Enone cancer therapeutics

#This invention relates to novel enones that can be used in enone-containing prodrugs for use as cancer therapeutics. It also relates to methods for synthesizing these novel enones and enone-containing prodrugs.

#The search for improved chemotherapeutic agents with novel mechanisms of action is driven by several, important, unmet clinical needs. First and foremost, almost all anticancer drugs cause extreme secondary side effects. Moreover, solid tumors, which are subject to abnormally high interstitial pressure, tend to be resistant to drug penetration by simple diffusion. Since diffusion rates correlate closely with molecular weights, the utility of anticancer strategies that rely on high molecular weight species such as monoclonal antibodies, tumor necrosis factor, interleukins, interferons, and other macromolecules for tumor therapy may ultimately be limited. For that reason, the development of new chemotherapeutic agents and strategies based on small molecules that can be targeted selectively to diseased tissues continues to be an important undertaking. Research over the past several decades indicates that some of the best new lead compounds for cancer chemotherapy have emerged from a better understanding of natural compounds possessing antitumor or anticancer activity.

#In 1975, the fungal metabolite 2-crotonyloxymethyl-(4R, 5R, 6R)-4,5,6-trihydroxy-2-cyclohexenone (COTC, 1a, Scheme 1) was found to display potent antitumor activity in vitro (Takeuchi, T.; Chimura, H.; Hamada, M.; Umezawa, H.; Yoshka, H.; Oguchi, N.; Takahashi, Y.; Matsuda, A. A Glyoxalase I Inhibitor of a New Structural Type Produced by Streptomyces. J. Antibiot. 1975, 28, 737-742). Besides that antitumor activity, it was noted that COTC reacted with reduced glutathione (GSH) to form a new product that inhibited glyoxalase I, a key enzyme in the detoxification of methylglyoxal. In 1975, Chimura et al attributed the antitumor activity of la to its putative glutathione (GSH) adduct 2a, which was proposed to inhibit the enzyme glyoxalase I (Gl×I) (Chimura, H.; Nakamura, H.; Takita, T.; Takeuchi, T.; Umezawa, M.; Kato, K.; Saito, S.; Tomisawa, T.; litaka, Y. The Structure of a Glyoxalase I Inhibitor and Its Chemical Reactivity with SH Compounds. J. Antibiot. 1975, 28, 743-748). In the 25 years since those findings were reported, the inhibition of Gl×I by 2a has come to be associated with its antitumor activity, although no direct evidence supports such a causal relationship, and the hypothesis has never been tested. 1

#FIG. 1 shows the reciprocal plot of the velocity of the glyoxalase I reaction (ΔOD240) versus the concentration of GSH-methylglyoxal-thiohemiacetal (S) in the absence and presence of different concentrations of 2b. In each kinetic run, the concentration of free GSH was maintained at 0.2 mM by varying the total concentration of GSH and methylglyoxal on the basis of the dissociation constant of the hemithioacetal (Kdiss=2.2 mM). Conditions: 50 mM phosphate buffer, pH 7.0, 25° C.

#FIG. 2 shows the spectrophotometrically determined rates of reaction of GSH (1.03 MM) with 1b (0.05 mM) (A) in the absence GST (k=0.070±0.0002 min−) (B) in the presence 1.8 units of GSTP1-1 (k1=0.882±0.055 min−1; k2=0.633±0.053 min−1),and (C) in the presence of 2.4 units of GSTP1-1(k1=1.33±0.037 min−1; k2=0.685±0.024 min−1).

#FIG. 3 shows the fractionation of product mixtures in the absence and presence of GSTP1-by reverse phase HPLC (Waters, μ-Bondapak C18, 0.78×30 cm) after 30 min incubation of reaction mixtures initially composed of 1b (0.1 MM), cysteine (0.5 mm), GSH (0.5 mM), 0.1 M KPi (pH 6.5) and EDTA (0.05 mM) at 25° C. Based on the integrated intensities of the well-resolved peaks corresponding to 4 (˜9.5 min) and 2b (˜11.5 min), the product ratio in the presence of GSTP1-1 was identical to that obtained from a nonenzymatic incubation of 1b with the same concentrations of cysteine and GSH (FIG. 3). That finding indicated that 3 dissociated from the enzyme prior to reacting with the free thiols in solution, and was consistent with the mechanism in Scheme 4. Adduct 2b (0.5 mM) was stable under the reaction conditions, undergoing less than 1% conversion to 4 in the presence of cysteine (2.5 mM) over 27 h, as determined by HPLC.

#In order to establish an alternative mode of tumor toxicity for COTC and COMC, we have now demonstrated that COMC is a substrate for glutathionyl transferase, an enzyme that is widely distributed in mammalian tissue. Glutathionyl transferase catalyzes a conjugate addition of GSH to 1b with concomitant elimination of crotonic acid leading to 3-glutathionyl-2-exomethylenecyclohexanone 3 (Scheme 2), a reactive intermediate that has now been isolated and characterized for the first time. Trapping studies with amino acids and nucleotides support the hypothesis that 3 can react with, and covalently modify, functionality on proteins and DNA that may be critical to cell viability. 2

#In the course of this work, a short and efficient synthesis of 1b was developed that is superior to the earlier reported seven-step procedure (Hamilton, D. S.; Ding, Z.; Ganem, B.; Creighton, D. J.; Org. Lett. accepted and in press). The improved synthesis is depicted in Scheme 3. Baylis-Hillman reaction of 2-cyclohexenone 4 with formaldehyde afforded 2-hydroxymethyl-2-cyclohexenone 5, (Rezgui, F.; El Gaied, M. M. DMAP-Catalyzed Hydroxymethylation of 2-Cyclohexenones in Aqueous Medium Through Baylis Hillman Reaction. Tetrahedron Lett. 1998, 39, 5965-5966) which was then crotonylated to 1b following the literature procedure. (Mirza, S.; Molleyres, L. -P.; Vasella, A. Synthesis of a Glyoxalase I Inhibitor from Streptomyces griseosporeus Niida et Ogasawara. Helv. Chim. Acta 1985, 68, 988-996). 3

#The GSH conjugate 2b was prepared from 1b by adapting the procedure for making 2a from 1a (Huntley, C. F. M.; Wood, H. B.; Ganem, B. A New Synthesis of the Glyoxalase-I Inhibitor COTC. Tetrahedron Lett. 2000, 41, 2031-2034). The NMR spectrum of 2b featured the expected glutathionyl resonances and the downfield resonance (δ 7.12, triplet) characteristic of H3 in β, gamma-unsubstituted 2-cyclohexenones.