Characterization of the 4-(Benzothiazol-2-yl)phenylnitrenium Ion from a Putative Metabolite of a Model Antitumor Drug

Abstract

The 4-(benzothiazol-2-yl)phenylnitrenium ion 11 is generated from hydrolysis or photolysis of O-acetoxy-N-(4-(benzothiazol-2-yl)phenyl)hydroxylamine 8, a model metabolite of 2-(4-aminophenyl)benzothiazole 1 and its ring substituted derivatives that are being developed for a variety of medicinal applications including anti-tumor, anti-bacterial, anti-fungal, and imaging agents. Previously we showed that 11 had an aqueous solution lifetime of 530 ns, similar to the 560 ns lifetime of the 4-biphenylylnitrenium ion 12 derived from the well known chemical carcinogen 4-aminobiphenyl. We now show that the analogy between these two cations extends well beyond their lifetimes. The initial product of hydration of 11 is the quinolimine 16 which can be detected as a long-lived reactive intermediate that hydrolyzes in a pH-dependent manner into the final hydrolysis product, the quinol 15. This hydrolysis behavior is equivalent to that previously described for a large number of ester metabolites of carcinogenic arylamines including 4-aminobiphenyl. The major azide trapping product (90% of azide products) of 11, 20, is generated by substitution on the carbons ortho to the nitrenium ion center of 11. This product is a direct analogue of the major azide adducts, such as 22, generated from trapping of the nitrenium ions of carcinogenic arylamines. The azide/solvent selectivity for 11, kaz/ks, is also nearly equivalent to that of 12. A minor product of the reaction of 11 with N3-, 21, contains no azide functionality, but may be generated by a process in which N3- attacks 11 at the nitrenium ion center with loss of N2 to generate a diazene 25 that subsequently decomposes into 21 with loss of another N2. The adduct derived from attack of 2′-deoxyguanosine (d-G) on 11, 28, is a familiar C-8 adduct of the type generated from the reaction of d-G with a wide variety of arylnitrenium ions derived from carcinogenic arylamines. The rate constant for reaction of d-G with 11, kd-G, is very similar to that observed for the reaction of d-G with 12. The similar lifetimes and chemical reactivities of 11 and 12 can be rationalized by B3LYP/6-31G(d) calculations on the two ions that show that they are of nearly equivalent stability relative to their respective hydration products. The calculations also help to rationalize the different regiochemistry observed for the reaction of N3- with 11 and its oxenium ion analogue, 13. Since 8 is the likely active metabolite of 1 and a significant number of derivatives of 1 are being developed as pharmaceutical agents, the similarity of the chemistry of 11 to that of carcinogenic arylnitrenium ions is of considerable importance. Consideration should be given to this chemistry in continued development of pharmaceuticals containing the 2-(4-aminophenyl)benzothiazole moiety.

Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology

“…Thioflavin S is a complex mixture of compounds and, originally, its biological activity was not definitively attributed to any one component; however, a subsequent study isolated and purified Thio-2 (Figure 5A), a component which retained the ability of Thioflavin S to block the BAG-1/HSC70 interaction (Enthammer et al., 2013). Despite this advance, the Thioflavin class of molecules, to which Thio-2 belongs, exhibit multiple sources of off-target activities, including CYP1A1-mediated generation of reactive intermediates and DNA-adduct formation (Chakraborty et al., 2010) as well as PAINS motifs (see next section), casting strong doubt upon the credentials of these compounds as high-quality chemical probes. Consultation with expert chemistry colleagues can avoid wasted time and effort.

Figure 5Thio-2, Lowering Lipophilicity and Pharmacophore Crossing(A) Chemical structure of Thio-2 with the reactive metabolite precursor highlighted in orange.(B) Lowering the calculated lipophilicity (cLogP) of the PAK1 inhibitor chemical tool G-5555 by 100-fold compared with starting compound FRAX1036 by removal of the lipophilic side chain (lilac) bearing a highly basic nitrogen and addition of a tolerated polar side chain (green) bearing a weakly basic nitrogen.(C) Pharmacophore crossing: the PLK1 kinase-binding motif of BI2536 is highlighted in purple and the BRD4 Asn140-binding motif is shaded blue.

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Chemistry of Ring-Substituted 4-(Benzothiazol-2-yl)phenylnitrenium Ions from Antitumor 2-(4-Aminophenyl)benzothiazoles

“…The smaller rate constant, required for fitting of the data at some wavelengths, is moderately pH-dependent in these buffers as was previously observed for 9a . 30 HPLC data taken during the hydrolysis of 9c confirmed that the appearance of the peak for 13c was governed by the slow decay of a transient HPLC peak identified as that of 14c . This result is similar to that previously observed by HPLC analysis of the formation of 13a during the decomposition of 9a .…”

“…29,30 Laser flash photolysis (lfp) of 9a led to the direct detection of 11a with λ max 570 nm and an aqueous solution lifetime of 530 ns. 29 The effect of N 3 − on the kinetics of decay of 11a monitored at 570 nm was consistent with N 3 − trapping data obtained during hydrolysis of 9a , demonstrating that the same intermediate was generated during photolysis and hydrolysis.…”

Synthesis and antitumor activity evaluation of new 2-(4-aminophenyl)benzothiazole derivatives bearing different heterocyclic rings

“…Furthermore, compounds 9-15 vary from compounds 3-9 due to including chlorine atom on phenyl ring at second position of the benzothiazole structure. Imidazole including compounds namely N-[4-(benzothiazole-2-yl)phenyl]-2-[(1,5-diphenyl-1H-imidazole-2-yl)thio]acetamide derivatives (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) have methyl, methoxy and fluoro substituents on phenyl groups at the first and fifth positions of the imidazole ring. Among the nine tested compounds, imidazole and non-substituted benzimidazole including compounds (3, 10 and 16) possessed higher activity.…”

“…This discovery was followed by the identification of the 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) and 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) and the evaluation of the analogue compounds with more potent and diverse activities [8][9][10][11][12][13][14] . Phortress (NSC 710305, dihydrochloride salt of the lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203)), the fluorinated water-soluble pro-drug, which has been synthesized to address formulation and bioavailability issues related to the desired parenteral administration [15][16][17][18][19] , was then chosen for phase 1 The mechanism of action involves formation of reactive intermediates that can bind covalently to DNA and can be metabolized only by sensitive cancer cell lines 21 . Conversely, in insensitive cell lines, neither retaining nor metabolization occurs, thereby selective antitumor properties appear due through to metabolism [22][23][24][25][26] .…”