Search for Potent and Selective Aurora A Inhibitors Based on General Ser/Thr Kinase Pharmacophore Model

Based on the data for compounds known from the literature to be active against various types of Ser/Thr kinases, a general pharmachophore model for these types of kinases was developed. The search for the molecules fitting to this pharmacophore among the ASINEX proprietary library revealed a number of compounds, which were tested and appeared to possess some activity against Ser/Thr kinases such as Aurora A, Aurora B and Haspin. Our work on the optimization of these molecules against Aurora A kinase allowed us to achieve several hits in a 3–5 nM range of activity with rather good selectivity and Absorption, Distribution, Metabolism, and Excretion (ADME) properties, and cytotoxicity against 16 cancer cell lines. Thus, we showed the possibility to fine-tune the general Ser/Thr pharmacophore to design active and selective compounds against desired types of kinases.


Introduction
Serine/threonine protein kinases are enzymes that phosphorylate the OH group of serine or threonine. Among more than 500 human protein kinases, at least 125 appeared to be serine/threonine kinases (STK) [1]. Inhibitors of Ser/Thr kinases can possess potential therapeutic uses, from treating cancer to immune disorders. Since they were found in a number of mycobacterial organisms, they can be also used for treatment of bacterial infections such as tuberculosis.
A number of attempts have been made to construct a pharmacophore model for various Ser/Thr kinase inhibitors, such as serine/threonine receptor kinase (STPK) inhibitors of tuberculosis, mTor kinase inhibitors, Aurora A and B inhibitors, B-Raf inhibitors, etc. [2][3][4][5][6][7][8]. Some of these models are based on the structure-based approach, i.e., docking [3]. Although docking is a promising tool for drug discovery, not all kinases (e.g., tuberculosis PknA) have an X-Ray-resolved structure, and, therefore, preliminary modeling of the binding site is necessary. This results in "double step prediction": first the modeling of the binding site is done, followed by docking, which in turn decreases the obtained hit rate. Another group of these models has been developed based on the ligand-based approach [4][5][6][7][8]. In general, a more-or less-wide group of molecules is to be aligned and the common structure features are elucidated. Usually, common structure features are developed into more general pharmacophore models [4][5][6][7].
All cited articles are devoted to the search for specific pharmacophore models targeted to a specific group of kinases. We hypothesized that it is possible to find some general features of all serine-threonine inhibitors and to construct a general pharmacophore model. Then this general model might be adjusted to any specific kind of serine-threonine kinase. To verify our hypothesis and the validity of the design, a series of compounds belonging to this scaffold were tested against several Ser/Thr kinases available in-house: Aurora A, Aurora B and Haspin kinases. The results for the most active compounds are shown in Table 1. Application of this general pharmacophore model to the ASINEX proprietary library allowed us to find a scaffold fitting to its requirements. This scaffold consisted of a thiazole group connected through an amino group to a nitrogen-containing six-member aromatic cycle, which in turn was bound to a pyrrolidine or piperidine ring directly or via a short linker. In this scaffold, thiazole and saturated cycles served as a hydrophobic group, while the middle-positioned six-member cycle presented a source for H-bond donor or acceptor projections (if one or more "A" moieties in the ring means N).
To verify our hypothesis and the validity of the design, a series of compounds belonging to this scaffold were tested against several Ser/Thr kinases available in-house: Aurora A, Aurora B and Haspin kinases. The results for the most active compounds are shown in Table 1.
To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific requirements. Compound 2, which showed the best activity against Aurora A (IC 50 100 nM), was taken as a starting point and a series of iterations was performed. During the optimization process, three series of compound 2 analogues were synthesized. During the first step we kept the methyl substituent on the thiazole ring and the 2-fluoro-3-chloro-benzoyl substituent on the aliphatic nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to the aromatic one either directly or through a methylene linker. After choosing the best compound in this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made. None of     To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific  To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific  To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific  To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific  To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific  To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific  To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific As a result we obtained several compounds with excellent potency in the biochemical assay (Table 2). nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to the aromatic one either directly or through a methylene linker. After choosing the best compound in this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made. None of these substituents demonstrated superiority compared to the 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole analogues.
As a result we obtained several compounds with excellent potency in the biochemical assay (Table 2). Compounds 11-14 were synthesized in accordance with Scheme 1. Amide 2′ was prepared from the corresponding Boc-protected amino acid. Compound 1′ was converted into amidine salt 3′ nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to the aromatic one either directly or through a methylene linker. After choosing the best compound in this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made. None of these substituents demonstrated superiority compared to the 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole analogues.
As a result we obtained several compounds with excellent potency in the biochemical assay (Table 2). Compounds 11-14 were synthesized in accordance with Scheme 1. Amide 2′ was prepared from the corresponding Boc-protected amino acid. Compound 1′ was converted into amidine salt 3′ nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to the aromatic one either directly or through a methylene linker. After choosing the best compound in this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made. None of these substituents demonstrated superiority compared to the 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole analogues.
As a result we obtained several compounds with excellent potency in the biochemical assay (Table 2). nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to the aromatic one either directly or through a methylene linker. After choosing the best compound in this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made. None of these substituents demonstrated superiority compared to the 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole analogues.
As a result we obtained several compounds with excellent potency in the biochemical assay (Table 2). nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to the aromatic one either directly or through a methylene linker. After choosing the best compound in this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made. None of these substituents demonstrated superiority compared to the 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole analogues.
As a result we obtained several compounds with excellent potency in the biochemical assay (Table 2). nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to the aromatic one either directly or through a methylene linker. After choosing the best compound in this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made. None of these substituents demonstrated superiority compared to the 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole analogues.
As a result we obtained several compounds with excellent potency in the biochemical assay (Table 2). the aromatic one either directly or through a methylene linker. After choosing the best compound in this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made. None of these substituents demonstrated superiority compared to the 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole analogues.
As a result we obtained several compounds with excellent potency in the biochemical assay (Table 2). Compounds 11-14 were synthesized in accordance with Scheme 1. Amide 2 1 was prepared from the corresponding Boc-protected amino acid. Compound 1 1 was converted into amidine salt 3 1 by reaction with triethyloxoniuntetrafluoroborate, followed by treatment with a solution of ammonia in methanol. Cyclization of the amidine obtained with acetoacetic ester in ethanol under reflux gave 6-methylpyrimidone 4 1 in 65% yield. This pyrimidones 4 1 were treated with three equivalents of phosphorus oxychloride and nine equivalents of dimethylaniline in toluene to give chloride 5 1 in 62% yield. A Buchwald reaction with aromatic amines (2-aminothiazoles or 3-aminopyrazoles) led to new derivatives 6 1 , and this reaction was performed in a toluene-water mixture with 2.5% mol Pd 2 dba 3 and 5% mol xantphos and potassium carbonate using a microwave reactor (65%-75% yield). For compound 13 with a 5-chloro-2-thiazole moiety, intermediate 6 1 (where R 1 -unsubstituted 2-aminothiazole) was treated with N-chlorosuccinimide in dichloroethane at room temperature (yield 70%). To synthesize compound 14 containing a 3-aminopyrazole moiety, the corresponding 3-amino-1H-pyrazole was protected by the tosyl group via reaction with tosyl chloride and sodium hydrocarbonate in acetonitrile. This protective group was easily removed from intermediate 6 1 by sodium hydroxide in methanol treatment. After Boc-deprotection, amines 7 1 were acylated by 3-chloro-2-fluorobenzoic acid using TBTU as a coupling reagent (yields 85% and more).
by reaction with triethyloxoniuntetrafluoroborate, followed by treatment with a solution of ammonia in methanol. Cyclization of the amidine obtained with acetoacetic ester in ethanol under reflux gave 6-methylpyrimidone 4′ in 65% yield. This pyrimidones 4′ were treated with three equivalents of phosphorus oxychloride and nine equivalents of dimethylaniline in toluene to give chloride 5′ in 62% yield. A Buchwald reaction with aromatic amines (2-aminothiazoles or 3-aminopyrazoles) led to new derivatives 6′, and this reaction was performed in a toluene-water mixture with 2.5% mol Pd2dba3 and 5% mol xantphos and potassium carbonate using a microwave reactor (65%-75% yield). For compound 13 with a 5-chloro-2-thiazole moiety, intermediate 6′ (where R1-unsubstituted 2-aminothiazole) was treated with N-chlorosuccinimide in dichloroethane at room temperature (yield 70%). To synthesize compound 14 containing a 3-aminopyrazole moiety, the corresponding 3-amino-1H-pyrazole was protected by the tosyl group via reaction with tosyl chloride and sodium hydrocarbonate in acetonitrile. This protective group was easily removed from intermediate 6′ by sodium hydroxide in methanol treatment. After Boc-deprotection, amines 7′ were acylated by 3-chloro-2-fluorobenzoic acid using TBTU as a coupling reagent (yields 85% and more). Synthesis of compound 15 is shown in Scheme 2. Alkene 10′ was obtained from ketone 9′ via reaction with methyltriphenylphosphonium iodide and sodium hydride in 75% yield. Subsequent treatment with 9-BBN in THF, and 2,6-dibromopyridine in Suzuki reaction conditions gave bromide 11′ in 78% yield. A Buchwald reaction led to intermediate 12′ (70% yield). Compound 15 was prepared by acylation reactions using TBTU as a coupling reagent.
Compound 16 was synthesized as shown in Scheme 3. A Buchwald reaction with 2,4-dichloropyrimidine 15′ in conditions described above gave a mixture of regioisomers that were separated by column chromatography in 45% yield of target compound 16′. This compound was Synthesis of compound 15 is shown in Scheme 2. Alkene 10 1 was obtained from ketone 9 1 via reaction with methyltriphenylphosphonium iodide and sodium hydride in 75% yield. Subsequent treatment with 9-BBN in THF, and 2,6-dibromopyridine in Suzuki reaction conditions gave bromide 11 1 in 78% yield. A Buchwald reaction led to intermediate 12 1 (70% yield). Compound 15 was prepared by acylation reactions using TBTU as a coupling reagent.
Compound 16 was synthesized as shown in Scheme 3. A Buchwald reaction with 2,4-dichloropyrimidine 15 1 in conditions described above gave a mixture of regioisomers that were separated by column chromatography in 45% yield of target compound 16 1 . This compound was involved in a Suzuki reaction with alkene 10 1 and 9-BBN, followed by Boc-deprotection, and this led to compound 18 1 which was further acylated to compound 16.
Then three of the most active compounds were tested for selectivity over a number of other kinases and for some of their ADME properties, where they showed rather good results (Table 3). involved in a Suzuki reaction with alkene 10′ and 9-BBN, followed by Boc-deprotection, and this led to compound 18′ which was further acylated to compound 16. Then three of the most active compounds were tested for selectivity over a number of other kinases and for some of their ADME properties, where they showed rather good results (Table 3).   involved in a Suzuki reaction with alkene 10′ and 9-BBN, followed by Boc-deprotection, and this led to compound 18′ which was further acylated to compound 16. Then three of the most active compounds were tested for selectivity over a number of other kinases and for some of their ADME properties, where they showed rather good results (Table 3).

General Procedure of Boc-Deprotection
A solution of Boc-protected compound in 18% HCl in 1,4-dioxane (20 mL) was stirred for 4 h at 25˝C (TLC control). The solvent was removed in vacuo, the residue was triturated with ethyl acetate; a precipitate was filtered off, washed with ethyl acetate and dried.