Synthesis of New 2-Phenylamino-4H-chromene-3-carbonitrile Derivatives and Their Effects on Tumor Cell Lines and against Protein Kinases

The synthesis of 2-phenylimino-4H-chromene-3-carbonitriles 6(a-d) in good overall yields using an efficient and practical methodology in 3 steps has been implemented in this present work. The first step was a heterocyclization between 2-hydroxybenzaldehyde 1 and propanedinitrile 2 which produced 2-iminocoumarin 3 which was submitted to nitrogen/nitrogen displacement in the presence of aromatic primary amine 4. In the third step, reduction of 5 led to the desired 2-phenylimino-4H-chromene-3-carbonitriles 6. Compounds 5(a-d) and 6(a-d) were evaluated for their potential in vitro cytotoxicity against six selected tumor cell lines (Huh7-D12, Caco2, MDA-MB231, HCT 116, PC3 and NCI-H727) and tested for their protein kinase inhibition on eight selected protein kinases. Among them, compounds 5c and 6b exhibited inhibition on HsCK1e (5c: 44% and 6b: 42% at 1 μM) and 5c for cytotoxicity on PC3 cell lines (63% at 25 μM).


Introduction
During the two last decades, the 2-amino-4H-chromene moieties ( Figure 1) emerged to be a promising platform in new chromene and benzo chromene derivatives, which exhibit a wide range of biological and pharmacological activities. For examples, Crolibulin™ (EPC 2407) (I) was currently in phase I/II of clinical trials in 2016 for the treatment of aggressive and advanced solid tumors [1][2][3][4]. During the randomized phase II trial (NTC01240590) [5], the authors compared the activity of the combination of Crolibulin™ plus cisplatin with cisplatin alone on a maximum of 40 enrolled patients with a focus on anaplastic thyroid cancer (ATC).
MX 58151 (II) or 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-4H-chromene-3-carbonitrile was identified as a tubulin inhibitor [6]. It induced apoptosis with an EC 50 of 50 nM and inhibited cell growth with a GI 50 of 37 nM in T47D breast cancer cells [7]. HA 14-1 (III) is another small molecule and nonpeptidic ligand of Bcl-2 surface pocket discovered by using the de novo computer-aided design strategy based on the predicted structure of Bcl-2 protein [8]. In vitro binding studies of HA 14-1 (III) against a set of Jurkat cells confirmed inhibition of Bcl-2 protein [9,10] and the authors of this study demonstrated that the 6-bromo on (III) is not essential for its bioactivity and the 6-position can accommodate a variety of alkyl and aryl functional groups [11]. SV30 or ethyl [2-amino-6-bromo-4-diethylmalonate]-4H-chromene-3carboxylate (IV), an analogue of pro-apoptotic molecule HA 14-1 (III), was explored in combination with lipid nanocapsules (LNCs) on F98 cells to improve its biological activities [12]. The authors demonstrated that SV30-LNCs were able to trigger cell death together with a potentiation of the mitochondrial membrane potential decrease. The naphthopyran LY 290181 (V) [2-amino-4-(pyridyl)-4H-naphtho [1,2-b]pyran-3-carbonitrile] was identified in 1997 as a potent antiproliferative compound blocking cells in the G 2 /M phase of the cell cycle associated to action on microtubules [13,14]. Finally, it's interesting to note that a QSAR (Qualitative Structure Activity Relationship) analysis was developed with novel 3Ddescriptors using Triplets Of Pharmacophoric Points (TOPP) on 80 virtual apoptosis inducing 3-amino-4-aryl-4H-chromene-3-carbonitriles [15], the underlying idea of this work was to optimize the pharmacological and pharmacokinetic properties via QSAR models prior to synthesis.  As part of our program aimed at developing new methods, new building-blocks and platforms for the preparation of heterocyclic compounds showing potential biological properties for central nervous system (CNS) (Alzheimer's disease and Down syndrome) [16,17], or for cancer (calcium ion channel inhibitors in cancer) [18], we were motivated in this work by the synthetic development of some new 2-amino-4H-[1]-chromene-3-carbonitriles without aryl substituent in C-4 position but with a phenyl fragment on the 2-amino function, to evaluate their biological activities on protein kinases (PKs) and also their anti-proliferative activities on tumor cell lines.

Chemistry
For this project, the desired 2-N-phenylamino-4H-chromene-3-carbonitriles 6(a-d) were prepared only in three steps (Scheme 1). The first step involved synthesis of iminocoumarins 3(a,b) or 2-imino-2H-[1]-benzopyran-3-carbonitriles and protocol of this classical heterocyclization was developed in our laboratory [19][20][21]. The reaction was realized from an equimolecular mixture of 2-hydroxybenzaldehyde 1 (1a: 2-hydroxy-3-methoxybenzaldehyde or o-vanillin, 1b: 2-hydroxynaphthaldehyde) and propanedinitrile 2 using 0.5% of piperidine in ethanol at room temperature. After a reaction time of 9 hours, the volatile compounds were eliminated in vacuo and we obtained these starting iminocoumarins in good yields (3a: 85% [19], 3b: 90% [22]). For the second step, the transformation of the 2-imino-2H-[1]benzopyran-3-carbonitriles 3(a,b) into 2-N-phenylimino-2H-[1]-benzopyran-3-carbonitriles 5(a-d) is the key step in our approach for introduction of molecular diversity in this C-2 position. In a preliminary approach, we studied this nitrogen/nitrogen displacement with volatile primary aliphatic amine 4 (from 1 to 2 equivalents) using solvent-less reaction conditions [23] in a mono-mode microwave cavity (Monowave® 300 Anton-Paar apparatus operating at a frequency of 2.45 GHz from 0 to 800 Watt). Initial attempts to obtain a good reproducibility for this nitrogen/nitrogen displacement with primary aliphatic amine 4 involving modification of the reaction conditions (reaction time range: 15-60 min., ratio of reagents 3 / 4: from 1 to 2, reaction temperature, use of non-polar or polar solvents) were unsuccessful. Therefore, we decided to abandon this solvent-less microwave approach. On the other hand, the use of an aromatic primary amine 4(a,b) associated with an acidic catalysis was much fruitful. Indeed, when this reaction was carried out in glacial acetic acid with an equimolecular mixture of 3 and 4 at room temperature, we observed that after 4 hours, the desired compound 5 obtained by nitrogen/nitrogen displacement (Scheme 2) becomes insoluble in the reaction medium. Thus recovery of this insoluble material by filtration on a Buchner funnel followed by washing with deionized water allows us to get very easily the four desired 2-N-phenylimino-2H-[1]-benzopyran-3-carbonitriles 5(a-d) in yields ranging from 55 to 80% (Table 1). 1 H NMR control in DMSO-d 6 of 5(a-d) after filtration showed that no need of supplementary purification and it can be used as it in the next step. Finally for the third step involving transformation of 2-N-phenylimino-2H-[1]-benzopyran-3-carbonitriles 5(a-d) into 2-N-phenylamino-4H-chromene-3-carbonitriles 6(a-d), we used an experimental protocol developed in our laboratory [19] but only changing the nature of the solvent reaction (THF vs MeOH) and the reaction temperature. From 0.5 equivalent of sodium borohydride in THF at 25°C during 45 min., the desired compounds 6(a-d) were obtained in good yields (Table 1) Scheme 2: A plausible suggested mechanism for nitrogen/nitrogen displacement via a nucleophilic attack of amino group of 4 to C-2 imino function of 3.
Before exploring their potential biological activities, the four products 5(a-d) and their four reduced derivatives 6(a-d) were characterized by 1 H, 13 C NMR, HRMS and FTIR. In the IR spectrum, the presence of carbonitrile function in C-3 position was detected at 2227 -2229 cm -1 for 5(a-d) and at 2189 -2195 cm -1 for 6(a-d) ( Table 1). The C-4 imino functions of 5(ad) were analyzed at 1650 -1662 cm -1 . Important absorption bands were observed at 3278 -3320 cm -1 for the NH stretching frequencies of 6(a-d). In 1 H NMR spectrum of compounds 6(a-d), a signal located at 3.54 < δ < 3.98 ppm is in agreement with the H-4 methylene fragment. In 13 C NMR spectrum, the signal for the CH 2 fragment in position C-4 of compounds 6(a-d) is located at 22.4 < δ < 25.3 ppm. For HRMS analysis, the [M+Na] + molecular ion signal for all products 5 and 6 were obtained as base signal. Table 1: Results obtained for the preparation of 2-N-phenylimino-2H-[1]-benzopyran-3carbonitrile 5(a-d) and 2-N-phenylamino-4H-chromene-3-carbonitrile 6(a-d).

Biology
For the second part of this project related to 2-N-phenylamino-4H-chromene-3-carbonitrile 6(a-d) and their precursors 5(a-d), we have been interested to exploring their potential biological properties, particularly their impact on protein kinase inhibition activity and their cytotoxic character against various tumor cell lines. Table 2: Effects of 2-phenylimino-2H-benzopyran-3-carbonitrile 5(a-d) and 2-Nphenylamino-4H-chromene-3-carbonitrile 6(a-d) on the catalytic activity of eight protein kinases.

Compound
Conc. The compound has an interest on the catalytic activity because the rate is less than or close to 50% The compound has a marked effect on the catalytic activity because the rate is less than 50% The new compounds 5 and 6 were evaluated on eight different in vitro kinase assays. The selected enzymes are very important in protein phosphorylation of serine, threonine and tyrosine residues, which are connected, in many cellular regulatory mechanisms; dysfunction of protein kinase activities is in many cases responsible of human diseases. The protein kinases used for these in vitro assays are respectively HsCDK5-p25 (Homo sapiens cyclindependent kinase 5p-25) [24], HsCDK9/cyclin T (Homo sapiens cyclin T dependent kinase 9 protein) [25], SscCK1ε (casein kinase 1ε, from porcine brain) [26], SscGSK3β (glycogen synthase kinase-3β, from porcine brain) [27], HsPim1 (Homo sapiens Pim1 proto-oncogene, serine/threonine kinase) [28], MmCLK1 (from Mus musculus, Dual specificity protein kinase) [29] and RnDYRK1A-kd (Rattus norvegicus, Dual specificity tyrosine phosphorylation regulated kinase 1A) [30].
Results for these in vivo kinase assays are reported in Table 2 with the rate (%) of residual activities at 10 µM and 1 µM. These results showed three categories of compounds. The first concerns compounds with no significant protein kinase activity (rate > 60-70% at 10 µM and 1 µM). The second category integrated compounds with moderate interest because the rate is less than or close to 50% and for the third category; the rate is less than 50% at 10 µM and 1 µM. In this last one, the tested compounds 5 or 6 have a marked effect on the catalytic activity. Examination of results given in table 2 showed that many compounds (Figure 2) exhibited inhibitory activity on HsCK1ε, HsHaspin, HsCDK9/cyclin T. This concerned respectively and particularly HsCK1ε with 5c (41% at 1 µM), 5d (57% at 1 µM) and 6c (46% at 10 µM and 59% at 1µM). For HsHaspin, the better result was obtained with 5c: 45% at 1 µM. For RnDYRK1A and HsGSK3β, good residual activities were observed only at 10 µM for 6a (RnDYK1A: 34%) and 6d (HsGSK3β: 31%). Pursuing this biological exploration, the 2-phenylimino-2H-[1]-benzopyran-3-carbonitrile 5(a-d) and 2-phenylamino-4H-chromene-3-carbonitrile 6(a-d) were also subjected for their in vitro cytotoxic potential on six selected human cancer cells which are respectively: Huh7 for hepatocellular carcinoma, Caco2 for colorectal adenocarcinoma, MDA-MB231 for human breast adenocarcinoma, HCT 116 for colon carcinoma, PC3 for human Caucasian prostate adenocarcinoma and NCI-H727 for human lung non-small cell carcinoma. For each tumoral cell line, the % of cell survival was measured at a single dose of 25 µM (after 48 h) in triplicate. Roscovitine, doxorubicin and taxol were used as references for positive control. It can be observed from these primary screening results reported in Table 3 that only two compounds exhibited antiproliferative activities in tumoral cell lines. This concerns 5c, which exhibited cytotoxic effect on HCT 116 (20% of survival), PC3 (63%) and NCI-H727 (41%). The presence of a methoxy group on the 2-phenylimino moiety in 5d increase the percentage of survival, excepted on Caco2 (44%). For compound 6d, which is the reduced derivative of 5d, it showed cytotoxicity against PC3 (61%). The compound has antiproliferative activity on tumor cell but the survival rate is less than or close to 50% The compound has a marked antiproliferative activity on tumor cell and the survival rate is less than 50%

Conclusion
This preliminary study described a practical approach to 2-N-phenylamino-4H-chromene-3-carbonitriles 6(a-d) in three steps. The key step in this methodology is the nitrogen/nitrogen displacement between 2-imino-2H-[1]-benzopyran-3-carbonitriles 5(a-d) and aromatic primary amines 4(a,b) in acetic medium on the 2-imino function. Insolubility of compounds 5(a-d) in the acetic acid mixture facilitated its separation by simple filtration, which offers a practical, attractive protocol and extension to a wide variety of aromatic primary amine 4 is possible. The explorations of biological activities were successively performed on a panel of six tumoral cell lines and against eight protein kinases. Among the bioactive compounds, 5c and 6b turned out to be interesting because they present a good percentage of residual activities at 1 µM for HsCK1ε. 5c showed also a marked antiproliferative activity on HCT 116 (20% of survival). The present work is the starting point of a new longer program through a next structure activity relationship (RSA) study for a complete identification of a better promising anticancer agent bearing a 2-amino-4H-chromene platform.

Chemistry
General information: Solvents were evaporated with a BUCHI rotary evaporator. All reagents and solvents were purchased from Acros Fisher, Sigma-Aldrich Chimie, and Fluka France and were used without further purification. 1 H NMR spectra were recorded on BRUKER Avance 300 (300 MHz) spectrometer and 13 C NMR spectra on BRUKER Avance 300 (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 or on a Thermo Fisher Scientific Q-Exactive spectrometers at the "Centre Régional de Mesures Physiques de l'Ouest" platform and centesimal analysis of the final compounds was performed on a microanalyzer Thermo Scientific Flash EA1112 CHNS/O at the CRMPO platform, ScanMAT UMS CNRS 2001, Rennes, France). IR spectra were registered on a Jasco FT-IR 420 spectrophotometer using KBr pellets. Melting points were determined on a Kofler melting point apparatus and were uncorrected. 2-Imino-2H-[1]-benzopyran-3- carbonitrile 3(a,b) were synthesized according to procedure described in literature [20,22]. 5(a-d).