Prekinamycin

Seaton and Gould isolated from Streptomyces murayamaensis (27, 110) prekinamycin (190), C18H10N2O4, m.p. 300°C (decomp). From its uv (X , 254, 288.4, 342, 574 nm, 8 5500, 21700, 5770, 37700) and ir spectrum it had a carbazole skeleton with a hydroxyl group chelated to a quinonoid function. The H-NMR spectrum revealed the presence of the three adjacent H-5, H-6, H-7 protons. It also showed the presence of two mefa-coupled H-2, H-4 protons (5 6.60 and 6.69, d, J = 1.5 Hz each) and an aromatic C-Me group (8 2.29) besides two protons (5 11.60 and 12.32) which were absent in its diacetate, 402 239, C22H16N2O6, m.p. above 300° (decomp). In the C NMR spectrum of the diacetate the presence of two quinonoid carbonyls (8 174.14, and 192.48), ester carbonyls (8 170.28 and 170.64) and a cynamide function (8 83.71) was discernible. From these data and the C-NMR spectrum of a sample 

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SCHEME 7.5 Generation of the prekinamycin quinone methide. The 13C label is designated with an asterisk ( ). 

The UV-Vis spectral detection of an intermediate in the catalytic reductive alkylation reaction provides only circumstantial evidence of the quinone methide species. If the bioreductive alkylating agent has a 13C label at the methide center, then a 13C-NMR could provide chemical shift evidence of the methide intermediate. Although this concept is simple, the synthesis of such 13C-labeled materials may not be trivial. We carried out the synthesis of the 13C-labeled prekinamycin shown in Scheme 7.5 and prepared its quinone methide by catalytic reduction in an N2 glove box. An enriched 13C-NMR spectrum of this reaction mixture was obtained within 100 min of the catalytic reduction (the time of the peak intermediate concentration in Fig. 7.2). This spectrum clearly shows the chemical shift associated with the quinone methide along with those of decomposition products (Fig. 7.3). 

Our studies of quinone methides and related species using 13C labeling and spectral global fitting started in the late 1990s and have continued to the present with two papers on prekinamycins slated for publication in 2009. In this section, we summarize what we learned from both our published and unpublished studies. 

FIGURE 7.23 Prototype diazobenzo[6]fluorene-based natural products kinamycin A and prekinamycin. Compounds prepared for this study are shown in the inset. 

Figure 7.23 shows the prototype diazobenzo[Z ]fluorene-based natural products kinamycin A and prekinamycin. The kinamycin A-D family were first isolated from Streptomyces murayamaensis, but the structures were incorrectly characterized as having a cyanobenzo[Z ]carbazole ring. Since the initial discovery of the kinamycins, many new analogues have been discovered from natural sources.88-92... 

The 13C-NMR spectrum of the products arising from prekinamycin reductive activation is shown in Fig. 7.3. The identity of the compounds giving rise to the... 

SCHEME 7.22 Products arising from prekinamycin reductive activation at pH 7.5. The 13C labels are designated with asterisks ( ). 

SCHEME 7.24 Hydrolysis mechanism of the prekinamycin quinone methide. 

FIGURE 7.25 pH-rate profile for prekinamycin quinone methide hydrolysis. 

The results of AE calculations shown in Scheme 7.26 show that internal hydrogen bonds can influence the thermodynamics of quinone methide tautomerization in some instances. For the prekinamycin quinone methide without internal hydrogen bonds... 

Table 7.3 shows the concentrations of 1-5 that result in 50% growth inhibition (GI50) of five human cancer cell lines. Inspection of these data reveals that cytostatic activity of 1 and 3-5 depends on the thermodynamic favorability of the quinone methide species compared to the corresponding keto form. The most cytostatic prekinamycins 1 and 5 are associated with the thermodynamically stable quinone methides. In contrast, the inactive prekinamycins 3 and 4 are associated with thermodynamically stable keto tautomers. The exception is prekinamycin 2, which is cytostatic and possesses a relatively stable keto tautomer 3 compared to its quinone methide. Although the AE value for quinone methide tautomerization can predict cytostatic properties, prekinamycin 2 shows that there must be other factors determining biological activity. 

Chart 3.2 13 Originally proposed structure for kinamycin C 14 structure an /V-cyanoindo-loquinone synthesized by Dmitrienko and coworkers 15 originally proposed structure for the metabolite prekinamycin... 

The matter was settled in 1994 in back-to-back communications by Gould [12] and Dmitrienko [13]. Gould showed that treatment of natural prekinamycin with dirhodium tetraacetate in methanol yielded the fluorene 16 (Scheme 3.1). The vinyl proton formed in this reaction (H-l) provided a critical spectroscopic handle and allowed unambiguous determination of the carbocyclic structure, excluding the presence of an indole heterocycle. In parallel, his research group obtained a high-quality crystal structure of a kinamycin derivative. The refined data set was shown to best accommodate a diazo rather than cyanamide (or isonitrile) function. 

Feldman and Eastman have suggested that the kinamycins may by reductively activated to form reactive vinyl radical (25) and orf/to-quinone methide (26) intermediates (Scheme 3.2c) [16]. The authors provided convincing evidence that the alkenyl radical 25 is generated when the model substrate dimethyl prekinamycin (24) is exposed to reducing conditions (tri-n-butyltin hydride, AIBN). Products that may arise from addition of this radical (25) to aromatic solvents (benzene, anisole, and benzonitrile) were isolated. The ort/io-quinone methide 26 was also formed,... 

Scheme 3.3 Hydrogenation of prekinamycin analogs by Skibo and Khdour...

The presence of 9-diazofluorene groups in kinamycin antitumor natural products would lead one to think of an active role for the diazo group. The hypothesis may be substantiated by the fact that one of the precursors in kinamycin biosynthesis, kinafluorenone 10 [44], which lacks the diazo moiety, shows no antibiotic activity against B. subtilis ATCC 6633, known to be very sensitive to the kinamycins. However, prekinamycin (9) [49], which is similar to kinafluorenone but retains the diazo group, shows activity to-... 

Prekinamycin (35), like kinamycins A-F, was isolated from Streptomyces murayamaensis. Carbazole 33, a regioisomer of 7-deoxyprekinamycin (34), was synthesized in only four steps utilizing Pd(OAc), promoted oxidative cyclization as the pivotal step [27]. Similar to Furukawa s approach, the anilino-l,4-naphthoquinone 32 was obtained via Michael addition of the corresponding 2-methoxy-4-methyl-aniline to 1,4-naphthoquinone. Oxidative cyclization proceeded in 84% yield. 

A number of studies on the palladium(n)-mediated oxidative cyclization of aniUno-quinones later appeared. Some of the compounds produced via this protocol are depicted in Figure 9.4. Bittner et al. [37b] and Furukawa and coworkers [37c] both described the application of the intramolecular cyclization chemistry toward the synthesis of analogues of the carbazole-l,4-quinone alkaloids. Furukawa and coworkers [37c] also reported the synthesis of murrayaquinone A (79) using this chemistry. Knolker and O Sullivan [37d,e] later demonstrated the utility of the palladium(ll)-mediated cycUzation in the synthesis of 83, which was initially anticipated to be a prekinamycin analogue precursor. In all... 

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