structure of 2 was related to mitomycin B (3) because 2 was derived from 3 under basic conditions. The ketonic form 4 was presumed to be involved in the generation of 2.

Mitomycin G (2) was derived from porfiromycin (10b) in 3 steps via the methanesulfonate (14b) in an overall yield of 39%. On the basis of the established method for the introduction of an exomethylene group in mitomycins with a 9a-methoxy group, the preparation of biologically more important 1a-demethylmitomycin G (5) from mitomycin C (1) was accomplished by the use of a protective acetyl group on the aziridine in an overall yield of 57%. 1a-Demethylmitomycin K (6) was obtained from 5 in a yield of 42%. In a preliminary evaluation of their antitumor activity, compound 5 showed superior activity against sarcoma 180 (sc-ip) to its 1a-methyl congener, i.e., mitomycin G (2).

The histamine H1 receptor has an aspartate (Asp) residue in transmembrane helix 3 (TM3), which is well-conserved among biogenic amine receptors. The Asp residue is one of the most crucial amino acids for ligand binding. The tested histamine H1 receptor antagonists with tri- and tetracyclic structures were not selective for histamine H1 receptors and showed affinity for several other biogenic amine receptors. In contrast, KW-4679 ((Z)-11-[3-(dimethylamino)propylidene]-6,11-dihydrodibenz[b, e]oxepin-2-acetic acid hydrochloride), a tricyclic compound, was a selective histamine H1 receptor antagonist. [3H]KW-4679 had high affinity (Kd value of 2.5 +/- 0.12 nM) for wild-type human histamine H1 receptors. In the [3H]KW-4679 binding assay, replacement of Asp107 by alanine by site-directed mutagenesis greatly reduced the affinities (280-2100-fold) of tri- and tetracyclic compounds, whereas this mutation led to a comparatively small reduction (14-fold) in KW-4679 affinity. These results demonstrate that the tested tri- and tetracyclic histamine H1 receptor antagonists which have a tight interaction with the Asp residue are not selective for the histamine H1 receptor. Furthermore, the high selectivity of KW-4679 might be explained by a unique binding pocket, which consists of the Asp residue and other acceptor sites, in the histamine H1 receptor.

The search for 3-D requirements for the adenosine A1 receptor affinity is useful to aid in the design of more potent and/or novel ligands as pharmacological tools and therapeutics for the receptor. To emboss 3-D requirements for adenosine A1 receptor affinity among adenosine receptor antagonists, adenosine and xanthine analogs, conformations for the N6-substituted adenosine analogues and related adenosine A1 receptor antagonists were thoroughly searched by semi-empirical quantum mechanics calculations. Newly established global minima for these compounds (C1'-N6-C6-N1 torsion: 10 degrees) are consistent with retrieved structures from the Cambridge Structural Database and previously published NMR data on the solution conformation of N6-substituted adenosine analogues. However, these newly studied global minima for adenosine analogues are found to be different from those previously reported (C1'-N6-C6-N1 torsion: +/-75 degrees).

The three-dimensional (3-D) requirements for A1 adenosine receptor affinity have been studied based on hydrogen-bonding functionality correlation between a group of twelve A1 adenosine receptor ligands representing ten structurally different classes of compounds. Electrostatic potential similarity indices and shape similarity indices strongly support the proposed receptor-bound orientations of the ligands. We conclude, in areas common to both agonist and antagonist binding at the A1 receptor, that the ligands are recognized by a similar physicochemical 3-D environment. The finding of similar 3-D requirements for agonists and antagonists suggests a fairly static receptor structure in the region common to agonist and antagonist binding. The ribose moiety is remote from antagonist binding site. Such a 3-D environment rationalizes the binding of a number of potent novel antagonists including KW-3902, not previously reported in modeling studies.

A series of C-6-substituted methyl mitomycins was synthesized and evaluated for anticellular and antitumor activities. These novel compounds were prepared by Michael addition of various alcohols or thiols to 6-demethyl-7,7-(ethylenedioxy)-6,7-dihydro-6-methylidenemitosanes followed by treatment with NH3 or MeOH/K2CO3. Most compounds were potent against HeLa S3, and some of them showed superior activity to that of mitomycin C (MMC) against P388 leukemia and sarcoma 180 in mice. In addition, some compounds exhibited remarkable activity against MMC-resistant P388 in mice. FAB-MS spectra of these mitomycin derivatives showed the elimination of the C-6-methyl substituents from the mitomycin skeletons to form quinonemethides. Interestingly, treatment of 6-demethyl-6-[[(2-pyrimidinyl)thio]methyl ]mitomycin C (12v) with diethylamine afforded 6-demethyl-6-[(diethylamino)methyl]mitomycin C (31) in good yield. These results suggested that the C-6-substituted methyl mitomycins would have different biological character from that of MMC.

A series of 6-alkyl-6-demethylmitomycins (1-5) was synthesized and eva- luated for anticellular and antitumor activities. These novel compounds were prepared by Michael addition of various carbanion species to 6-de- methyl-7,7-(ethylenedioxy)-6,7-dihydro-6-methylidenemitosane (6 and 9) followed by treatment with NH 3 or MeOH/K 2 CO 3 . Alkylation at the C-6 position of 6-demethyl-6-selenide (7) was also useful for the alterna- tive synthesis of 6-alkyl-6-demethylmitomycins. The antitumor activity of these derivatives was evalulated, and 6-demethyl-6-ethylmitomycin A (2a) was found to exhibit excellent activity against S-180 solid tumor in mice. The structure-activity relationship is also discussed

The reaction between mitomycin A (1) and cysteamine afforded 7-N,7'-N'-bis(2-thioethyl)dimitomycin C (7), 7-N-[2-[(2-aminoethyl)dithio]ethyl]mitomycin C (8), and 7-methoxy mitosenes (10, 11). The structures of 7 and 8 were elucidated on the basis of spectroscopy and reactions between 1 and 8, and 1 and cystamine. The observation of the time course for the reaction revealed the mechanism of the formation of 7 and 8. The rapid oxidation of cysteamine by the quinone of 1 gave cystamine, which was trapped by 1 to give 8, and 8 was additionally reacted with 1 to give 7. Since 7 showed significant antitumor activities, related 7-N,7'-N'-bis(omega-thioalkyl)dimitomycins were synthesized. They also showed remarkable antitumor activities against HeLa-S3 in vitro, sarcoma 180 (sc-ip), leukemia P388 (ip-ip) in vivo. In these evaluations, compound 7 demonstrated unique potency.

KW-2149 is a new derivative of mitomycin C (MMC). The plasma concentrations, distribution, metabolism, and excretion of [3H]-KW-2149 in normal and tumor-bearing mice after i. v. administration of 16.6 mg/kg were investigated. The plasma radioactivity decreased biexponentially after i. v. administration in normal mice. However, the unchanged drug disappeared rapidly, showing a half-life (t1/2) of 9.7 min, which was shorter than MMC's (18 min). The radioactivity was excreted in mouse urine (33%) and feces (58%) within 144 h. High radioactivity was distributed in the gallbladder, liver, kidney, pancreas, and lung at 1 h after i. v. administration to normal mice. The tumor concentration was lower than the plasma or blood concentration. The lowest radioactivity was observed in the brain. The metabolic rate of KW-2149 was very rapid. The methyl sulfide form (M-16), the symmetrical disulfide dimer (M-18), and the albumin conjugate were detected in plasma, which possessed anticellular activity. The specific anticellular activity of these compounds against uterine carcinoma (HeLa S3) was 1/100, 1, and 1/20 respectively, as compared with that of KW-2149.

L'epimere obtenu a la meme stereochimie en C-9 que la mitomycine C. L'epimerisation s'est faite via une decarbamoylation/amination/carbamoylation

The Raman and infrared spectra of 2-chloro-, 2-bromo, and 2-iodoethyl methyl ethers, CH3OCH2CH2X (X=Cl, Br, and I), were measured for the liquid, glassy, and crystalline states. The gaseous-state spectra of the chloride were also measured. The vibrational frequencies of these molecules were calculated by the use of the force constants transferred directly from unbranched ethers and alkyl halides. The rotational isomerism was studied and the following conclusions were obtained. (1) In the crystalline state, the molecule takes the form having the trans conformation about the CO–CC axis and the gauche conformation about the OC–CX axis. (2) In addition to this form (TG), three other forms (GG, TT, and GT) exist in the gaseous, liquid, and glassy states. (3) In the liquid state, the TG form is the most stable and the GT form is the least stable. (4) The less polar TT and GT forms are more favored and the more polar TG and GG forms are less favored in the gaseous state than in the liquid state.

Barriers to exchange between dl and meso forms via rotation about the C9–Csubst bond of 9-(arylmethyl)-1,4-dimethoxytriptycenes are obtained by the use of the methoxyl signals in the 1H NMR spectra. It is found that, although the population ratios are affected by the substituent in the 9 position, the enthalpy of activation for the rotation is almost invariant at ca. 10 kcal/mol.

Benzyl methylene signals, which split into an AB quartet and a singlet and show temperature dependence, are obtained at various temperatures and are simulated with the use of two independent rates of rotation. Certain limitations of accuracy are found in this method. The uncertainty becomes less if additional data are provided from other sources, but never becomes negligible. Contribution of the slower process is found difficult to detect especially when the population involved in the process are the less.

Mitomycin G 2 was derived from mitomycin B 5 by 3 steps in an overall yield of 43 %.

L'epimere obtenu a la meme stereochimie en C-9 que la mitomycine C. L'epimerisation s'est faite via une decarbamoylation/amination/carbamoylation

Introducing the mercaptoethyl group at the 7-N position of mitomycin C 1 has led to the isolation of 7-N,7'-N'-dithiodiethylenedimitomycin C 2. The compound 2 showed excellent antitumor activity against sarcoma 180 (sc-ip) and leukemia P388 (ip-ip) in mice. As an extension of this study, we synthesized mitomycin dimers with symmetrical disulfide and mitomycin derivatives with unsymmetrical disulfide at the 7-N side chain. Among these compounds, the water soluble conjugate 3 with ethyl gamma-L-glutamyl-L-cysteinylglycinate was far more effective against sarcoma 180 and leukemia P388 than 1. During the subsequent stage of inquiry for the potent congeners of 3, the compound 4 (water solubility: greater than 500 mg/ml), designated as KW2149, with the gamma-L-glutamylcystamino group at the 7th position was finally selected for further evaluation.

The Raman and infrared spectra of 2-chloro-, 2-bromo, and 2-iodoethyl methyl ethers, CH3OCH2CH2X (X=Cl, Br, and I), were measured for the liquid, glassy, and crystalline states. The gaseous-state spectra of the chloride were also measured. The vibrational frequencies of these molecules were calculated by the use of the force constants transferred directly from unbranched ethers and alkyl halides. The rotational isomerism was studied and the following conclusions were obtained. (1) In the crystalline state, the molecule takes the form having the trans conformation about the CO–CC axis and the gauche conformation about the OC–CX axis. (2) In addition to this form (TG), three other forms (GG, TT, and GT) exist in the gaseous, liquid, and glassy states. (3) In the liquid state, the TG form is the most stable and the GT form is the least stable. (4) The less polar TT and GT forms are more favored and the more polar TG and GG forms are less favored in the gaseous state than in the liquid state.