Microwave-assisted practical synthesis of 4-imino-3-phenyl-3,4-dihydro-1 H - chromeno[2,3 -d ]pyrimidine-2(5 H )-thione derivatives and exploration of their biological activities

The synthesis of a series of new 4-imino-3,4-dihydro-1 H -chromeno[2,3 -d ]pyrimidine-2(5 H )-thiones 6(a-f) without substituent in C-5 position using microwave dielectric heating is reported. These new compounds were obtained in three steps with good overall yields (21-50%)


Results and Discussion
Access to the planned 4-imino-3,4-dihydro-1H-chromene [2,3-d]pyrimidine-2(5H)-thiones 6 and their 2-amino-4H-1-chromene-3-carbonitrile precursors 4 is outlined in Scheme 1. Owing to that our synthetic strategy involved the preparation of iminocoumarin-3-carbonitriles 3 as starting materials, we selected a series of aromatic aldehydes 1(a-f) bearing diethylamino-or methoxy-groups at various positions for the introduction of molecular diversity on the expected compounds.The desired iminocoumarins 3(a-f) or 2-imino-2H-[1]benzopyran-3-carbonitriles were easily prepared by classical heterocyclization from an equimolecular mixture of substituted 2-hydroxybenzaldehyde 1 and malonitrile 2 in the presence of 0.5% piperidine in EtOH according to a procedure developed in our laboratory. 12After a reaction time varying from 10 min.to 10 hours and elimination of volatile compound in vacuo, the desired compounds 3(a-f) were obtained in yields ranging from 60 to 96% (Table 1).For the second step, transformation of the 2-imino-2H-[1]-benzopyran-3-carbonitrile 3(a-f) into 2-amino-4H-1-chromene-3-carbonitrile 4(a-f) could be readily accomplished in good yields (50 to 90%) using 0.5 equivalent of NaBH4 in MeOH at 0 °C during 45 min.Separation of the desired compound 4 from the crude reaction mixture was easily realized by addition of deionized water and the resulting precipitated product 4 was collected by simple filtration.With the 2-amino-4H-1-chromene-3-carbonitrile 4(a-f) in hand, we decided to explore the addition of phenylisothiocyanate 5 to 4 under microwave irradiation 13,14 for the preparation of 4-imino-3,4-dihydro-1H-chromene [2,3-d]pyrimidine-2(5H)-thiones 6.The underlying idea for the use of microwave dielectric heating in organic synthesis is that commercial laboratory apparatus favored significant rate enhancements compared to reactions, which run with conventional heating, (i.e. in oil bath) 15 and higher product yields.
The experiments were realized in the mono-mode microwave cavity of Monowave® 300 Anton-Paar apparatus operating with continuous power from 0 to 800 Watt at a frequency of 2.45 GHz and, the reactions were conducted in a glass reactor-vial closed with a snap-cap.Reaction optimization for the synthetic microwave process of compounds 6 consisted in varying the following parameters: -the nature of the solvent to obtain a homogeneous reaction mixture under microwave heating (toluene, DMF, DMSO, AcOEt, MeCN, pyridine, DIPEA, Et3N), -the reaction time (20-45 min.),-and the reaction temperature (90-150 °C).After a complete screening of the reaction conditions, we observed a good reproducibility when we applied microwave irradiation during 30 min.at 120 °C from a stoechiometric mixture of reagents 4 and 5 in pyridine (1 mmol./mL,total conc.).The desired 4-imino-3-phenyl-3,4-dihydro-1H-chromeno [2,3-d]pyrimidine-2(5H)thione 6 was isolated from the crude reaction mixture after a simple precipitation from cooled AcOEt followed by successive washings with deionized water, diethyl ether and finally recrystallized from absolute ethanol to increase the quality of the precipitated product 6 before biological tests.Inspection of the data presented in Table 1 showed that this protocol for heterocyclocondensation under microwave irradiation can be applied to the 2-amino-4H-1-chromene-3-carbonitrile 4(a-f) with commercial phenyl isothiocyanate 5 and the resulting 4-imino-3-phenyl-3,4-dihydro-1H-chromeno [2,3-d]pyrimidine-2(5H)-thione 6(a-f) were synthesized in yields ranging (Table 1) from 55 to 75% and also in good overall yields (21-50%).Before entering the biological tests, the six products 6 were characterized by 1 H, 13 C NMR, IR and HRMS.Examination of their 1 H NMR spectra in DMSO-d6 solution showed signal for the CH2 (H-5) in the range 3.56 < δ < 3.96 ppm.It is noteworthy that the NH of imino group (C-4 position) and thiourea function (N-1 position) does not appear in the 1 H NMR spectra due to exchange with DMSO-d6 solvent for analysis.The presence of the C-4 imino function of 6 in the IR spectrum was detected at 1623-1626 cm -1 and most important absorption bands were observed at 3133 and 3401 cm -1 (for 6a) and attributed to the two N-H stretching frequencies.The presence of thiourea (C=S) and imino (C=NH) functions were confirmed respectively in the 13 C NMR spectra by a C-4 signal at δ 160.21 ppm and a C-2 signal at δ 179.14 ppm for 6a.In mass spectrometry (MS) analysis, the [M+Na] + molecular ion signal for all products 6 were easily obtained as base signal.Exploring the biological properties of this new family of 4-imino-3-phenyl-3,4-dihydro-1H-chromeno[2,3d]pyrimidine-2(5H)-thione 6(a-f) and their intermediates 3(a-f), 4(a-f), we tested these 18 products on six different in vitro kinase assays because these enzymes play an important role in protein phosphorylation of serine, threonine and tyrosine residues, all part of important cellular regulatory mechanism which are frequently deregulated in human diseases.The protein kinases used for these assays are respectively HsCDK5-p25 (Homo sapiens cyclin dependant kinase 5p-25), 16 GSK3α/β (glycogene synthase kinase-3α/β), 17 CLK1 (cdc2-like kinase-1), HsHaspin (Homo sapiens Haploid Germ Cell-Specific Nuclear Protein Kinase), 18 HsPim1 (Homo sapiens Pim1 proto-oncogene, serine/threonine kinase) 19 and HsAurora B. The IC50 values were determined from the dose-responses curves (Sigma Plot) and Harmine was used as reference for positive control.Results are given in Table 2. Analysis of the results for in vitro kinase assays highlighting three categories of compounds.The first category prove to be inactive products with IC50 > 10 μM and to our surprise, no 4-imino-3-phenyl-3,4-dihydro-1H-chromeno [2,3-d]pyrimidine-2(5H)-thione 6 exhibited inhibitory activity on protein kinases.The second category integrate compounds (Figure 2), which presents selective bioactivity in the micromolar range against CLK1.This is the case for iminocoumarin 3c (IC 50 1.5 μM) and its 2aminochromene derivative 4c (IC50 0.9 μM).The last category concerns compounds with selective submicromolar bioactivity against CLK1: this concerns 3e (IC50 0.5 μM) bearing a methoxy group in C-8 position and the most interesting compound is 3d (with MeO in C-7 position) showed an IC50 0.25 μM.Continuing this biological exploration, these 18 compounds were also subjected to in vitro cancer assays 20,21 against a panel of 7 tumor cell lines, HuH7-D12, Caco2, MDA-HB231, HCT116, PC3, NCI-H727, which are representative of different cancers (leukemia, melanoma and cancers of the liver, colon, breast, prostate, lung and kidney respectively).HaCat keratinocytes and diploid skin fibroblasts were also used as normal cell lines.Taxol, Doxorubicine and Roscovitine were also used as references for positive controls and their IC50 values are compared with those obtained for compounds 3(a-f), 4(a-f) and 6(a-f).All results obtained were reported in Table 3. Again, none of compounds 6(a-f) and also the 2-aminochromene 4(a-f) presented a significant cytotoxic activity on the seven tumoral cell lines.For the other compounds 3(a-f), the most active compounds were clearly 3b, 3c and 3f (Figure 2).They exhibited anti-tumor activities against the Huh7-D12, Caco2, HCT116 cell lines with IC50 values lower than 10 mM (Caco2, 3b: IC50 6 μM and 3f: IC50 9 μM; HCT116, 3f: 9 μM, Huh7-D12, 3b: IC50 2 μM) and did not inhibit the growth of normal fibroblasts (IC50 > 25 μM).

Conclusions
This preliminary study described a new route to 4-imino-3-phenyl-3,4-dihydro-1H-chromeno[2,3-d]pyrimidine-2(5H)-thione 6(a-f) with good overall yields ranging from 21 to 50% using a solution-phase microwave dielectric heating protocol and 2-amino-4H-1-chromene-3-carbonitrile 4(a-f) without substituent in C-4 position via iminocoumarins as starting materials.This protocol under microwave dielectric heating offered the possibility of an extension to a wide variety of substrates.The in vitro inhibition of cell proliferation was carried out on a panel of seven representative tumoral cell lines and the compounds 3(a-f), 4(a-f) and 6(a-f) were also evaluated against six protein kinases.Among all of these compounds, the iminocoumarin 3d and the 2-amino-4H-1-chromene-3-carbonitrile 4c turned out to be interesting because they presented selective submicromolar inhibition activity on CLK1 (3d: IC50 0.25 μM and 4c: IC50 0.9 μM).The iminocoumarin 3b, without substituent, exhibited antitumor activities against Huh7-D12 cell lines with IC50 2 μM.These current results are the starting point of a new larger program within our group to investigate intensively the biological properties of these new inhibitors through a complete structure-activity relationship (SAR) with potential applications in cancer or diseases of the central nervous system, using the present protocol under microwave dielectric heating.

Experimental Section
General.Melting points were determined on a Kofler melting point apparatus and were uncorrected.Infrared (IR) spectra were registered on a Jasco FT-IR 420 spectrophotometer apparatus using KBr pellets. 1 H NMR spectra were recorded on BRUKER AC 300 P (300 MHz) spectrometer and 13 C NMR spectra on BRUKER AC 300 P (75 MHz) spectrometer.The high resolution mass spectra (HRMS) were recorded in positive mode using direct Electrospray infusion, respectively on a Waters Q-Tof 2 and on a Thermo Fisher Scientific Q-Exactive spectrometers at the "Centre Régional de Mesures Physiques de l'Ouest" SFS ScanMAT (CRMPO SFS ScanMAT, Rennes, France).Reactions under microwave irradiations were realized in the Anton Paar Monowave 300® microwave reactor (Anton Paar France) using borosilicate glass vials of 10 mL equipped with snap caps (at the end of the irradiation, cooling reaction was realized by compressed air).The microwave instrument consists of a continuous focused microwave power output from 0 to 800W for this Anton Paar Monowave 300® apparatus.All the experiments were performed using stirring option.The target temperature was reached with a ramp of 3 minutes and the chosen microwave power stay constant to hold the mixture at this temperature.The reaction temperature is monitored using calibrated infrared sensor and the reaction time included the ramp period.The microwave irradiation parameters (power and temperature) were monitored by the Monowave software package for the Anton Paar Monowave 300® reactor.All reagents and solvents were purchased from Acros Fisher or Sigma-Aldrich and were used without further purification.The 2-imino-2H-1-benzopyran-3-carbonitrile 3(a-c) and 3f were synthesized according to literature. 12andard procedure for the synthesis of 2-imino-2H-1-benzopyran-3-carbonitriles 3(d,e).In a 50 mL twonecked round-bottomed flask, provided with magnetic stirrer and condenser, containing a solution of aromatic aldehyde 3 (10 mmol.) in absolute ethanol (20 mL) was added dropwise during 15 min.commercial malonitrile 2 (66.1 mg, 10 mmol.) in the presence of a catalytic amount of piperidine (0.5%).The resulting reaction mixture was stirred vigorously (500 rpm) for an appropriate reaction time at room temperature.Then, the reaction mixture was concentrated in a rotary evaporator under reduced pressure for elimination of volatile solvent.The desired product 3 was collected by filtration on a Büchner funnel (porosity N°4) and washed with 3 x 10 mL of absolute ethanol.The desired 2-imino-2H-1-benzopyran-3-carbonitrile 3 was dried under high vacuum (10 -2 Torr) at 25°C for 1 h that gave a yellowish powder and was further used without purification.

4(a-f)
thione 6(a-f) on seven representative tumor cell lines and fibroblasts a IC50 values are expressed in μM and are the average of three assays, standard error ± 0.5 μM.b ND: Not determined.