Synthesis and biological evaluation of new thiazolo [5,4-f]quinazolines as serine/threonine kinases inhibitors

In our continuous effort aiming at preparing novel heterocyclic scaffolds able to modulate the activity of kinases in signal transduction, thiazolo[5,4f]quinazolines were particularly studied. This presentation describes a novel strategy for a convenient structure-activity-relationship study towards five serine/threonine kinases (CDK1/cyclin B, CDK5/p25, DYRK1A, CK1, and GSK-3α/β) involved in Alzheimer’s disease. The chemical highlight of this work was the use of Appel salt (4,5-dichloro-1,2,3dithiazolium chloride) for the conception of 6-amino-2-cyanobenzo[d]thiazole-7carboxylate derivatives as a versatile molecular platform from the 5-nitroanthranilic acid. Thus, introduction of various aliphatic, aromatic or amino substituents at position 8 was best achieved by one-pot DMFDMA-mediated cyclisation. Transformation of carbonitrile group into various chemical functions (e.g. imidate, ester, amidine...) allowed the efficient preparation of a library of novel thiazoloquinazoline derivatives. The first biological results have identified great and selective inhibition against DYRK1A and DYRK1B. The more active compounds are imidate derivatives exhibiting inhibitory activity in a subnanomolar range against DYRK1A.


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. 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, and especially Ser/Thr kinases (CDK5, GSK3, CLK1 and CK1) and dual-specificity kinases (DYRK family), selected for their strong implication in various human pathologies, especially in Alzheimer disease and cancer.

Introduction
In the course of our work, we described ten years ago the synthesis of the 8Hthiazolo[5,4-f]quinazolin-9-ones (A). Brief studies of their structure-activity relationships as dual CDK1/GSK-3 kinases inhibitors were described. More recently, the synthesis and the kinase inhibitory potency of various benzo-, pyrido-and pyrazinothieno [3,2d]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.
Pursuing our studies, we conceived 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).
The aromatic amine groups linked to the main thiazoloquinazoline structure were selected because of their frequent presence in drugs or drug candidates. General retrosynthetic pathways envisioned for this work.

First route
Second route Despite its effectiveness, the synthesis presented above has some limitations. A) Each modification of the substituent in N 3 of the pyrimidine ring generates three intermediates for which biological significance is not established. B) Reduction and bromination steps require being adapted to the aromatic substituent of the intracyclic N 3 -nitrogen atom.
C) It implied synthesis of a versatile platform: Possible transformations of benzothiazole 16 as a versatile molecular platform.
Chemical structures and yields obtained for the synthesis of the four series (7a-g-10a-g) 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 (cyclindependent 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.
The DYRK1A IC 50 values obtained for 7i, 8i and 9i are situated in the doubledigit 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.
The previous part of this showed that lead compounds possess a methylcarbimidate function in position 2 of the thiazole ring, associated with an N-aryl substituent on position 9 of the thiazolo[5,4-f]quinazoline scaffold (compounds C).
The overall potential therapeutic interest of these compounds encouraged us to extend this series of thiazolo[5,4-f]quinazolines by substituting the position 4 of the pyrimidine ring with various aromatic amines and by leaving a methyl carbimidate group in position 2 of the thiazole moiety..  Table 1; (c) NaOMe (0.5 M in MeOH), MeOH, 65°C (μw), 30 min, for yields see Table. Synthesis of 7, 8 and 9 series (C) via transformation of 4, 5 and 6 series Our choice was also guided by the tendency of pressure to accumulate when a product as DMF/DMA was heated into pressurized vials, especially under microwaves.
In the main part of reactions studied, 600-800 W irradiation 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.

Note concerning microwave-assisted methods used in this work
Microwave heating in this work was mainly performed at atmospheric pressure in a controlled multimode cavity with a microwave power delivery system ranging from 0 to 1200 W (Milestone). Open vessel microwave experiments have some advantages, such as the possibility of easier scale-up and the possibility to use current laboratory glassware.
Results concerning EHT 5372 and other derivatives on the inhibition of DYR1B/Mirk and quiescence of cancer cells : Results concerning EHT 1610 and DYRK1A : Conclusion 25 These results confirm that the thiazolo[5,4-f]quinazoline scaffold has a great potential in the development of novel and highly potent dual inhibitors of DYRK1A and DYRK1B kinases that are involved in many neurodegenerative diseases (AD and other tauopathies), in genetic disease (DS), in oncology, and in diseases involving abnormal pre-mRNA splicing.