Design and Synthesis of Thiazolo[5,4-f]quinazolines as DYRK1A Inhibitors, Part I

The convenient synthesis of a library of novel 6,6,5-tricyclic thiazolo[5,4-f] quinazolines (forty molecules) was achieved mainly under microwave irradiation. Dimroth rearrangement and 4,5-dichloro-1,2,3,-dithiazolium chloride (Appel salt) chemistry were associated for the synthesis of a novel 6-aminobenzo[d]thiazole-2,7-dicarbonitrile (16) a versatile molecular platform for the synthesis of various bioactive derivatives. Kinase inhibition of the final compounds was evaluated on a panel of four Ser/Thr kinases (DYRK1A, CDK5, CK1 and GSK3) chosen for their strong implications in various regulation processes, especially Alzheimer’s disease (AD). In view of the results of this preliminary screening, thiazolo[5,4-f]quinazoline scaffolds constitutes a promising source of inspiration for the synthesis of novel bioactive molecules. Among the compounds of this novel chemolibrary, 7i, 8i and 9i inhibited DYRK1A with IC50 values ranging in the double-digit nanomolar range (40, 47 and 50 nM, respectively).


Introduction
Kinases are one of the largest enzyme families of the genome. More than 500 kinases play an important role in the regulation of most cellular processes. These enzymes are involved in all major diseases, including cancer, neurodegenerative disorders and cardiovascular diseases [1][2][3]. Our research groups are mainly invested in the synthesis of C,N,S-or C,N,O-containing heterocyclic precursors of bioactive molecules able to modulate the activity of kinases in signal transduction [4][5][6][7][8].
In the course of our work based on microwave-assisted chemistry, we described ten years ago the multistep synthesis of the 8H-thiazolo [5,4-f]quinazolin-9-ones (A) [9,10]. Brief studies of their structure-activity relationships as dual CDK1/GSK-3 kinases inhibitors were described [7]. At that time, the inhibitory potency of the final products was evaluated and some products showed a micromolar range affinity against DYRK1A [11]. More recently, the synthesis and the kinase inhibitory potency of various benzo-, pyrido-and pyrazinothieno [3,2-d]pyrimidines derivatives (B), have been published. Kinase inhibition of the compounds was evaluated on Ser/Thr kinases (CDK5, GSK3, DYRK1A, CLK1 and CK1) selected for their strong implications in various human pathologies, especially in AD [3].
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 groups in position 2 of the thiazole moiety (see general formula C in Scheme 1).

Scheme 1. Structures of previous molecules which inspired the current work.
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 [11]. The aromatic amine groups linked to the main thiazoloquinazoline structure were selected because of their frequent presence in drugs or drug candidates [11]. On the other side of the target molecules, the aliphatic chains of the carboximidamide groups were also chosen because of their frequent presence in many drugs.
This paper describes the development of a simple and reliable method that allows the preparation of a library of new thiazolo [5,4-f]quinazolines for which interesting 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 which consists to design adapted reactants and techniques offering operational, economic, and environmental benefits over conventional methods [12].

Synthesis
The target molecules we studied were thiazolo [5,4-f]quinazolines (C) substituted in position 4 of the pyrimidine ring (which corresponds to position 9 of the tricyclic compound) by an aromatic amine. The retrosynthetic pathway depicted in Scheme 2 was directly inspired by our previous work on the synthesis of various thiazoloquinazoline isomers [13,14] and on general access to pyrimidine-condensed heterocyclic compounds [15]. It suggested introducing the thiazole ring via a copper(I)-mediated cyclization of ortho-brominated N-arylimino-1,2,3-dithiazoles intermediates. The latter would be isolated after condensation of 4,5-dichloro-1,2,3-dithiazolium chloride (Appel salt) [16] with a key N 2 -protected brominated aminoanthranilonitrile. The synthesis of the final pyrimidinic structures was envisioned via a microwave-assisted thermal-sensitive Dimroth rearrangement. [17] A nucleophilic attack of intermediate amidines by various aromatic amines would give the expected tricyclic compounds. This fast and convenient procedure was recently explored for the design of novel bioactive 4-anilinoquinazolines [4,5,17]. Scheme 2. General retrosynthetic pathways envisioned for this work.
The synthetic route described in Scheme 2 was applied to the preparation of some of our target molecules. It started from 2-amino-5-nitroanthranilonitrile (1) which was treated for 15 min at 105 °C with N,N-dimethylformamide dimethyl acetal (DMFDMA) under microwave irradiation. The resulting N,N-dimethylformamidine 2 was heated with the appropriate aromatic amine in the presence of acetic acid at 118 °C for 20 min. Compounds 3a-d were then obtained in two steps in a good average yield of 85%.
In order to isolate the desired aryliminodithiazoles, 4-substituted-6-nitroquinazolinones 3a-c were first reduced into 6-amino derivatives 4a-c by transfer hydrogenation using ammonium formate in refluxing ethanol. Expectedly, in these conditions, the palladium-catalyzed reduction of the nitro group of intermediate 3d also provoked the protodehalogenation of the chloride atom of the 3-chloro-4-fluoroaniline moiety. This difficulty was circumvented when the reduction of 3d was achieved with iron and acetic acid in refluxing ethanol to give 4d in excellent yield (99%).
Compounds 4a-d were treated with bromine in acetic acid to yield the ortho brominated imines 5a-d. In the case of 4b, a polybrominated by-product 5e was obtained along with 5b whatever the conditions tested. Unfortunately both derivatives 5b and 5e were inseparable under convenient conditions. Another route consisting in preliminary bromination of the starting 5-nitroanthranilonitrile (1) was experimented. Unfortunately, whatever the bromination method, only 3-bromo-5-nitroanthranilonitrile (1b) was detected (see alternative route in Scheme 3).

Scheme 3.
Synthetic routes experimented for the access to the target compounds (series 7-10).
Despite its effectiveness, this synthesis presents some limitations. Each modification of the substituent in N 3 of the pyrimidine ring (e.g., 3a-d) generates three intermediates (e.g., 4a-d, 5a, c and d and 6a-d in this study) for which synthetic and biological significance are not really established yet. The second drawback of this synthetic route lies in the reduction and bromination steps both of which require being adapted to the aromatic substituent of the intracyclic N 3 -nitrogen atom. As an example, compound 5b was never isolated in analytically pure form and the synthesis of 8 via this intermediate was judged infeasible.
On a practical aspect, this synthetic sequence is easily upscalable and 10 g of 2-amino-5-nitrobenzonitrile (1) led to 2 g of 16 in an average 23% yield. This new compound 16 can be considered as a molecular platform that can be employed in new areas of investigation and prove its utility for the synthesis of innovative molecular systems with potent biological applications. Indeed, the versatile carbonitrile function in position 2 of the thiazole ring may allow the synthesis of various amidine, imidazoline and imidate derivatives. On the other side the 2-aminobenzonitrile moiety offers a large panel of possibilities for extension of the aromatic structure with a heterocyclic core such as a pyrimidine (Scheme 5). The synthesis of the target molecules was carried on by treatment of 16 with DMFDMA under microwave irradiation at 70 °C to give (E)-N'- (2,7-dicyanobenzo[d]thiazol-6-yl)-N,Ndimethylformimidamide (17) in good yield (86%). Cyclization of formimidamide 17 into thiazolo [5,4f]quinazoline-2-carbonitriles was accomplished via thermal Dimroth rearrangement using 1.5 eq of the appropriate aniline in acetic acid under microwave irradiation at 118 °C for short times and gave compounds 7-10 in good yields (71%-85%). This method constitutes a versatile route to various compounds, especially 8 which was obtained in excellent yield (98%).
In order to enhance the chemodiversity of the thiazolo [5,4-f]quinazolines studied, the reactivity of the aromatic carbonitriles 7-10 was tested against a panel of substituted amines (mainly alkylamines) inspired by our previous studies [4,5]. A new set of 27 novel carboximidamides 7a-g, 8a-f, 9a-g and 10a-g was prepared by stirring overnight at room temperature carbonitriles 7-10 with the appropriate amines (1.2 eq) in dry THF under argon. The chemical structures and yields obtained for the synthesis of the four prepared series (7a-g-10a-g) are shown in Table 1.
The key molecules 7-10 were also heated with sodium hydroxide (2.5 N in water) or with a solution of sodium methoxide in methanol to give respectively amides 7h-10h and methyl imidates 7i-10i in good to excellent yields (Scheme 6).
Note that microwave heating was mainly realized at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Concerning the technical aspect, the choice of a reactor able to work at atmospheric pressure was guided by our previous experience in microwave-assisted heterocyclic synthesis, especially in the chemistry of quinazolines [6,9]. Open vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the possibility to use current laboratory glassware. Our choice was also guided by a recent work describing the tendency of pressure to accumulate when a product as DMFDMA was heated in pressurized vials, especially under microwaves [18]. In most cases of our study, an irradiation of 800 W was enough to efficiently reach the programmed temperature. This parameter was mainly monitored via a contactless-infrared pyrometer which was calibrated in control experiments with a fiber-optic contact thermometer. Scheme 6. Synthesis of thiazolo [5,4-f]quinazoline-2-carbonitriles 7-10 and their derivatives via transformation of the carbonitrile functions in carboxamidines (a-g), amides (h) or imidates (i).

Biological Studies
Compounds of series 7 (7, 7a-i), series 8 (8, 8a-i), series 9 (9, 9a-i) and series 10 (10, 10a-i) were tested on four different in vitro kinase assays (CDK5/p25 (cyclin-dependent kinase), CK1δ/ε (casein kinase 1), GSK3α/β(Glycogen Synthase Kinase 3) and DYRK1A (dual-specificity, tyrosine phosphorylation regulated kinase) to evaluate their inhibition potency [19][20][21][22][23]. These four kinases are all involved in Alzheimer's disease (AD), a multi-kinase inhibitor able to target two or three of them could be quite desirable. This is linked to the fact that it is still not known whether any of these four kinases plays a more prominent role in Alzheimer's disease than the others and, consequently, which one should therefore preferably be targeted. In pathological situations such kinases are overexpressed and-activated, this fact justify the interest of multi-target-directed ligands (MTDLs) while complete inhibition is likely to be detrimental.
All compounds were first tested at a final concentration of 10 µM. Compounds showing less than 50% inhibition were considered as inactive (IC50 > 10 µM). Compounds displaying more than 50% inhibition at 10 µM were next tested over a wide range of concentrations (usually from 0.01 to 10 µM), and IC50 values were determined from the dose-response curves (Sigma-Plot). Harmine is a β-carboline alkaloid known to be a potent inhibitor of DYRK1A [24]. Leucettine L41 is also a potent DYRK1A inhibitor derived from a marine natural product, Leucettamine B [25,26]. They were tested as positive controls and their IC50 values were compared with those obtained for the compounds under study.
On a general aspect, compounds a-f of series 7, 9 and 10 were also judged inactive against DYRK1A, except for some compounds (8b-8e) of series 8 for which micromolar IC50 values were observed. Note that among the four families of tested molecules, all compounds of a-f series were found relatively active (mainly in the micromolar range) against GSK3α/β. All these compounds were substituted with bulky aminoethyl groups on the carboximidamide function of the final structures. The large size of these groups appeared to influence the reactivity of the products against the four kinases tested, conferring them a relative selectivity for GSK3α/β (1.10 μM < IC50 < 7.00 μM). Table 2. Kinase inhibitory activity a,b,c of the four thiazolo [5,4- Among the compounds tested, the two most interesting series are 8 and 9 which showed submicromolar values against GSK3 α/β. From this point of view, series 8 is really promising with micromolar range activities against DYRK1A (6.5 μM < IC50 < 1.05 μM) and submicromolar IC50 values against GSK3α/β (0.25 μM < IC50 < 0.97 μM).
Undoubtedly, the most active molecules prepared in this study were series g-i of the four family of thiazolo [5,4-f]quinazolines (7)(8)(9)(10). The latter showed spectacular submicromolar activities against DYRK1A (0.04 μM < IC50 < 0.70 μM) and GSK3α/β kinases (0.16 μM < IC50 < 0.77 μM) with a marked preference for the first one, respectively. The DYRK1A IC50 values obtained for 7i, 8i and 9i are situated in the double-digit nanomolar range (40, 47 and 50 nM, respectively) demonstrating that small-sized groups linked to the thiazole ring were able to induce a dramatic enhancement of the inhibitory activity against DYRK1A.
Taking into account these preliminary results, defining a specific role of the aromatic substituents of the amine located at position 4 of the pyrimidine ring remains challenging. Nevertheless, the presence of substituting groups on the aromatic moiety seemed to have a positive effect on the inhibitory activity of the studied compounds. Without being able to establish a general rule, the presence of substituents in positions 2 and 4 of the aromatic chain (series 9) has a rather beneficial effect compared to substituents in position 3 and 4 (series 8 and 10).
The most active thiazoloquinazolines were less potent and specific against DYRK1A (Table 2) compared to harmine and leucettine L41 [25][26][27] but constitute a promising source of inspiration for the synthesis of novel bioactive molecules. Our results confirm that the thiazolo [5,4-f]quinazoline scaffold has a great potential in the development of potent inhibitors of DYRK1A and GSK3α/β kinases that are involved in many neurodegenerative diseases and cancers. Lead compounds 7i, 8i and 9i presented in this paper allow us to consider further structure-activity relationships for the design of more efficient and selective inhibitors of these targeted kinases.

General Information
All reactions were carried out under inert atmosphere of argon or nitrogen and monitored by thin-layer chromatography with silica gel 60 F254 pre-coated aluminum plates (0.25 mm). Visualization was performed with a UV light at 254 and 312 nm. Purifications were carried out on an Armen Instrument Spot 2 Flash System equipped with a dual UV-Vis spectrophotometer (200-600 nm), a fraction collector (192 tubes), a dual piston pump (1 to 250 mL/min, Pmax = 50 bar/725 psi) allowing quaternary gradients and an additional inlet for air purge. Samples can be injected in liquid or solid mode. Purification was edited and monitored on an integrated panel PC with a touch screen controlled by Armen Glider Flash v3.1d software [28]. Biotage SNAP flash chromatography cartridges (KP-Sil, normal phase, 10 to 340 g) were used for the purification process. Melting points of solid compounds were measured on a WME Köfler hot-stage with a precision of +/−2 °C and are uncorrected. IR spectra were recorded on a PerkinElmer Spectrum 100 Series FT-IR spectrometer. Liquids and solids were applied on the Single Reflection Attenuated Total Reflectance (ATR) Accessories. Absorption bands are given in cm −1 .
1 H/ 19 F/ 13 C-NMR spectra were recorded on a Bruker DXP 300 spectrometer at 300, 282 and 75 MHz respectively. Abbreviations used for peak multiplicities are s: singlet, d: doublet, t: triplet, q: quadruplet and m: multiplet. Coupling constants J are in Hz and chemical shifts are given in ppm and calibrated with DMSO-d6 or CDCl3 (residual solvent signals). Mass spectra analysis was performed by the Mass Spectrometry Laboratory of the University of Rouen. Mass spectra (EI) were recorded with a Waters LCP 1er XR spectrometer.
Dichloromethane was distilled from CaH2 under argon. NBS was recrystallized in water. Other reagents and solvents were used as provided by commercial suppliers.
Appel salt was prepared according to literature procedure [16] by addition of chloroacetonitrile (1 eq) to a solution of sulfur dichloride (5 eq) in dichloromethane (50 mL). Adogen™ (3-4 drops Microwave experiments were conducted at atmospheric pressure in a commercial microwave reactors especially designed for synthetic chemistry. Time indicated in the various protocols is the time measured when the mixtures reached the programmed temperature after a ramp period of 2 min. RotoSYNTH™ (Milestone S.r.l. Italy) is a multimode cavity with a microwave power delivery system ranging from 0 to 1200 W. Open vessel experiments were carried out in round bottom flask (from 25 mL to 4 L) fitted with a reflux condenser. The temperature was monitored via a contact-less infrared pyrometer (IRT) and fiber-optic contact thermometer (FO). Temperature, pressure and power profiles were edited and monitored through the EASY-Control software provided by the manufacturer . Compounds 7a-i, 8a-i,  9a-i and 10a-i, 16 and 17 were described in a previous patent application [29]; to help readers, physicochemical data of these compounds are added in the following experimental part.

Conclusions
The convenient synthesis of a forty molecule library of novel 6,6,5-tricyclic thiazolo [5,4-f]-quinazolines was realized under microwave irradiation associating Dimroth rearrangement for construction the pyrimidine part and 4,5-dichloro-1,2,3,-dithiazolium chloride (Appel salt) chemistry for introducing the thiazole ring to its quinazoline partner. Our work allowed to prepare in an efficient and reproducible multistep synthesis a novel 6-aminobenzo[d]thiazole -2,7-dicarbonitrile (16) which can be considered as a very powerful molecular platform for the synthesis of various bioactive derivatives. On chemical and practical aspects this article is a further example illustrating how microwave heating can be a very powerful tool for medicinal chemistry.
The inhibitory potency of the final products against a panel of four kinases was evaluated. In view of the results of this preliminary study, we consider that the thiazolo [5,4-f]quinazoline derivatives (series 7-10) constitute a promising source of inspiration for the synthesis of novel bioactive molecules. Novel synthetic transformations will be explored and factors governing the dual activity of the compounds toward DYRK1A and GSK3 will be further investigated.