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Exploring the Synthesis of New 1-(4-Substitutedphenylamino) imidazo[1,5-a]indol-3-one Derivatives as Cyclized Analogs of Leucettines

New 1-arylaminoimidazo[1,5 -a ]indol-3-ones were synthesized as cyclized derivatives of leucettine L41, a low molecular weight inhibitor of the DYRKs/CLKs protein kinases with potential applications in Alzheimer's disease and Down syndrome. In this first approach, access to the desired 1-aminoimidazo[1,5- a ]indol-3-ones involved 5 steps and was explored with a series of various primary amines and polar secondary amines in order to introduce molecular diversity on N -1 position. The 5 step synthesis of the 1-arylaminoimidazo[1,5 -a ]indol-3-ones was achieved and the limiting step of this process was the final cyclization via a sulphur/nitrogen displacement from methylsulfanyl thiourea intermediates. Good results were obtained for isothioureas derived from primary amines. The 1-arylaminoimidazo[1,5 -a ]indol-3-ones were evaluated on a panel of five protein kinases (DYRK1A, CK1, CDK5/p25, GSK3   and CLK1).

. A detailed structure/activity relationship (SAR) study led to the selection of leucettine L41 ( Figure 1) as a potent inhibitor of DYRKs and CLKs. To understand the kinase/inhibitor selectivities and to generate models for structure-based optimization, leucettine L41, was co-crystallized with DYRK1A and found to bind in the ATP pocket of this kinase by two direct polar interactions and hydrogen bonds (Debdab et al., 2011). Considering these biological results, particularly the hydrogen bonding network in the co-crystal structure, it was of interest to explore the building of 1-phenylamino imidazo [1,5-a]indol-3-one ( Figure 1) which can be considered as an analog of leucettine L41 obtained by the ring closure of N-C atoms between the N-1 position of the 2-aminoimidazoline-4-one platform and the C-6 position of the 1,3-benzodioxol-5-ylidene moiety. Here, we describe the synthesis of new 2-phenylamino imidazo[1,5-a]indol-3-ones without substituents on the indolone moiety and evaluate their effects on five purified protein kinases.

Materials and instruments
All reagents and solvents were purchased from Acros, Aldrich Chimie, and Fluka France and were used without further purification. Solvents were evaporated with a BUCHI rotary evaporator. Melting points were determined on a Kofler melting point apparatus and were uncorrected. 1 H NMR spectra were recorded on BRUKER AC 300 P (300 MHz) spectrometer, 13 C NMR spectra on BRUKER AC 300 P (75 MHz) spectrometer. Chemical shifts are expressed in parts per million downfield from tetramethylsilane as an internal standard. Data are given in the following order: d value, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), number of protons, coupling constants J is given in Hertz. The mass spectra (HRMS) were taken respectively on a MS/MS ZABSpec Tof Micromass (EBE TOF geometry) at an ionizing potential of 8 eV and on a VARIAN MAT 311 at an ionizing potential of 70 eV in the "Centre Régional de Mesures Physiques de l'Ouest" (CRMPO, Rennes). Elemental analyses were performed on a Carlo Erba 1106 apparatus.
In a 50 ml two-necked round-bottomed flask provided with a magnetic stirrer and condenser, a suspension of commercial indole-2-carboxylic acid 1 (1 g, 6.2 mmol) was vigorously stirred at room temperature in 25 ml of benzene. To this mixture was added dropwise phosphorous trichloride [0.85 g, 0.54 ml, 6.2 mmol for compounds 6(a-e) or 1.7 g, 1.08 ml, 12.4 mmol for compounds 6(f,g)] during 30 min. at 25°C under a stream of Ar. The reaction mixture was heated in oil bath at 80°C during 1 hr. After cooling down to room temperature, the reaction mixture was filtered on a Buchner funnel (porosity N°4) and the resulting filtrate was concentrated until a final volume of 5 ml in a rotary evaporator under reduced pressure. This concentrated solution was poured in a 50 ml round bottomed flask provided with a magnetic stirrer and condenser. To this solution were added successively 30 ml of acetone p.a. and potassium thiocyanate (603 mg, 6.2 mmol). This suspension was stirred at 25°C during 1 hr and primary amine 4 (6.2 mmol) or morpholine 5a or 4-methylpiperazine 5b (6.2 mmol) was added in one portion. The reaction mixture was stirred vigorously at 25°C during 1 hr, then the solvent was removed in a rotary evaporator under reduced pressure. The resulting crude oil crystallized at standing room temperature that gave the desired product 6 as yellowish powder, which was submitted to purification by recrystallization in absolute ethanol (1-3 ml).

Standard procedure for the synthesis of methylsulfanyl derivatives 7(a-g).
To a solution of compound 6 (1.81 mmol) in acetone p.a. (5 ml) mixed in a 50 ml round-bottomed flask provided with a magnetic stirrer and a condenser, was added successively methyl iodide MeI (770 mg, 335 µl, 5.43 mmol, 3 equiv) and anhydrous potassium carbonate K 2 CO 3 (250 mg, 1.81 mmol). This reaction mixture was heated at 56°C in oil bath during 60 min. under vigorous magnetic stirring. After cooling down to room temperature, 35 ml of deionized water was poured in the reaction mixture, then the resulting suspension was stored in a refrigerator (4°C) during 12 hr. The crystallized compound 7 was collected by filtration on a Buchner funnel (porosity N°4) and was dried under high vacuum (10 -2 Torr) at 25°C for 1 hr. The product 7 was further used without purification.
In a 50 ml round-bottomed flask provided with a magnetic stirrer and condenser was added successively compound 7 (3 mmol), 5 ml of anhydrous dimethylformamide Me 2 NCHO and lithium hydride LiH [powder, 30 mesh, 95%] (50 mg, 6.3 mmol, 2.1 equiv). The reaction mixture was stirred vigorously at room temperature under a stream of argon during 6 hr. After addition of cold deionized water (35 ml), the resulting reaction mixture was stored in a refrigerator (4°C) during 12 hr. The insoluble compound 8 was collected by filtration on a Buchner funnel (porosity N°4) and washed with deionized water (3x10 ml). The crude product 8 was purified by recrystallization in absolute ethanol and dried under high vacuum (10 -2 Torr) at 25°C for 2 hrs that gave the desired product 8 as yellowish powder.

Kinase preparations and assays
Kinase activities for each enzyme were assayed in buffer A or B, with their corresponding substrates, in the presence of 15 µM ATP in a final volume of 30 µL. After 30 min incubation at 30°C, the reaction was stopped by harvesting, using a FilterMate harvester (Packard), onto P81 phosphocellulose papers (GE Healthcare) which were washed in 1% phosphoric acid. Scintillation fluid was added and the radioactivity measured in a Packard counter. Blank values were subtracted and activities calculated as pmoles of phosphate incorporated during the 30 min incubation. The activities were expressed in % of the maximal activity, i.e. in the absence of inhibitors. Controls were performed with appropriate dilutions of DMSO.
CDK5/p25 (human, recombinant) was prepared as previously described (Leclerc et al., 2001). Its kinase activity was assayed in buffer B, with 1 mg histone H1/ml, in the presence of 15 µM [γ-33 P] ATP (3,000 Ci/mmol; 10 mCi/ml) in a final volume of 30 µl. After 30 min incubation at 30°C, 25 µl aliquots of supernatant were spotted onto 2.5 x 3 cm pieces of Whatman P81 phosphocellulose paper, and, 20 s later, the filters were washed five times (for at least 5 min each time) in a solution of 10 ml phosphoric acid/liter of water. The wet filters were counted in the presence of 1 ml ACS (Amersham) scintillation fluid.
DYRK1A (rat, recombinant, expressed in Escherichia coli as a GST fusion protein, provided by Dr. W. Becker) was purified by affinity chromatography on glutathione-agarose and assayed as described for CDK5/p25 using myelin basic protein (1 mg/ml) as a substrate.

RESULTS AND DISCUSSION
For the preparation of the cyclized derivative of leucettine L41, the planned retrosynthesis of 1-aminoimidazo[1,5a]indol-3-ones ( Figure 2) involved firstly, a ring closure by intramolecular sulphur/nitrogen displacement; secondly, activation of the carbon sulphur double bond by classical S-alkylation with an halogenoalcane; thirdly, thiourea synthesis by addition of primary or secondary amine on isothiocyanate function and fourthly, nucleophilic addition of thiocyanate anion on the indole-2-carbonyl halide In order to synthesize the desired new 1aminoimidazo[1,5-a]indol-3-ones 8 (Scheme 1), commercial indole-2-carboxylic acid 1 was treated successively by phosphorous trichloride (Katritzky et al., 2004) under reflux at 85°C during 2 hrs followed by addition of potassium isothiocyanate in acetone at room temperature for a supplementary reaction time of 1 hr (Abu-El-Halawa et al., 2008). Initial attempts to isolate the indole-2-carbonyl isothiocyanate 3 for complete caracterization by 1 H, 13 C NMR and HRMS analyses failed. This can be explained by the instability of intermediate 3 in the reaction medium during its preparation (Kutschy et al., 2000).
To obtain the desired product N,N'-disubstituted thioureas 6 from indole-2-carboxylic acid 1 in a reasonable yield, we decided to avoid isolation and purification of the indole-2carbonyl chloride 2 and indole-2-carbonyl isothiocyanate 3 intermediates due to their instability under the hydrolytic work-up conditions. With the aim of introducing diversity on the N-1 position of the desired 1-aminoimidazo[1,5-a]indol-3-ones 8, we examined the reactivity of indole-2-carbonyl isothiocyanate 3 with five primary amines 4(a-c) (i.e. p-toluidine 4a, aniline 4b, 4fluoroaniline 4c, 3-fluoroaniline 4d and benzylamine 4e) and two polar secondary cyclic amines 5(a,b) (i.e. morpholine 5a and Nmethylpiperazine 5b). The reaction of the intermediate 3 with the amines 4 and 5 afforded the corresponding thioureas 6(ag) as yellowish solids, which were purified by recrystallization from EtOH. As shown in Table 1, the N,N'-disubstituted thioureas 6 were synthesized in low to good yields (9-80%).
In order to prepare the desired 1-aminoimidazo[1,5-a]indol-3-ones 8 by cyclization, via intramolecular sulphur/nitrogen displacement, it was necessary to activate the C=S bond of the N,N'-disubstituted thioureas 6 with a halogenoalcane. S-alkylation of these N,N'disubstituted thioureas 6 was realized with 3 equivalents of methyl iodide in refluxed acetone in the presence of powdered potassium carbonate. Compounds 7(a-g) were obtained in 52-69% yields. To investigate the scope and limitation of the cyclization depicted in Scheme 1, we screened the reactivity of various bases in appropriate solvent (Et 3 N, pyridine, piperidine in acetonitrile, tert-BuOK in tert-BuOH, AcONa in AcOH, NaH in THF or 1,4dioxane). No cyclization took place and starting compounds were recovered, thus indicating, that the choice of the base and the solvent was essential for successful ring closure. In fact, the lithium hydride (2 equivalents) in dry dimethylformamide mediated cyclization of compounds 7. This cyclization proceeded MeI 3 equiv, K2CO3 1 equiv, acetone, 1 hr, reflux. (v) LiH 2 equiv, dry DMF, 6 hrs, 25°C. via intramolecular nucleophilic attack of the deprotonated indole nitrogen on the highly electrophilic carbon atom of the enamide bond C=N bearing the methylsulfanyl leaving group of the isothioureas 7.
The 1-aminoimidazo[1,5-a]indol-3-ones 8 were easily isolated after quenching the crude reaction mixture with cold deionized water and the insoluble compound 8 was collected by filtration and was recrystallized from EtOH.
As can be seen in Table 1, cyclization worked well with methylsulfanyl isothioureas 7(a-e) derived from primary amines 4(a-e) while, on the contrary, cyclization failed with compounds 7(f,g) derived from cyclic secondary amines 5(a,b) even in the presence of ten-fold excess of lithium hydride. This might be explained by the decrease of the electrophilic character on the carbon atom of the enamide C=N bond or by the bulky effect of morpholine-1-yl or N-methylpiperazine-1-yl moiety in the possible transient intermediate (Figure 3) which decreases the leaving character of the methylsulfanyl group. Table. 1: Results for the preparation of compounds 6(a-g), 7(a-g) and 1-amino imidazo[1,5-a]indol-3-one derivatives 8(a-g).
Compound R n X Yield a (%) 6a As can be seen in Table 1, compounds 8(a-e) were isolated in yields ranging from 27 to 52%. The structure identification of these five new 1-aminoimidazo[1,5-a]indol-3ones 8(a-e) were based on the 1 H and 13 C assignments and was performed extensive 1D and 2D NMR spectroscopy. Examination of the 1 H NMR spectra in DMSO-d 6 showed that the CH= proton of the indolyl moiety of 8(a-e) appears at low field (7.13 ppm < δ indol < 7.21 ppm).
It is interesting to note that there is only one signal for the NH proton in the 1 H NMR spectra of 8(a-e) (11.22 ppm < δ NH < 11.60 ppm), which strongly indicates the cyclized structure of these compounds. For the methylsulfanyl precursors 7(a-e), we observed two separated signals for the NH protons. As example, in the 1 H NMR spectrum of 7a in DMSO-d 6 , the first NH broad singulet appears at 11.17 ppm and the second shifted to 11.51 ppm. The five new 1-aminoimidazo [1,5-a]indol-3-ones 8(a-e) were evaluated against five different and representative kinases. Harmine (a natural βcarboline alkaloid known to be a potent inhibitor of DYRK1A (Tahtouh et al., 2012 andGöckler et al., 2009)) was also used as positive control and its IC 50 values were compared with those obtained for the compounds 8. Results given in Table 2 demonstrated that 1-aminoimidazo[1,5-a]indol-3-ones 8 are inactive against DYRK1A, CK1, CDK5/p25, GSK3  and CLK1, except for compound 8b for which an IC 50 value of 9 µM was determined.

CONCLUSION
In summary, we worked out the synthesis in 5 steps of new 1-aminoimidazo[1,5-a]indol-3-ones 8(a-e) without substituents on the indole moiety. The key step of this sequence is the ring closure by intramolecular sulphur/nitrogen displacement in basic medium with S-methylsulfanyl thioureas 7 derivatives issued from primary amines.
Among these cyclized compounds 8, only 8b presented modest micromolar inhibition activity on DYRK1A (IC 50 9 mM). Now, we are interested in further explorations for introduction of molecular diversity with appropriate groups on the indole moiety of compounds 8, in order to increase the biological properties of these new 1-aminoimidazo[1,5-a]indol-3-ones with potential applications in Alzheimer's disease/Down syndrome.