Synthesis of 3-indolylazoles and meridianin derivatives from indolyl enaminonitriles

The reaction of indole derivatives with cyanoacetic acid followed by treatment with DMFDMA gave the intermediate indolyl enaminonitriles (3). Further reaction with aminoguanidine yielded 5 ́-cyanomeridianin analogues (4). The same intermediate reacted with pmethoxyphenylhydrazine to give the pyrazolyl derivative (8). Treatment of (2E)-3-dimethylamino-2-(1H-indol-3-yl)-propenoate (3a) with hydroxylamine hydrochloride in basic medium afforded (5-amino-isoxazol-4-yl)-(1H-indol-3-yl)-methanone (5) and the acrylic acid derivative (6) after a short or a long heating, respectively. Unequivocal structural elucidation of the latter compound was achieved from single-crystal X-ray diffraction studies.


Introduction
Enaminonitriles are versatile reagents which have been efficiently utilized for the synthesis of heteroaromatics. 1 Meridianin alkaloids, isolated from the south atlantic tunicate Aplidium meridianum, 2 are indole derivatives substituted at the C-3 position by a 2-aminopyrimidine ring.
Since abnormal protein phosphorylation is related to many human diseases interest on the search for inhibitors of kinases has increased.A vast number of inhibitors of CDKs have been discovered, however, only very few GSK-3 inhibitors have been described.Meridianins [2][3][4] (Fig. 1) were described as potent kinase inhibitors, 4 inhibiting CDKs, GSK-3, PKA and other protein kinases in the low micromolar range.Some derivatives displayed also antitumor activity. 5here are three reported approaches for the synthesis of meridianins from indole derivatives based on a Suzuki cross-coupling with indole-3-boronic acid derivatives. 6A Bredereck synthesis was also described from -enaminones, which were, in turn, obtained from 3-acetylindole derivatives. 5,7,8More recently, meridianins were also obtained from trimethylsilylynone indole derivatives. 9

Results and discussion
Following our interest in the chemistry of -enaminonitriles, [10][11][12][13] in this manuscript we describe our most recent results that explore the potential of 3(2E)-3-dimethylamino-2-(1H-indol-3-yl)propenoate in the synthesis of 5´-cyanomeridianin C, G and 3-heteroarylindoles.Commercially available indole and indole-3-carboxaldehyde were employed as the starting materials giving access to the meridianin analogs 4a-c in a straightforward three-step synthesis following the Bredereck approach 7 (Scheme 1), and to 14 from one-pot synthesis as shown in Scheme 3. Recently, a facile procedure for the cyanoacetylation of indoles leading to compounds 2a-c has been reported. 14This one-step approach, applying cyanoacetic acid in acetic anhydride for the inclusion of the cyanoacetyl functionality, provides an easy access to cyanoacetylated indoles 2a-c.The Bredereck protocol 7 was used for the formation of the 2-aminopyrimidine ring.When compounds 2a-c were treated with dimethylformamide dimethylacetal (DMFDMA), 15 without solvent at room temperature, the corresponding enaminonitriles 3a-c were obtained in yields ranging from 78 to 88%.
Direct conversion of 3a-c into 5´-cyano meridianin C and meridianin G derivatives 4a-c, involving the formation of the 2-aminopyrimidine ring, was achieved in 70-78% by treatment with guanidine hydrochloride in the presence of anhydrous potassium carbonate (Scheme 1).The structure of compounds 4a-c was established on the basis of elemental analysis, IR, mass, 1 H and 13 C NMR spectral data studies (cf.Experimental Section).
Heating compound 3a for 7 hours, with hydroxylamine hydrochloride in the presence of base, did not produce the isoxazole 5, but led instead to the formation of the indole derivative 6.
The alternative structure 5 was excluded based on FT-IR and NMR data.The 1 H NMR spectrum of 6 showed two signals that are D 2 O exchangeable at 7.90 and 12.20 and a singlet for H-2 at 8.48 ppm, while 13 C NMR showed a carbonyl signal at 181.55 ppm (C-3).Unequivocal structural elucidation was achieved from single-crystal X-ray diffraction studies.Compound 6 crystallises in the centrosymmetric monoclinic C2/c space group, with the asymmetric unit being composed of a whole molecular unit (Figure 2) with typical geometrical features for the bond lengths and angles (see caption of Figure 2).When the reaction proceeded under mild heating conditions, for 30 minutes at 60ºC, the product isolated (26%) was the oxazole derivative 5.The singlet due to H-3 may be seen at 8.97 and the NH 2 signal is located at 8.18 ppm and readily exchanges with D 2 O.It is assumed that 5 may be formed initially and with prolonged heating, in basic medium, yields 6. Removal of the proton 3 from 5, which is known to happen in basic medium, would open the isoxazole ring to an amide whose hydrolysis would give the corresponding carboxylic acid derivative (6).Ring opening always occurs in the same fashion leading to the stereoisomer E depicted in Figure 2.
This configuration has important consequences in the crystal packing as it permits the existence of a vast network composed of strong and highly directional hydrogen bonding interactions (Figure 3a): (i) the carboxylic acid groups of two neighbouring units are engaged in a typical R 2 2 (8) graph set motif 16   As anticipated, the treatment of compound 3a with p-methoxyphenylhydrazine, in refluxing ethanol, in a basic medium, afforded as single product only pyrazole 7 (Scheme 1).The structure of the pyrazole 9 was excluded due to the absence of the typical signals of cyano groups in both the FT-IR and 13 C NMR spectra.The 1 H NMR of compound 7 showed all the expected signals which was not sufficient to differentiate between structures 7 and 8.For this reason, we collected the HMQC and HMBC NMR spectra and performed an unambiguous assignment in the 1 H and 13 C NMR spectra (see experimental section).In the HMBC spectrum, no correlation peak between the pyrazole H-3 at 8.13 ppm and carbon signals at 130.67 (C-1´´) was observed.This occurrence is characteristic only for structure 7 but not for 8. 17 Compound 3a reacted with malononitrile in the presence of piperidine as a catalyst to give in 75% yield.The structure of compound 10 was readily established from 1 H NMR data, which revealed all the expected protons of the indole moiety alongside with a singlet at 8.59 ppm for the H-4 of the pyridinone ring and a NH broad signal at 13.10 ppm.Two signals due to CN groups were found in the 13 C NMR spectrum at 115.44 and 116.96 ppm.The formation of the pyridine-3,5-dicarbonitrile 10 from the reaction of 3a with malononitrile is assumed to take place through the sequence depicted in Scheme 2. Compound 3a reacted with malononitrile in the presence of piperidine as a catalyst to give in 75% yield.The structure of compound 10 was readily established from 1 H NMR data, which revealed all the expected protons of the indole moiety alongside with a singlet at 8.59 ppm for the H-4 of the pyridinone ring and a NH broad signal at 13.10 ppm.Two signals due to CN groups were found in the 13 C NMR spectrum at 115.44 and 116.96 ppm.The formation of the pyridine-3,5-dicarbonitrile 10 from the reaction of 3a with malononitrile is assumed to take place through the sequence depicted in Scheme 1.
The reactivity of the methylene group in compound 2 was exploited by reacting this compound with trichloroacetonitrile in ethanol in the presence of sodium acetate to obtain the new enamine 11 (Scheme 1).The structure of compound 11 was established by 1 H NMR data which revealed the absence of a signal for the methylene group and the emergence of another at 11.98 ppm corresponding to two protons of the amino group.

Conclusion
Two novel indolyl enaminonitriles (3b and 3c) were prepared and reacted with guanidine hydrochloride yielding two new meridianine derivatives (4b and 4c).
The reactivity of the enaminonitrile 3a was tested with p-methoxyphenylhydrazine and malononitrile giving a pyrazole derivative 7 and a pyridone derivative 10, respectively.
The reaction of 3a with hydroxylamine hydrochloride in basic medium produced, as expected, the oxazole derivative 5.When the heating period was extended a different product, whose structure, 6, was elucidated from single-crystal X-ray diffraction.

Experimental Section
General.Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected.FT-IR spectra were registered on a Perkin Elmer FT-IR 1600 using Nujol emulsions between NaCl plates. 1 H NMR (300 MHz) and 13

General procedure for the preparation of compounds 2a-c
To a solution of cyanoacetic acid (5.0 g, 50 mmol) in Ac2O (50 mL) at 50 °C, indole (5.85 g, 50 mmol) was added.The resulting solution was heated at 85 °C for 5 min.During that period 3cyanoacetylindole started to crystallize.The mixture was allowed to cool and the solid was collected, washed with MeOH, and dried.The same procedure was applied with 2-methylindole to give 2b and with 5-bromoindole to produce 2c.Compounds 2a, b were found to be identical to those described in the literature. 14

General procedure to prepare compounds 3a-c
A mixture of 2a-c (10 mmol) and DMFDMA (11 mmol) was stirred for 5 min.at room temperature, and the yellow solid that formed was filtered off and crystallized from EtOH to afford 3a-c in 78-88% yield.Compound 3a was found to be identical to that previously described in the literature. 5

General procedure to prepare meridianin derivatives 4a-c
A mixture of enaminonitrile 3a-c (10 mmol), guanidine hydrochloride (12.0 mmol), anhydrous K 2 CO 3 (2.0 g, 15.0 mmol), and absolute ethanol (20 mL) was heated at reflux for 7 h.After cooling, the mixture was poured into ice-water and the solid formed was filtered off to afford 5´cyano-meridianin derivatives 4a-c as yellow solids.Recrystallization from EtOH led to overall yields of 70-78%.Spectral data for compound 4a are in good agreement with those reported in the literature. 5

Single-crystal X-ray Diffraction
A single-crystal of compound 6 was manually harvested from the crystallization vial and mounted on a Hampton Research CryoLoop using FOMBLIN Y perfluoropolyether vacuum oil (LVAC 25/6) purchased from Aldrich 20 with the help of a Stemi 2000 stereomicroscope equipped with Carl Zeiss lenses.Data were collected at 150(2) K on a Bruker X8 Kappa APEX II charge-coupled device (CCD) area-detector diffractometer (Mo K  graphite-monochromated radiation,  = 0.71073 Å) controlled by the APEX2 software package, 21 and equipped with an Oxford Cryosystems Series 700 cryostream monitored remotely using the software interface Cryopad. 22Images were processed using the software package SAINT+, 23 and data were corrected for absorption by the multi-scan semi-empirical method implemented in SADABS. 24he crystal structure was solved by employing the direct methods implemented in SHELXS-97. 25,26This strategy allowed the immediate location of the vast majority of the atoms.All the remaining non-hydrogen atoms were directly located from difference Fourier maps calculated from successive full-matrix least squares refinement cycles on F 2 using SHELXL-97. 26,27All non-hydrogen atoms have been successfully refined using anisotropic displacement parameters.
Hydrogen atoms bound to carbon/nitrogen and oxygen were placed at their idealized positions using the HFIX 43 or 147 instructions in SHELXL and included in subsequent refinement cycles in riding-motion approximation with isotropic thermal displacements parameters (U iso ) fixed at 1.2 or 1.5×U eq of the carbon/nitrogen or oxygen atom to which they are attached, respectively.The last difference Fourier map synthesis showed the highest peak (0.494 eÅ -3 ) and deepest hole (-0.635 eÅ -3 ) located at 0.40 Å and 0.51 Å from O1 and O2, respectively.

Figure 3 .
Figure 3. (a) Schematic representation of the one-dimensional zigzag supramolecular tape assembled by adjacent molecules of compound 6 engaged in strong and highly directional (N,O)-H•••(N,O) hydrogen bonding interactions (dashed green linessee main text for Scheme 2
13rian Unity Plus Spectrometer at 298 K or on a Bruker Avance III 400 spectrometer (400 MHz for 1 H and 100.6 MHz for 13 C).Chemical shifts are reported in ppm relative to solvent peak or TMS; coupling constants (J) are given in Hz.Double resonance, HMQC (heteronuclear multiple quantum coherence) and HMBC (heteronuclear multiple bond correlation) experiments were carried out for complete assignment of 1 H and13C signals in the NMR spectra.ESI mass spectrum was obtained on a LC-MS Finnigan LXQ spectrometer.High-resolution mass spectra were obtained on a Bruker FTMS APEXIII (ESI-TOF).Elemental analyses were obtained on a Leco CHNS-932 instrument.
C NMR (75.4 MHz) spectra were recorded on a