Guareschi Synthesis Essay

1. Introduction

Class I phosphatidylinositol-3-kinases (PI3Ks) [1] are lipid kinases able to phosphorylate the hydroxyl group at the three position of the phosphatidylinositol-4,5-biphosphate (PdtIns(4,5)P2). They exert a pivotal role on several cellular processes such as proliferation, survival, motility, metabolism, and differentiation [2].

Class I PI3Ks are usually heterodimers consisting of a catalytic subunit and an adaptor/regulator subunit. All class I catalytic subunits share substantial homology and are referred to as p110 subunits. The contribution of each of the four isoforms of class I PI3Ks has been elucidated in detail, as their tissue localization [3]. For example, in mammals, whereas the expression of PI3Kδ is restricted to the immune system, PI3Kα and PI3Kβ are ubiquitously expressed, and the p110γ catalytic isoform is mainly expressed in leukocytes. For these reasons, the identification of PI3K inhibitors with a different profile of selectivity can be useful for the treatment of different diseases [4].

Over the last decades, pan-PI3K inhibitors [5,6] were extensively investigated for the treatment of cancer; however, the use of isoform-selective PI3K inhibitors seems to be a better strategy in cancer therapy in order to reduce the side effects associated with pan-PI3K inhibitors [7,8]. Alterations in the PI3K pathway, such as PI3Kα mutations, over-expression, or gene amplification, and PTEN (phosphatase and tensin homologue) phosphatase deletion, have been identified in several types of cancer (mammary gland, colon, prostate, brain) [5,6]. Moreover, recent evidence demonstrated that PI3Kα selective inhibitors [7] are more effective against tumors with mutations in the PI3Kα gene. PI3Kβ-selective inhibitors have been proposed in the treatment of PTEN-deficient tumors [9,10,11], while PI3Kδ inhibitors are currently under evaluation in clinical trials for the treatment of some specific lymphoproliferative diseases including chronic lymphocytic leukemia (CLL), multiple myeloma, and Hodgkin’s lymphoma [12,13,14,15]. In this context, a more specific targeted therapy instead of the use of pan-PI3K inhibitors could help to maximize the antitumor effects, reducing the potential side effects (hematological complications, insulin resistance) associated with an indiscriminate inhibition of all the PI3K isoforms.

The identification of selective PI3K inhibitors is still a topic of research both in industry and academia and, to date, several potent PI3K inhibitors, able to compete with the ATP binding site, have been reported [16,17,18,19].

The intellectual property of ATP-competitive inhibitors is highly over-crowded, rendering the identification of novel molecular entities a daunting task for medicinal chemists. One important feature present in most of the ATP-competitive PI3K inhibitors is the presence of the so-called clamp motif [20] formed by a morpholine, a hinge-binder typical for lipid kinases, and a meta-substituted phenol (or one of its bioisosteres) grafted onto a heteroaromatic ring (Figure 1).

The strategy of the clamp motif grafted to a heteroatomatic ring has been utilized by several research groups. For example, cyanuric chloride was used as the starting material, exploiting the different reactivity of chlorine atoms in the nucleophilic aromatic substitution to add different hinge-binders [21].

With this in mind, looking for novel PI3K inhibitors, we focused our attention on the neglected 3-cyano-2,6-dihydroxypyridine scaffold, which can be obtained via Guareschi reaction [22], as a valuable chemical platform. Indeed, it contains all the chemical functions to build the clamp motif and a group pointing towards the rim of the binding pocket. Furthermore, the pyridine ring can increase the solubility, while the cyano group is an added bonus as it might play an active role in the kinase inhibition [23] or it can be considered as a handle for further chemical manipulations (Figure 2). As far as we know, this is the first attempt to use this chemical scaffold in the kinase inhibitor field.

Figure 1. The clamp motif strategy for the identification of novel PI3K inhibitors (in the case of PI3Kγ, the phenol group interacts with Tyr867, while morpholine interacts with Val882).

Figure 1. The clamp motif strategy for the identification of novel PI3K inhibitors (in the case of PI3Kγ, the phenol group interacts with Tyr867, while morpholine interacts with Val882).

Figure 2. The Guareschi pyridine scaffold can be advantageously used for the design of novel PI3K inhibitors.

Figure 2. The Guareschi pyridine scaffold can be advantageously used for the design of novel PI3K inhibitors.

As the amino acid residues located at the entrance to the catalytic site are not conserved in the different PI3K isoforms [24], we decided to maintain the morpholine and phenol clamp motifs, exploring the C4-position of the pyridine ring introducing different aromatic groups. It would be expected that these groups, which protrude outside the ATP-binding pocket, might give additional contacts with the less conserved amino acid residues, altering the profile of selectivity.

The synthesis, the biological activity, and the PI3K subtype selectivity of the compounds synthesized are presented herein.

2. Results and Discussion

The 4-aryl-3-cyano-2,6-dihydroxypyridines (3ah) were synthesized using the improved Guareschi pyridine synthesis reported by Bobbit and Scola [25]. This methodology consists of the condensation of aryl β-keto esters (1ah) and cyanoacetamide (2) in potassium hydroxide at reflux using methanol as solvent (Scheme 1).

Aryl β-keto esters 1ah were prepared from the corresponding commercially available aryl methyl-ketones 4ah by deprotonation with sodium hydride and quenching with dimethylcarbonate (5) [26] (Scheme 2).

Scheme 1. The Guareschi pyridine reaction.

Scheme 1. The Guareschi pyridine reaction.

Scheme 2. Synthesis of aryl β-keto esters.

Scheme 2. Synthesis of aryl β-keto esters.

Condensation of these crude dicarbonyl compounds with cyanoacetamide under basic conditions afforded the corresponding 4-aryl-3-cyano-2,6-dihydroxypyridines (3ah) (Scheme 1). Despite our effort to optimize this reaction, the compounds were obtained in modest yields (Table 1).

Table 1. Yields of 4-aryl-3-cyano-2,6-dihydroxypyridines.

CompoundYield (%)
3a41
3b15
3c14
3d28
3e9
3f27
3g26
3h10

This result can be ascribed to the behavior of β-ketoesters which decarboxylate at high temperatures, and then they underwent ester hydrolysis to β-ketoacids under these basic conditions. In confirmation of this situation, we recovered aryl methyl-ketones from the reaction mixture.

Unfortunately, under room temperature, the Guareschi reaction did not take place and the use of a milder reported procedure (1,4-diazabicyclo[2.2.2]octane or DABCO®, r.t.) failed to afford the desired products [27].

The key intermediates (3ah) were then converted to the corresponding dichlorides (6ah) with phosphorus oxychloride (Scheme 3).

Scheme 3. Synthesis of the dichloride analogues.

Scheme 3. Synthesis of the dichloride analogues.

The regioselective reaction with morpholine in methanol afforded the derivatives 7ah (Scheme 4). A recent paper demonstrated that regioselectivity at the six position can be easily achieved using a polar solvent like methanol [28].

Scheme 4. Regioselective addition of morpholine at the six position.

Scheme 4. Regioselective addition of morpholine at the six position.

Finally, the desired tetrasubstituted pyridines 9ah were obtained via Suzuki coupling with 3-hydroxyphenylboronic acid (8) (Scheme 5).

Scheme 5. Synthesis of compounds 9ah.

Scheme 5. Synthesis of compounds 9ah.

All the synthesized compounds were then tested in vitro for their ability to inhibit the kinase activity of the PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ isoforms in a luminescent kinase assay (ADP-GloTM kinase assay) using purified, recombinant human enzymes and ATP. IC50 values were subsequently determined.

As shown in Table 2

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Мысли Сьюзан перенеслись в прошлое, и глаза ее непроизвольно упали на листок бумаги возле клавиатуры с напечатанным на нем шутливым стишком, полученным по факсу: МНЕ ЯВНО НЕ ХВАТАЕТ ЛОСКА, ЗАТО МОЯ ЛЮБОВЬ БЕЗ ВОСКА.

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