Synthesis of Thiazolo[5,4-f]quinazolin-9(8H)-ones as Multi-Target Directed Ligands of Ser/Thr Kinases

A library of thirty novel thiazolo[5,4-f]quinazolin-9(8H)-one derivatives belonging to four series designated as 12, 13, 14 and 15 was efficiently prepared, helped by microwave-assisted technology when required. The efficient multistep synthesis of methyl 6-amino-2-cyano- benzo[d]thiazole-7-carboxylate (1) has been reinvestigated and performed on a multigram scale. The inhibitory potency of the final products against five kinases involved in Alzheimer’s disease was evaluated. This study demonstrates that some molecules of the 12 and 13 series described in this paper are particularly promising for the development of new multi-target inhibitors of kinases.


Introduction
Major human diseases such as cancer, neurodegenerative disorders and cardiovascular diseases have been closely associated with the deregulation of kinases [1][2][3]. Consequently, protein kinases represent pertinent targets for academic and industrial chemists searching for kinase inhibitors as potential new therapeutic agents [4][5][6]. Most kinases phosphorylate both serine and threonine residues, others phosphorylate tyrosines, and a small number phosphorylate all three amino acids (dual-specificity kinases). Our research groups are mostly invested in the synthesis of sulfur-nitrogen heteroaromatic molecules able to modulate the activity of deregulated kinases thought to be involved in Alzheimer's disease (AD) [7][8][9][10][11][12][13][14][15]. These five important kinases used in this study are the Ser/Thr kinases (CDK5, GSK-3, CLK1 and CK1) and the dual-specificity kinases (DYRK1A family) [16][17][18][19]. The important impact of these kinases in various key cellular regulatory mechanisms is justifying recent approaches consisting in the design of multi-target-directed ligands (MTDLs) [20][21][22][23] able to target more than one kinase. This highly pertinent therapeutic strategy may allow the development of new tools or therapies to better understand and treat patients suffering of neurodegenerative diseases.
In the course of our work, we previously described the synthesis of a small library of 8H-thiazolo [5,4-f ]quinazolin-9(8H)-ones (A in Figure 1) as dual CDK1/GSK-3 kinases inhibitors. Brief studies of their inhibitory potency were realized with a small panel of kinases and showed two compounds (I and II in Figure 2) with a micromolar range inhibitory effect on CDK1 and GSK-3 [7,8]. More recently, the synthesis and the kinase inhibitory potency of various N-arylbenzothieno [3,2-d] pyrimidin-4-amines, and their pyrido and pyrazino analogues (B in Figure 1), have been published.  The overall pharmaceutical interest of all these compounds encouraged us to conceive new series of thiazolo [5,4-f]quinazolines substituted in position 4 of the pyrimidine ring by an aromatic amine and by carboximidamide or amidine groups in position 2 of the thiazole moiety [6,[12][13][14][15] (see general formula C in Figure 1). These compounds were conceived as 6,6,5-tricyclic homologues of the basic 4-aminoquinazoline pharmacophore which is present in approximately 80% of ATP-competitive kinase inhibitors that have received approval for the treatment of cancer [4,5]. Five of the novel thiazolo [5,4-f]quinazoline derivatives prepared displayed single-digit nanomolar or subnanomolar IC50 values and are among the most potent and selective DYRK1A/1B inhibitors disclosed to date [13][14][15].
Returning to our initial work [7,8] and extending the list of targeted kinases, we discovered that compounds I and II exhibit micromolar IC50 values against DYRK1A ( Figure 2). This result suggested the possibility to extend the scope of pertinent kinases that these ligands are able to target.
The results of various docking studies realized to understand the structure-activity relationships (SARs) are represented in Figure 2 and concern the ATP-binding site of GSK-3β [7,8,24]. They suggest that the unencumbered nitrogen in position 6 of the tricyclic core can form a hydrogen bond with the backbone NH-residue of Val135 in the hinge segment. This region of kinases ATP-binding site is considered as a critical H-bonded system with the majority of inhibitors that have been published to date. As described in Figure 2, a benzylcarbimidate function may form polar interaction between nitrogen atom of the imidate and the ammonium group of Lys85. In this hypothesis a bulky substituent at the R 2 position may not fit into the hydrophobic back-pocket of GSK-3β. This region of kinases ATP-binding site is known to not be conserved among kinases and can thus be used to gain affinity as well as selectivity. Figure 1. General formulae A, B and C of previously described kinase inhibitors [6][7][8][9][10][11][12][13][14]. These original heteroaromatics provide new means to target and inhibit some of the abovementioned kinases in the nanomolar range [9][10][11]. Figure 1. General formulae A, B and C of previously described kinase inhibitors [6][7][8][9][10][11][12][13][14]. The overall pharmaceutical interest of all these compounds encouraged us to conceive new series of thiazolo [5,4-f]quinazolines substituted in position 4 of the pyrimidine ring by an aromatic amine and by carboximidamide or amidine groups in position 2 of the thiazole moiety [6,[12][13][14][15] (see general formula C in Figure 1). These compounds were conceived as 6,6,5-tricyclic homologues of the basic 4-aminoquinazoline pharmacophore which is present in approximately 80% of ATP-competitive kinase inhibitors that have received approval for the treatment of cancer [4,5]. Five of the novel thiazolo [5,4-f]quinazoline derivatives prepared displayed single-digit nanomolar or subnanomolar IC50 values and are among the most potent and selective DYRK1A/1B inhibitors disclosed to date [13][14][15].
Returning to our initial work [7,8] and extending the list of targeted kinases, we discovered that compounds I and II exhibit micromolar IC50 values against DYRK1A ( Figure 2). This result suggested the possibility to extend the scope of pertinent kinases that these ligands are able to target.
The results of various docking studies realized to understand the structure-activity relationships (SARs) are represented in Figure 2 and concern the ATP-binding site of GSK-3β [7,8,24]. They suggest that the unencumbered nitrogen in position 6 of the tricyclic core can form a hydrogen bond with the backbone NH-residue of Val135 in the hinge segment. This region of kinases ATP-binding site is considered as a critical H-bonded system with the majority of inhibitors that have been published to date. As described in Figure 2, a benzylcarbimidate function may form polar interaction between nitrogen atom of the imidate and the ammonium group of Lys85. In this hypothesis a bulky substituent at the R 2 position may not fit into the hydrophobic back-pocket of GSK-3β. This region of kinases ATP-binding site is known to not be conserved among kinases and can thus be used to gain affinity as well as selectivity. The overall pharmaceutical interest of all these compounds encouraged us to conceive new series of thiazolo [5,4-f ]quinazolines substituted in position 4 of the pyrimidine ring by an aromatic amine and by carboximidamide or amidine groups in position 2 of the thiazole moiety [6,[12][13][14][15] (see general formula C in Figure 1). These compounds were conceived as 6,6,5-tricyclic homologues of the basic 4-aminoquinazoline pharmacophore which is present in approximately 80% of ATP-competitive kinase inhibitors that have received approval for the treatment of cancer [4,5]. Five of the novel thiazolo [5,4-f ]quinazoline derivatives prepared displayed single-digit nanomolar or subnanomolar IC 50 values and are among the most potent and selective DYRK1A/1B inhibitors disclosed to date [13][14][15].
Returning to our initial work [7,8] and extending the list of targeted kinases, we discovered that compounds I and II exhibit micromolar IC 50 values against DYRK1A ( Figure 2). This result suggested the possibility to extend the scope of pertinent kinases that these ligands are able to target.
The results of various docking studies realized to understand the structure-activity relationships (SARs) are represented in Figure 2 and concern the ATP-binding site of GSK-3β [7,8,24]. They suggest that the unencumbered nitrogen in position 6 of the tricyclic core can form a hydrogen bond with the backbone NH-residue of Val135 in the hinge segment. This region of kinases ATP-binding site is considered as a critical H-bonded system with the majority of inhibitors that have been published to date. As described in Figure 2, a benzylcarbimidate function may form polar interaction between nitrogen atom of the imidate and the ammonium group of Lys85. In this hypothesis a bulky substituent at the R 2 position may not fit into the hydrophobic back-pocket of GSK-3β. This region of kinases ATP-binding site is known to not be conserved among kinases and can thus be used to gain affinity as well as selectivity. As depicted in Figure 3, modulating the size and/or the nature of R 1 and R 2 may have an effect on the affinity of the ligands by allowing cisor trans-spatial positions of these groups in the targeted binding sites. In the latest case, R 2 group will be able to fit into the back-pocket of kinases and concomitantly, the R 2 group present on N 8 may also influence the position of the inhibitor into the enzyme site. As depicted in Figure 3, modulating the size and/or the nature of R 1 and R 2 may have an effect on the affinity of the ligands by allowing cis-or trans-spatial positions of these groups in the targeted binding sites. In the latest case, R 2 group will be able to fit into the back-pocket of kinases and concomitantly, the R 2 group present on N 8 may also influence the position of the inhibitor into the enzyme site. Considering all these facts, the synthetic route to the thiazolo[5,4-f]quinazolin-9(8H)-one scaffold was optimized with the aim of modulating the R 1 and R 2 groups. This was expected by substituting the position 8 of the pyrimidine ring with various alkyl and aryl groups and by introducing various alkyl substituents on the carbimidate groups in position 2 of the thiazole moiety. Concerning this last point, the choice of the aliphatic chains for R 1 was inspired by previous results (see compounds C in Figure 1) showing that small size groups can help to enhance the inhibitory activity against kinases [13].
This paper describes the development of a simple and reliable method allowing the preparation of a library of new thiazolo [5,4-f]quinazolin-9(3H)-ones for which interesting multi-target kinase inhibitory activities were observed. The main part of the chemistry described in this paper was achieved under microwave irradiation as a continuation of our global strategy consisting in the design of appropriate reagents and techniques offering operational, economic, and environmental benefits over conventional methods [25][26][27].

Chemistry
The target molecules were thiazolo [5,4-f]quinazolin-9(8H)-ones substituted in position N 8 (which corresponds to position 4 of the pyrimidine ring) by an aliphatic chain or an aromatic substituent (Scheme 1). In order to reach an efficient route to these various 8-substituted thiazolo [5,4-f] quinazolin-9(8H)-ones, a rational multistep synthesis of a novel polyfunctionalized benzothiazole (see 1 in Scheme 1) has been developed [28][29][30]. This molecular system was conceived to be a versatile efficient precursor to various target molecules. The presence of the carbonitrile function in position 2 of the thiazole ring should allow easy access to (alkyl)carbimidate function and the ortho-aminobenzoïc ester part should access to the target N 8 -substituted pyrimidin-4-one derivatives. Considering all these facts, the synthetic route to the thiazolo[5,4-f ]quinazolin-9(8H)-one scaffold was optimized with the aim of modulating the R 1 and R 2 groups. This was expected by substituting the position 8 of the pyrimidine ring with various alkyl and aryl groups and by introducing various alkyl substituents on the carbimidate groups in position 2 of the thiazole moiety. Concerning this last point, the choice of the aliphatic chains for R 1 was inspired by previous results (see compounds C in Figure 1) showing that small size groups can help to enhance the inhibitory activity against kinases [13].
This paper describes the development of a simple and reliable method allowing the preparation of a library of new thiazolo [5,4-f ]quinazolin-9(3H)-ones for which interesting multi-target kinase inhibitory activities were observed. The main part of the chemistry described in this paper was achieved under microwave irradiation as a continuation of our global strategy consisting in the design of appropriate reagents and techniques offering operational, economic, and environmental benefits over conventional methods [25][26][27].

Chemistry
The target molecules were thiazolo [5,4-f ]quinazolin-9(8H)-ones substituted in position N 8 (which corresponds to position 4 of the pyrimidine ring) by an aliphatic chain or an aromatic substituent (Scheme 1). In order to reach an efficient route to these various 8-substituted thiazolo [5,4-f ]quinazolin-9(8H)-ones, a rational multistep synthesis of a novel polyfunctionalized benzothiazole (see 1 in Scheme 1) has been developed [28][29][30]. This molecular system was conceived to be a versatile efficient precursor to various target molecules. The presence of the carbonitrile function in position 2 of the thiazole ring should allow easy access to (alkyl)carbimidate function and the ortho-aminobenzoïc ester part should access to the target N 8 -substituted pyrimidin-4-one derivatives. Based on our previous studies [25][26][27], the synthesis of the key intermediate 1 was revised and optimized in six steps according to the procedure depicted in Scheme 2. N 2 -Protection of methyl 5-nitroanthranilate (2) [31] provided methyl 2-[di(tert-butoxycarbonyl)amino]-5-nitrobenzoate (3), which was reduced by treatment with ammonium formate in the presence of a catalytic amount of 10% palladium charcoal. The resulting aromatic amine (4) was treated with N-bromosuccinimide (NBS) in DMF to give 5 and 6 (ratio 5/6: 9/1) in a quantitative yield. The mixture of ortho-bromo anilines 5 and 6 was reacted with Appel's salt (4,5-dichloro-1,2,3-dithiazolium chloride) to give intermediate imino-1,2,3-dithiazoles 7 and 8 which were separated and purified by flash-column chromatography on silica gel. The intermediate imine 7 was transformed into the target methyl 6-amino-2-cyanobenzo[d]thiazole-7-carboxylate (1) after N 2 -deprotection (giving 9, 96%) and microwave-assisted copper-mediated cyclization. This synthetic route allowed an efficient and reproducible preparation of 1, in a good overall yield of 43%, helped in some steps by microwave-assisted heating. In terms of efficiency, 20 g of 2-methyl 5-nitroanthranilate (2) may lead to 6 g of polyfunctionalized benzo[d]thiazole 1.  (10) in excellent yield (90%). This efficient synthesis can be performed at the multi-gram scale, enabling preparation of several grams of the key precursor 10. The aforementioned formimidamide was heated at 100 °C under microwave irradiation, in the presence of 1.05 equiv of appropriate amines, in acetic acid. After irradiation times of 6 to 10 min, cyclization of the pyrimidin-4-one part of the tricyclic product allowed access to the expected N 8 -substituted-9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbonitriles (series 11) in moderate to good yields (58%-84%). Considering previous kinetics studies [32], it may be assumed that the mechanism of cyclization occurred via a first attack of the amine on the activated carbon of the amidine. The intermediate triamine species released dimethylamine and cyclized into the expected quinazolin-4-one derivatives (Scheme 3). Based on our previous studies [25][26][27], the synthesis of the key intermediate 1 was revised and optimized in six steps according to the procedure depicted in Scheme 2. N 2 -Protection of methyl 5-nitroanthranilate (2) [31] provided methyl 2-[di(tert-butoxycarbonyl)amino]-5-nitrobenzoate (3), which was reduced by treatment with ammonium formate in the presence of a catalytic amount of 10% palladium charcoal. The resulting aromatic amine (4) was treated with N-bromosuccinimide (NBS) in DMF to give 5 and 6 (ratio 5/6: 9/1) in a quantitative yield. The mixture of ortho-bromo anilines 5 and 6 was reacted with Appel's salt (4,5-dichloro-1,2,3-dithiazolium chloride) to give intermediate imino-1,2,3-dithiazoles 7 and 8 which were separated and purified by flash-column chromatography on silica gel. The intermediate imine 7 was transformed into the target methyl 6-amino-2-cyanobenzo[d]thiazole-7-carboxylate (1) after N 2 -deprotection (giving 9, 96%) and microwave-assisted copper-mediated cyclization. This synthetic route allowed an efficient and reproducible preparation of 1, in a good overall yield of 43%, helped in some steps by microwave-assisted heating. In terms of efficiency, 20 g of 2-methyl 5-nitroanthranilate (2) may lead to 6 g of polyfunctionalized benzo[d]thiazole 1. Based on our previous studies [25][26][27], the synthesis of the key intermediate 1 was revised and optimized in six steps according to the procedure depicted in Scheme 2. N 2 -Protection of methyl 5-nitroanthranilate (2) [31] provided methyl 2-[di(tert-butoxycarbonyl)amino]-5-nitrobenzoate (3), which was reduced by treatment with ammonium formate in the presence of a catalytic amount of 10% palladium charcoal. The resulting aromatic amine (4) was treated with N-bromosuccinimide (NBS) in DMF to give 5 and 6 (ratio 5/6: 9/1) in a quantitative yield. The mixture of ortho-bromo anilines 5 and 6 was reacted with Appel's salt (4,5-dichloro-1,2,3-dithiazolium chloride) to give intermediate imino-1,2,3-dithiazoles 7 and 8 which were separated and purified by flash-column chromatography on silica gel. The intermediate imine 7 was transformed into the target methyl 6-amino-2-cyanobenzo[d]thiazole-7-carboxylate (1) after N 2 -deprotection (giving 9, 96%) and microwave-assisted copper-mediated cyclization. This synthetic route allowed an efficient and reproducible preparation of 1, in a good overall yield of 43%, helped in some steps by microwave-assisted heating. In terms of efficiency, 20 g of 2-methyl 5-nitroanthranilate (2) may lead to 6 g of polyfunctionalized benzo[d]thiazole 1. Treatment of 1 with 1.5 equiv of Vilsmeier-Haack reagent in dichloromethane at room temperature gave (E)-methyl 2-cyano-6-([(dimethylamino)methylene]amino)benzo[d]thiazole-7-carboxylate (10) in excellent yield (90%). This efficient synthesis can be performed at the multi-gram scale, enabling preparation of several grams of the key precursor 10. The aforementioned formimidamide was heated at 100 °C under microwave irradiation, in the presence of 1.05 equiv of appropriate amines, in acetic acid. After irradiation times of 6 to 10 min, cyclization of the pyrimidin-4-one part of the tricyclic product allowed access to the expected N 8 -substituted-9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbonitriles (series 11) in moderate to good yields (58%-84%). Considering previous kinetics studies [32], it may be assumed that the mechanism of cyclization occurred via a first attack of the amine on the activated carbon of the amidine. The intermediate triamine species released dimethylamine and cyclized into the expected quinazolin-4-one derivatives (Scheme 3). Treatment of 1 with 1.5 equiv of Vilsmeier-Haack reagent in dichloromethane at room temperature gave (E)-methyl 2-cyano-6-([(dimethylamino)methylene]amino)benzo[d]thiazole-7-carboxylate (10) in excellent yield (90%). This efficient synthesis can be performed at the multi-gram scale, enabling preparation of several grams of the key precursor 10. The aforementioned formimidamide was heated at 100˝C under microwave irradiation, in the presence of 1.05 equiv of appropriate amines, in acetic acid. After irradiation times of 6 to 10 min, cyclization of the pyrimidin-4-one part of the tricyclic product allowed access to the expected N 8 -substituted-9-oxo-8,9-dihydrothiazolo [5,4-f ]quinazoline-2-carbonitriles (series 11) in moderate to good yields (58%-84%). Considering previous kinetics studies [32], it may be assumed that the mechanism of cyclization occurred via a first attack of the amine on the activated carbon of the amidine. The intermediate triamine species released dimethylamine and cyclized into the expected quinazolin-4-one derivatives (Scheme 3). Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware.

Compound Yield b (%)
Molecules 2016, 21,578 Scheme 3. Synthesis of series 11 compounds and suggested mechanism of c the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile grou was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f] (12a-n) were thus obtained in good to excellent yields ( Table 2). In the cour of various carbimidates were extended to ethyl, isopropyl, and benzyl d procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9 performed with success. This process was helped by the use of methyl 6-amino 7-carboxylate (1) [27], a molecular platform conceived to be a versatile various target molecules. Note that microwave heating was mainly p pressure in a controlled multimode cavity with a microwave power deliver Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. Scheme 3. Synthesis of series 11 compounds and suggested mechanism of cyclization after attack of the primary amine. For yields see Table 1. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d]thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware. At the last stage of the synthesis, transformation of the carbonitrile group into a methylcarbimidate was realized by stirring compounds 11a-n with sodium hydroxide (2.5 M in water) in methanol at room temperature for 1 h (Scheme 4). Methyl 9-oxo-8,9-dihydrothiazolo[5,4-f ]quinazoline-2-carbimidates (12a-n) were thus obtained in good to excellent yields ( Table 2). In the course of SAR studies, libraries of various carbimidates were extended to ethyl, isopropyl, and benzyl derivatives using the same procedure and the appropriate alcohol (Scheme 4).
The synthesis of forty-four 8-susbtituted thiazolo[5,4-f ]quinazolin-9(8H)-one derivatives was performed with success. This process was helped by the use of methyl 6-amino-2-cyano-benzo[d] thiazole-7-carboxylate (1) [27], a molecular platform conceived to be a versatile and efficient precursor to various target molecules. Note that microwave heating was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspects, open-vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the use of common laboratory glassware.  Table 2).
Results given in Table 3 demonstrate that none of the tricyclic derivatives prepared in this work showed significant inhibitory activity against CDK5/p25 and CK1δ/ε. Only three compounds of 11a-c series are described in Table 3 and none of them showed any significant activity against the targeted kinases. These results are consistent with previous studies [7,8].
The results of the kinases inhibition potential obtained with compounds of the 14a-e and 15a-c series were also quite disappointing. Apart from product 14e, which exhibits a good and selective inhibition of GSK-3 (IC50 = 0.029 μM) and product 15a, which revealed a fairly good inhibition of CLK1 (IC50 = 0.043 μM), the IC50 values obtained for the other compounds of these two series (14a-d,  15b and 15c) were not significant.
In general, interesting and significant biological activity of the tested compounds was oriented towards three kinases of the initial panel: CLK1, DYRK1A and GSK-3α/β. Undoubtedly, the most active molecules prepared in this study were series 12 and 13 in which the final carbimidate function was obtained after attack of methanol or benzyl alcohol. Most of these compounds from these two series showed submicromolar activities against CLK1, DYRK1A and GSK-3α/β, except product 12m which was completely inactive (IC50 > 10 μM for the five kinases tested). Curiously, despite the size modification of the carbimidate substituents, similar activity profiles can be observed for the 12 and 13 series (see Table 3 in which the most significant results are underlined in grey and significant IC50 values in the nanomolar range written in bold). It appears that the most significant results were obtained with compounds of the a and d-g series in the two family products (12 and 13).  Table 2).
Results given in Table 3 demonstrate that none of the tricyclic derivatives prepared in this work showed significant inhibitory activity against CDK5/p25 and CK1δ/ε. Only three compounds of 11a-c series are described in Table 3 and none of them showed any significant activity against the targeted kinases. These results are consistent with previous studies [7,8].
The results of the kinases inhibition potential obtained with compounds of the 14a-e and 15a-c series were also quite disappointing. Apart from product 14e, which exhibits a good and selective inhibition of GSK-3 (IC 50 = 0.029 µM) and product 15a, which revealed a fairly good inhibition of CLK1 (IC 50 = 0.043 µM), the IC 50 values obtained for the other compounds of these two series (14a-d,  15b and 15c) were not significant.
In general, interesting and significant biological activity of the tested compounds was oriented towards three kinases of the initial panel: CLK1, DYRK1A and GSK-3α/β. Undoubtedly, the most active molecules prepared in this study were series 12 and 13 in which the final carbimidate function was obtained after attack of methanol or benzyl alcohol. Most of these compounds from these two series showed submicromolar activities against CLK1, DYRK1A and GSK-3α/β, except product 12m which was completely inactive (IC 50 > 10 µM for the five kinases tested). Curiously, despite the size modification of the carbimidate substituents, similar activity profiles can be observed for the 12 and 13 series (see Table 3 in which the most significant results are underlined in grey and significant IC 50 values in the nanomolar range written in bold). It appears that the most significant results were obtained with compounds of the a and d-g series in the two family products (12 and 13). Table 3. Kinase inhibitory activity a,b of the thiazolo [5,4-f ] quinazoline series 11a-c, 12a-n, 13a-h,  14a-e, 15a- Results obtained with compounds 12h-l (CLK1: 0.691 µM < IC 50 < 7.9 µM), DYRK1A (0.2 µM < IC 50 < 3.0 µM) and GSK-33 α/β (0.3 µM < IC 50 < 1.8 µM) demonstrated the necessity to conserve a cyclic alkyl group in position 8 of the tricyclic core (12a and 12d-g), in order to maintain good to very good affinity. The presence of basic groups in N 8 (12h-j) may be correlated with an rather important decrease of affinity, in the same way as the presence of aromatic substituents in position 8 of the thiazolo[5,4-f ]quinazolin-9(8H)-one core (compounds 12k-m). In this last case, it appeared that the nature of the substituents present in the para-position of the phenyl group in N 8 may influence and decrease the IC 50 values measured for the various kinases. Compared with data obtained for compounds in the 12d-g series, it seems to be the consequence to steric influence rather than electronic effects . Compounds 12b, 12c and 13b, 13c were also analyzed and although their inhibitory activity towards the three kinases (CLK1, DYRK1A and GSK-3α/β) was characterized by submicromolar values of IC 50 , their global activity was considered not significant enough to continue further studies.
Comparison of the substituents present on N 8 position of a and d-f series shows mainly a small-size ring (e.g., cyclopropyl for 12a and 13a series) or its equivalent such as isopropyl (12d and 13d), cyclobutyl (12f and 13f) and cyclopentyl (12e and 13e). The size of the cycle present in position N 8 seems to be extremely important, whilst 3-, 4-and 5-membered rings exhibit the best affinity for the target kinases.
Concerning the activity against CLK1, comparison of the results obtained for compounds 12a and 12f (IC 50 = 0.031 µM and 0.091 µM, respectively) with those obtained for 13a and 13f (IC 50 = 0.06 µM and 0.071 µM, respectively), demonstrates that the presence of methyl or benzyl carbimidates at C 2 may have a slightly beneficial effect on the inhibitory activity, accompanied by the same cyclopropyl substituent on N 8 . The same phenomenon is observed when data concerning DYRK1A and GSK-3α/β kinases are compared. The DYRK1A-IC 50 values obtained for 12a, 12d-f and 13a, 13d-f series are mainly in the submicromolar range (0.13 µM < IC 50 < 0.39 µM) except for compounds 12a and 13a (IC 50 = 0.091 µM and 0.06 µM, respectively), 12e and 13e (IC 50 = 0.072 µM and 0.062 µM, respectively) and 13f (IC 50 = 0.00.59 µM) for which nanomolar IC 50 values were observed. These five products show interesting activity against DYRK1A. As described above for CLK1, the interesting results concern the fact that two different carbimidate groups gave similar affinity for the same kinase despite changes in the size of their substituents in position N 8 . This may suggest the existence of a spatial zone in the front pocket of the active site of the kinases which is relatively tolerant to various sizes of the molecules with 3-, 4-or 5-membered cyclolalkyl groups.
In the case of GSK-3α/β, the nanomolar values obtained for compounds 12a, 12d-h and 13a, 13d-h are spectacular and the list of compounds able to show very good affinity for the target kinase was extended to 12d-13d (IC 50 = 0.041 µM and 0.030 µM, respectively) and 12g-13g (IC 50 = 0.030 µM and 0.083 µM, respectively). For GSK-3α/β in particular, the fact that its hydrophobic back-pocket was recognized to be larger than in the case of the two other kinases (CLK1 and DYRK1A) may explain why 12d and 12g show very good affinity for this kinase. It may be suggested that the space available in this back-pocket allowed the molecules to shift and to have a better interaction with GSK-3α/β than in the other enzymes in which hydrophobic back-pocket are known to have a reduced size. In the same time the displacement of the tricyclic core into the site may allow the front pocket to be more tolerant and accept other substituents in N 8 .
All these results demonstrate that it is difficult to define any role for the various substituents located at position 8 of the thiazolo[5,4-f ]quinazolin-9(8H)-one. The presence of small-sized cycloalkyl groups linked to the front pocket is favorable to the development of further SAR studies, whilst the other side of the molecule (carbimidate function) will perhaps serve to discriminate the pertinent kinases such as CLK1, DYRK1A and GSK-3α/β (Scheme 5). Although this needs to be confirmed, the results described in this study indicate clearly that the carbimidate function remains crucial for the multi-target inhibitory activity of kinases with such heterocyclic structures [13][14][15]. towards the three kinases (CLK1, DYRK1A and GSK-3α/β) was characterized by submicromolar values of IC50, their global activity was considered not significant enough to continue further studies.
Comparison of the substituents present on N 8 position of a and d-f series shows mainly a smallsize ring (e.g., cyclopropyl for 12a and 13a series) or its equivalent such as isopropyl (12d and 13d), cyclobutyl (12f and 13f) and cyclopentyl (12e and 13e). The size of the cycle present in position N 8 seems to be extremely important, whilst 3-, 4-and 5-membered rings exhibit the best affinity for the target kinases.
Concerning the activity against CLK1, comparison of the results obtained for compounds 12a and 12f (IC50 = 0.031 μM and 0.091 μM, respectively) with those obtained for 13a and 13f (IC50 = 0.06 μM and 0.071 μM, respectively), demonstrates that the presence of methyl or benzyl carbimidates at C 2 may have a slightly beneficial effect on the inhibitory activity, accompanied by the same cyclopropyl substituent on N 8 . The same phenomenon is observed when data concerning DYRK1A and GSK-3α/β kinases are compared. The DYRK1A-IC50 values obtained for 12a, 12d-f and 13a, 13d-f series are mainly in the submicromolar range (0.13 μM < IC50 < 0.39 μM) except for compounds 12a and 13a (IC50 = 0.091 μM and 0.06 μM, respectively), 12e and 13e (IC50 = 0.072 μM and 0.062 μM, respectively) and 13f (IC50 = 0.00.59 μM) for which nanomolar IC50 values were observed. These five products show interesting activity against DYRK1A. As described above for CLK1, the interesting results concern the fact that two different carbimidate groups gave similar affinity for the same kinase despite changes in the size of their substituents in position N 8 . This may suggest the existence of a spatial zone in the front pocket of the active site of the kinases which is relatively tolerant to various sizes of the molecules with 3-, 4-or 5-membered cyclolalkyl groups.
In the case of GSK-3α/β, the nanomolar values obtained for compounds 12a, 12d-h and 13a, 13d-h are spectacular and the list of compounds able to show very good affinity for the target kinase was extended to 12d-13d (IC50 = 0.041 μM and 0.030 μM, respectively) and 12g-13g (IC50 = 0.030 μM and 0.083 μM, respectively). For GSK-3α/β in particular, the fact that its hydrophobic back-pocket was recognized to be larger than in the case of the two other kinases (CLK1 and DYRK1A) may explain why 12d and 12g show very good affinity for this kinase. It may be suggested that the space available in this back-pocket allowed the molecules to shift and to have a better interaction with GSK-3α/β than in the other enzymes in which hydrophobic back-pocket are known to have a reduced size. In the same time the displacement of the tricyclic core into the site may allow the front pocket to be more tolerant and accept other substituents in N 8 .
All these results demonstrate that it is difficult to define any role for the various substituents located at position 8 of the thiazolo[5,4-f]quinazolin-9(8H)-one. The presence of small-sized cycloalkyl groups linked to the front pocket is favorable to the development of further SAR studies, whilst the other side of the molecule (carbimidate function) will perhaps serve to discriminate the pertinent kinases such as CLK1, DYRK1A and GSK-3α/β (Scheme 5). Although this needs to be confirmed, the results described in this study indicate clearly that the carbimidate function remains crucial for the multi-target inhibitory activity of kinases with such heterocyclic structures [13][14][15]. The first kinase CLK1 is one of the four isoforms (CLK1-4) of the cdc2-like kinase family. In humans, the highest levels of CLK1 expression were found in the brain. It was described that CLK inhibitors may alter the splicing of microtubule-associated protein tau implicated in AD and Parkinson's disease [39]. The second kinase targeted by the compounds described in this study is DYRK1A. Evidence for the role of overexpressed DYRK1A in various neurodegenerative diseases and Down syndrome is now well established and it has become an attractive drug target for numerous The first kinase CLK1 is one of the four isoforms (CLK1-4) of the cdc2-like kinase family. In humans, the highest levels of CLK1 expression were found in the brain. It was described that CLK inhibitors may alter the splicing of microtubule-associated protein tau implicated in AD and Parkinson's disease [39]. The second kinase targeted by the compounds described in this study is DYRK1A. Evidence for the role of overexpressed DYRK1A in various neurodegenerative diseases and Down syndrome is now well established and it has become an attractive drug target for numerous research groups [40][41][42]. The third kinase inhibited by the lead molecules in this study was GSK-3α/β which has definitely gained the attention of research groups and industry. This very versatile kinase has become a key target in type II diabetes, bipolar disorder, cancer, chronic inflammatory and immune disorder and, more importantly, in neurodegenerative diseases, making this enzyme a main target for AD [43].
Because drugs focusing their activity against a single target may generate low benefits, new studies are encouraged in the direction of multi-targeting strategies. Therefore, developing molecules showing submicromolar affinities on a panel of three kinases seems to be of great interest [17]. In this sense the new thiazolo[5,4-f ]quinazolin-9(8H)-one series described in this paper need to be developed for the discovery of valuable and pertinent multi-kinases inhibitors.

General Information
Starting materials were obtained commercially and used without further purification. All reactions were monitored by thin-layer chromatography with silica gel 60 F254 precoated aluminium plates (0.25 mm). Visualization was performed with a UV light at wavelengths of 254 and 312 nm. Purifications were conducted with a flash column chromatography system equipped with a dual UV/Vis spectrophotometer (200-600 nm), a fraction collector (176 tubes), a dual piston pump (1 to 200 mL/min, P max = 15 bar), which allowed quaternary gradients, and an additional inlet for air purge. Melting points of solid compounds were measured with a SMP3 Melting Point instrument (STUART, Bibby Scientific Ltd, Roissy, France) with a precision of˘1.5˝C. IR spectra were recorded with a Spectrum 100 Series FTIR spectrometer (PerkinElmer, Villebon S/Yvette, France). Liquids and solids were investigated with a single-reflection attenuated total reflectance (ATR) accessory; the absorption bands are given in cm´1. NMR spectra ( 1 H and 13 C) were acquired at 295 K using a WP 300 spectrometer an AVANCE 300 MHz spectrometer (Bruker, Wissembourg, France) at 300 and 75.4 MHz, using TMS as an internal standard. Coupling constants J are in Hz, and chemical shifts are given in ppm. Signals in 13 C spectra were assigned based on the result of 13 C DEPT135 experiments (see Supplementary Materials). Mass spectrometry was performed by the Mass Spectrometry Laboratory of the University of Rouen. The mass spectra [ESI, EI, and field desorption (FD)] were recorded with a LCP 1er XR spectrometer (WATERS, Guyancourt, France). Microwave experiments were conducted in a commercial microwave reactor especially designed for synthetic chemistry. Start S TM (Milestone S.r.l., Bergamo, Italy) is a multi-mode cavity with a microwave power delivery system ranging from 0 to 1200 W. The temperatures of the reactions were mainly monitored via contact-less infrared pyrometer which was calibrated in control experiments with a fibre-optic contact thermometer protected in a Teflon coated ceramic well inserted directly in the reaction mixture. Open vessel experiments were carried out in a 50-250 mL round bottom flask fitted with a reflux condenser. The vessel contents were stirred by means of an adjustable rotating magnetic plate located below the floor of the microwave cavity and a Teflon-coated magnetic stir bar inside the vessel. Temperature and power profiles were monitored in both cases through the EASY-Control software provided by the manufacturer (Milestone S.r.l., Bergamo, Italy). The times indicated in the various protocols are the times measured when the mixtures reached the programmed temperature after a ramp period of 2 min.  (20 mL) was added Vilsmeier-Haack reagent (0.824 g, 6.44 mmol, 1.5 equiv) at room temperature. The resulting mixture was stirred at room temperature for 2 h. On completion, the crude mixture was diluted with methylene chloride (40 mL) and a saturated aqueous solution of NaHCO 3 (40 mL). After 15 min of stirring, the product was extracted twice with methylene chloride.
8-Cyclopropyl-9-oxo-8,9-dihydrothiazolo [5,4- 12a-n, 13a-h, 14a-e and 15a-c from 11a-n To a stirred solution of 11a-n (0.25 mmol, 1 equiv) in appropriate alcohol (2.5 mL) was added an aqueous solution of sodium hydroxyde (2.5 M, 100 µL, 1.0 equiv) and the resulting mixture was stirred at room temperature for 1 h under argon atmosphere. The solvent was removed under vacuum and the crude residue was adsorbed on Celite ® and purified by flash chromatography on silica gel with methylene chloride/methanol (100:0 to 2:98; v/v) as eluent to furnish the desired carbimidate compound. particularly promising for the development of new multi-target inhibitors of kinases. The most active compounds showed nanomolar IC 50 values for CLK1, DYRK1A and GSK-3α/β over the other tested enzymes. Although affinities of these compounds for these three kinases are not negligible, optimization is still needed and this paper allows us to consider further SAR studies for the design of more efficient inhibitors of these targeted kinases.