A Novel Transition State Analogue Inhibitor of Guanase Based on Azepinomycin Ring Structure: Synthesis and Biochemical Assessment of Enzyme Inhibition

Abstract

Synthesis and biochemical inhibition studies of a novel transition state analogue inhibitor of guanase bearing the ring structure of azepinomycin have been reported. The compound was synthesized in five steps from a known compound and biochemically screened against the rabbit liver guanase. The compound exhibited competitive inhibition profile with a K_i of 16.7 ± 0.5 µM.

Guanine deaminase (EC 3.5.4.3, Guanase, or GDA), is an enzyme that hydrolyzes guanine to form xanthine that is unsuitable for DNA/RNA buildup.¹ This enzyme has been found in normal or transformed human organs and sera.² One of the approaches for antiviral/anticancer therapy is to design structural mimics of natural guanine as nucleic acid building blocks, with an anticipation that such analogues would be incorporated into DNA/RNA of virus or cancer cells, interrupting their normal replicative processes.³ Indeed, there are some known guanine mimics, such as 6-thioguanine^{4, 5} and 8-azaguanine,⁶ both of which are potent anticancer compounds. However, they both are believed to be substrates for the enzyme guanase, which converts them into their respective inactive forms 6-thioxanthine and 8-azaxanthine, respectively.⁷ A potent guanase inhibitor would restore the original potency of these anticancer compounds.

A potent guanase inhibitor will also be an excellent tool for studying a number of disease-related metabolic processes in which the enzyme is involved either as an indicative or as a causative factor. There are several documented reports on detection of abnormally high levels of serum guanase activity in patients with liver diseases like hepatitis.^8–10 Therefore, the elevated enzyme activity has been suggested as a marker of hepatitis and hepatoma.¹⁰ There are also various reports of patients developing hepatitis C when they are transfused with blood containing high levels of serum guanase activity.^{8, 10, 11} Even though no direct cause and effect relationship has been established between increased guanase activity and post-transfusional hepatitis (PTH), it is reasonable to assume that the guanase level is responsible, in part, for the development of PTH. There are only a few and scattered reports on the specific physiological or metabolic role of guanase. Nonetheless, there are important observations made on this enzyme as reported in the literature. For example, it is known that high serum guanase activity is a clear biochemical indicator of organ rejection in liver transplant patients.¹² It has also been shown that patients with multiple sclerosis (MS) have significantly elevated levels of guanase activity in their cerebral spinal fluid (CSF), and that there is a clear correlation between the extent of disability and the level of guanase activity.¹³

Another important aspect of guanase activity is its involvement in cancerous tissues. It has long been known that carcinogenic processes and the activities of some enzymes in cancer tissues and cells are strongly interrelated. In this regard, it is important to consider reports of abnormal levels of guanase activity in various cancer tissues. An increase in guanase activity has been reported in lung,¹⁴ gastric,¹⁵ kidney,^16–18 and breast cancer tissues.¹⁹ It is suggested that this difference in activity is a physiological attempt of the cancer cell to regulate the guanine and/or xanthine level, which is needed by cancer cells to accelerate their salvage metabolic pathway activity. Since the de novo pathway is mostly employed by normal cells for replication, a guanase inhibitor can preferentially check the growth of cancer cells without affecting the normal cells. Furthermore, guanase plays an important role in the detoxification process of high amounts of toxic guanine produced from accelerated metabolism in the cancerous tissues. In any event, a guanase inhibitor can trigger cell death either due to cytotoxicity of the accumulating metabolites as proposed in the case of adenosine deaminase (ADA)^20–23 or through depletion of the nucleotide pool necessary for cell growth.

While many studies of guanase inhibition have been reported both from this lab^24–30 and others,^31–35 guanase is still one of the least studied enzymes of the nucleic acid metabolism, although it has been isolated from rabbit liver,¹ human liver,³⁶ and rat brain³⁷ years ago. Azepinomycin^{38, 39} is a naturally occurring inhibitor of guanase, which has so far been studied only in tissue culture systems. So, while its IC₅₀ value (the concentration of the drug required to inhibit 50% of the guanase activity) is known to be ~10⁻⁵ M,³⁸ the K_i of azepinomycin against guanase has never been determined biochemically against the isolated pure enzyme. In our continued efforts to discover a potent guanase inhibitor, we report here the synthesis and biochemical studies of an analogue azepinomycin that more closely resembles the putative transition state of the enzyme-catalyzed reaction.

The recently solved crystal structure of guanase⁴⁰ from Bacillus subtilis suggests that the enzyme-catalyzed reaction (Figure 1) is assisted by an active site zinc metal ion (Zn⁺²), which forms a tetrahedral complex with His-53, Cys-83, and Cys-86 of the protein, along with an isolated water molecule. Glutamate-55 serves as a proton shuttle, abstracting a proton from the zinc-activated water to form the necessary hydroxide nucleophile, while also enabling protonation at the N-3 site of guanine, thus resulting in the formation of the tetrahedral intermediate 2, as shown. Glu-55 also assists in protonation of the NH₂ group of 2, facilitating the elimination of a molecule of ammonia to form the final product xanthine.

Figure 1.

Zn⁺²-Assisted Hydrolysis of Guanine to Xanthine, Catalyzed by Guanase

Azepinomycin may interrupt the above hydrolytic process via coordination of its own OH group at position −6 with the active site zinc, displacing the crucial active site water molecule involved in hydrolysis. The observed weak inhibitory characteristics of azepinomycin may involve multiple factors, including, but not limited to, the fact that it does not represent the true transition state analogue lacking the geminal hydroxy-amino groups attached to its 6-position so as to effectively mimic the tetrahedral intermediate (2) of the guanase-mediated conversion of guanine (1) to xanthine (3).

With the above rationale, we report here the design, synthesis, and biochemical evaluation of our current target molecule, namely, 3-benzyl-4,5,7,8-tetrahydro-6-hydroxymethyl-6-[(benzyloxycarbonyl)amino]imidazo [4,5-e][1,4]diazepine-5,8-dione (10). Compound 10 meets the criteria outlined above. It has the required geminal carbon at position-6 as described except that the OH group had to be removed one atom further from the geminal junction in order to avoid any potential elimination of water with anchimeric assistance from one of the two NH groups attached to the junction. For the same reason, the exonuclear NH₂ had to be protected by a benzyloxycarbonyl (CBZ) group. A benzyl group has been introduced at position-3 in order to create a hydrophobic environment in its vicinity, which has been suggested to enhance the enzyme-ligand interactions, based on our earlier studies on this enzyme.^{26, 28} Attachment of a benzyl group has further scope for introduction of a variety of substituents on the phenyl ring with defined dimensions, and thus can act as both hydrophobic and dimensional probes. Finally, the introduction of a carbonyl group at position-5 of the heterocycle, coupled with movement of the 6-hydroxy group away from the ring by an additional carbon atom, would allow excellent coordination of the inhibitor with Zn⁺² to form a stable, 6-membered ring structure. The latter would use two of the four metal coordination sites, while the other two would be occupied by two of the three original amino acid residues at the enzyme active site. This arrangement would would further strengthen the interaction of azepinomycin with the active site Zn⁺² ion.

The target 10 was synthesized in five steps commencing from compound 5, which has been reported from this laboratory several years ago.⁴¹ The sequential in situ bromination of the malonate carbon of 5, elimination of HBr, followed by conjugate addition of ammonia, gave 6 in 74% yield. The free NH₂ of 6 was protected as a benzyloxycarbonyl (CBZ) derivative by treatment with CBZ-Cl in the presence of Hünig’s base to produce 7 in 84% yield. The nitro group of 7 was reduced with zinc and acetic acid to obtain 8 in 87% yield. Compound 8 was ring-closed using potassium t-butoxide in dimethylformamide to yield 9 in 46% yield. Finally, the ester group of 9 was selectively reduced to a primary alcohol using LiH-9BBN⁴² to obtain the target 10 in 39% yield. All new compounds were completely characterized by ¹H- and ¹³C-NMR and mass spectral data, coupled with either elemental microanalyses or high resolution mass spectral (HRMS) data.⁴³

Guanase inhibition studies with target 10 were conducted using guanase from rabbit liver (MP Biochemicals) at 25 °C and pH 7.4 by spectroscopically measuring the rate of hydrolysis of the substrate guanine at λ_max 245 nm. The change in optical density at λ_max 245 nm per unit time is a measure of the guanase activity. A total of seven different concentrations of the substrate, ranging from 5 to 40 µM, was employed for each inhibitor concentration that was either 20 or 40 µM, while the amount of enzyme in each assay was 7.7 × 10⁻³ unit. The Lineweaver-Burk plots (1/V versus 1/S) (see Figure 2) were used to calculate K_M, V_max, and K_i. Our biochemical results showed that K_m of enzyme with guanine as substrate was 9.5 × 10⁻⁶ M. The target compound 10 showed competitive inhibition with a K_i of 16.70 ± 0.48 × 10⁻⁶ M.

Fig. 2.

Lineweaver-Burk plot of target compound 10

In conclusion, we have designed, synthesized, and biochemically evaluated a novel, putative transition state analogue inhibitor of guanase. While the observed K_i in micromolar range is respectable, additional structure-activity relationship (SAR) studies would be necessary to further enhance the inhibitory potential. Such studies could involve introduction of various alkyl, aryl, or aralkyl substituents at N-4, N-6, and/or N-7 of 10 or attachment of atoms or groups with + or − inductive effects at various positions of the two exonuclear phenyl rings.

Scheme 1.

Synthesis of the Target Compound 10