Synthesis of novel pyrroloazepinones by Schmidt expansions of 6-indolones

New derivatives of pyrroloazepinones were synthesized. The synthesis route consisted of three stages: the formation of a dimedone-derived tricarbonyl compound, the formation of a pyrrole ring resulting from the use of the Paal-Knorr method to generate tetrahydroindole-6-ones, and the expansion of the ketone by following the Schmidt method to generate lactams. The obtained 6-indolones were used to generate new derivatives: the pyrrolo[2,3-c]azepin-6-one and the pyrrolo[2,3-d]azepin-7-one ring systems. The synthesized pyrroloazepinones were evaluated for inhibitory activity in cancer cell lines and they did not show activity and cytotoxic effects on the non-tumor cells HEK239, with IC 50 ≥ 215 ± 5.41 μM.


Introduction
Pyrroloazepinones are biheterocyclic compounds that display an important diversity of biological properties, which have been used for the treatment of Alzheimer's, 1 arthritis and many types of cancer [2][3][4][5][6] (Scheme 1).They have been found in derivatives of natural products such as paullones 6 , hymenialdisine 1 , and stevensine 7 .Their mechanism of action is very versatile, involving mainly the inhibition of kinases (CDK1 or MEK1) 1,6 , specifically via competitive inhibition of ATP binding. 4However, few reported works have focused on obtaining pyrroloazepinone derivatives.Scheme 1. Natural and synthetic products with a pyrroloazepinone structure.
The most common key step for the syntheses of pyrroloazepinone derivatives is the construction of the bicyclic ring system.For example, typical aromatic electrophilic substitution and acid hydrolysis reactions are used to obtain hymenialdisine derivatives 8,9 , which have also been obtained by carrying out catalytic cyclizations of coordination compounds. 10On the other hand, pyrroloazepinones can be obtained by carrying out ring-expanding Beckmann rearrangements 11,12 and Schmidt reactions of tetrahydroindole-4-ones. 13 Tetrahydroindolones are aromatic heterocycles formed by fusing benzene and pyrrole rings.Their syntheses are based on methodologies such as the application of the Paal-Knorr method to synthesize pyrroles; this method is carried out with 1,4-dicarbonyl compounds and aliphatic or aromatic amines in an acidic medium. 14lso, the routes of formation of pyrrole rings have been developed using different types of metal catalysts used for the formation of indoles 15 and even tetrahydroindole-4-one. 16To the best of our knowledge, the synthesis of a pyrroloazepinone from the expansion of a tetrahydroindole-6-one has not yet been reported.
In the current work, we set out to develop a route for the syntheses of new indole derivatives, as well as the formation in one step of novel isomers of pyrrolo [2,3-d]azepin-7-ones and pyrrolo [2,3-c]azepin-6-ones using the Schmidt expansion.These products may be considered to be hymenialdisine analogues, with therapeutic potential for various diseases.

Results and Discussion
To carry out the synthesis of 6-indole and later of the pyrroloazepinones, the commercial compound 5,5dimethyl-1,3-cyclohexanodione (5, see Scheme 2) was first converted to the enol ether 6 17 , which was then Calkylated at position 6 by carrying out a nucleophilic substitution of 6 with allyl bromide to obtain the alkyl ketone 7. Subsequently, compound 7 was hydrolyzed with an aqueous-acid solution of p-toluenesulfonic and water to form the compound 8 18 .The preparation of the tricarbonyl compound 9 was performed by using a Wacker-type process to oxidize the terminal alkene group of 8. Here, various tests were performed by modifying the variables of the reaction forming compound 9 to obtain the best working conditions.The results of these experiments are listed in Table 1, with a 37% yield of tricarbonyl 9 being the best case (from the relative percentage of the area under each gas chromatography-mass spectrum (GC-MS) curve) and achieved when using 0.5 equiv. of PdCl 2 , 1.5 equiv. of CuCl, an O 2 atmosphere, and 1,4-dioxane as a solvent for 2 hours of reaction.The yields corresponded to the percentage relative to 100% of the area under the curve of the GC-MS peaks of the reaction mixture.
From the crude compound 9, the syntheses of tetrahydroindole-6-ones (10a-e) were performed based on the Paal-Knorr methodology.For each of these five syntheses, a different p-R-aniline was used, as was glacial acetic acid and infrared light as a source of energy, all in a nitrogen atmosphere.From each reaction mixture, a viscous black liquid product was obtained, and was purified using column chromatography.Table 2 lists the appearance, melting point, and yield of each of the tetrahydroindole-6-one (10a-e) products.Presence of other significant size peaks on the chromatogram obtained by GC-MS showed that other compounds with a higher molecular ion signal where present along with compound 9, this suggest that side reactions between products and/or reagents happened.Since compound 9 is a liquid, purification by and the column chromatography purification was not possible because it decomposes.These two facts indicate that compound 9 is unstable in acidic medium, the above explains the use of the reaction mixture to obtain compounds 10a-e, as well as the low yield of compound 9 and consequently the Paal-Knorr reaction efficiency to obtain compounds 10a-e.
As a final synthesis step, the Schmidt method was used for the formation of the new pyrroloazepinones from compounds 10a-e.Here, in situ-generated hydrazoic acid induced the direct ring expansion of each tetrahydroindole-6-one.The advantage of the Schmidt method here was that two isomers of pyrroloazepinones, specifically pyrrolo [2,3-d]azepin-7-one and pyrrolo [2,3-c]azepin-6-one, were generated in a single step with yields above 61% (Table 3).After the reaction, the mixture of pyrroloazepinones was purified, and the purified material was characterized using IR, MS, HETCOR, DEPT and FLOCK.
The Beckmann expansion of ketones in order to obtain lactams, is the alternative method for Schmidt method, the Beckmann method was also used to obtain the pyrroloazepinones 11 and 12 in polyphosphoric acid medium, however the obtained products are from decomposition.The ratio between the amounts of the pyrroloazepinone isomers was obtained using GC-MS HETCOR, DEPT AND FLOCK Scheme 3. Carbon assignments for the NMR signals of the pyrroloazepinones 11c and 12c.
Isomerism of the pyrroloazepinones compounds 11c and 12c was analyzed by assigning the carbon-carbon connectivity of the annular pyrroloazepinone systems from the results of mono-nuclear NMR experiments, specifically 1 H-NMR, 13  ppm from C-4 to two sigma bonds and with the signal at ∂ = 48.04ppm from C-5 to three sigma bonds.The latter signal correlated, in the HETCOR experiment, with the simple signal at ∂ = 2.67 ppm in the 1 H spectrum, and was hence assigned to C-5.The connectivity of C-5 with a carbonyl was also indicated by the observed correlation of the signal at ∂ = 2.67 ppm of the 1 H spectrum with the signal at ∂ = 175.94ppm of the 13 C spectrum to two bonds.

Cytotoxic activity
To assess in vitro the cytotoxicities of the isomeric pyrroloazepinone products, the MTT assay was used to determine their abilities to inhibit the growth of cancer cells such as cervical carcinoma (SiHa), lung adenocarcinoma (SKLU1), breast carcinoma (ZR-75-1), and colorectal adenocarcinoma (SW480) cells, and of cells of the non-tumor human embryonic kidney 293 (HEK293) cell line.Cisplatin was used as a positive control and cell growth inhibition was evaluated after 48 h of exposure with compounds 11a-e and 12a-e each at a concentration of 100 μM for the cancer cells and of up to 800 μM for the HEK239 cells.These compounds showed no cytotoxic effects on the non-tumor cells (HEK239), with the IC 50 determined to be above 215 ± 5.41 μM, and they did not show cytotoxic activity on the evaluated cancer cells.

Conclusions
Five novel 6-indole derivatives were subjected to Schmidt ring expansions to produce new derivatives of pyrroloazepinones.These products included the pyrrolo[2,3-c]azepin-6-one and pyrrolo [2,3-d]azepin-7-one ring system analogs of hymenialdisine.The pyrroloazepinones obtained were indicated to not be cytotoxic.

Experimental Section
General.All reagents and solvents were purchased from Sigma-Aldrich Co.The melting points were determined in a Melt-Temp apparatus, using open and uncorrected capillary tubes.The purification of the products was carried out by performing column chromatography, using E. Merck Kieselgel 60 silica gel (230-400 mesh), grade 9385, and hexane-ethyl acetate as eluent.The progress of the reactions and purity of the products were monitored by performing thin layer chromatography (TLC), using 60 F254 silica gel plates (Merck) and ultraviolet light as a developer.The FT-IR spectra were obtained using a Thermo Nicolet NEXUS 470 FT-IR spectrometer, with thin films on a KBr disk for solids and ATR with germanium crystals for liquids.The mass spectra (MS) were obtained using a Jeol AX505HA mass spectrometer.The purity levels of compounds 11a-e and 12a-e were determined by using an Agilent Technologies 6890N chromatograph coupled to an Agilent Technologies 5973 Network mass spectrometer operated at 70 eV and equipped with a DB-5HT capillary column (15 m, 0.25 i.d.) with (5%-phenyl)-methylpolysiloxane (0.10-μm-thick film).Here, helium was used as the carrier gas at a flow rate of 1 ml/min.The injector and MS transfer line temperatures were set at 320 °C.Diluted samples (1:10 v/v, in acetone) 2.0 μL in volume were manually injected.After sample injection, the initial temperature in the oven (50°C) was held constant for 2 min, then increased to 320 °C (at a rate of 15°C/min).This temperature was then maintained for 5 min.After a delay of 1.8 min to permit passage of the solvent, the mass spectra were obtained by scanning from 15 to 800 m/z.The 1 H-NMR and 13 C-NMR spectra were obtained using a Varian-Unity 300 MHz apparatus with deuterated chloroform (CDCl 3 ) as the solvent and tetramethylsilane (TMS) as the internal reference.Chemical shifts were reported in parts per million (ppm) relative to the residual peak.Multiplicities (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and coupling constants in Hertz (Hz) were reported for the Individual peaks.HRMS-EST spectra were obtained with a JEOL The AccuTOF JMS-T100LC mass spectrometer.The cytotoxicity activity levels of the newly synthesized compounds were assessed by testing these compounds against cells of a human liver carcinoma cell line, against cervical carcinoma (SiHa), lung adenocarcinoma (SKLU1), mammary breast carcinoma (ZR-75-1), and colon adenocarcinoma (SW480) cells, and against cells of the non-tumor human embryonic kidney 293 (HEK293) cell line.
C-NMR, and DEPT experiments, and from the results of correlative 2D-NMR heteronuclear experiments, specifically HETCOR and FLOCK experiments.The DEPT experiment for 11c showed signals at ∂ = 33.68ppm and ∂ = 52.80ppm, corresponding to secondary carbons.And in the HETCOR experiment, a correlation was observed between a signal at ∂ = 3.36 ppm and that at ∂ = 3.28 ppm of the 1 H spectrum.So these secondary carbons in turn corresponded to C-8 and C-5.The connectivity of the C-5, C-6, C-7 and C-8 atoms with other atoms was confirmed from the results of the FLOCK experiment: the signal at ∂ = 1.24 ppm from the C-4 methyl protons showed correlations with the signals at ∂ = 28.34ppm from the methyl carbons, at ∂ = 35.26ppm from the two sigma bonds made with C-4, and at ∂ = 52.79ppm from C-5.In turn, the doublet 1 H signal at ∂ = 3.28 ppm correlated with the signal assigned to C-5 in the HETCOR experiment.For compound 12c, the DEPT experiment showed the signals of the secondary carbons at ∂ = 38.19ppm and ∂ = 48.03ppm, which were found to correlate with the signals at ∂ = 3.97 ppm and ∂ = 2.67 ppm, respectively, in the HETCOR experiment; these C signals were assigned to C-8 and C-5.Similar to the case for the 11c isomer, the connectivity of the C-5, C-6, C-7 and C-8 atoms of 12c was confirmed from the results of the FLOCK experiment.The signal at ∂ = 1.34 ppm of the 1 H spectrum correlated with the signal at ∂ = 32.58AUTHOR(S)

Table 1 .
Screening of the reaction conditions a for the formation of the tricarbonyl 9

Table 1 .
Continued b

Table 2 .
Appearance, melting point, and yield of each of the tetrahydroindole-6-ones.