Synthesis of fused pyranocarbazolones with biological interest

Fused pyranocarbazolones were synthesized from the reactions of hydroxycarbazoles with ethyl propiolate in the presence of catalytic amount of InCl 3 . Carbalkoxypyranocarbazolones and methylenefurocarbazolones were obtained from the reactions of hydroxycarbazoles with dialkyl acetylene dicarboxylates in the presence of Ph 3 P in refluxing toluene. Furanone 10a (IC 50 = 10 µM) followed by pyranocarbazolones 9a and 11b are interesting lipoxygenase inhibitors. Pyranocarbazolones 9b , 11a-b are potent anti-lipid peroxidation agents and this is correlated to the presence of the coumarin ring.

There are many methods for the synthesis of coumarins including Pechmann, Perkin, Knoevenagel, Reformatsky, Wittig and Ring Closing Metathesis reactions. 3,14One of the most attractive methods is the Pechmann condensation, 15 which starts from phenols and β-ketoesters or malic acid or alkynoates [16][17][18] in the presence mainly of concentrated H 2 SO 4 or Lewis acids.The synthesis of carbazoles is achieved 12,13 mainly by multistep sequences involving coupling and cyclization reactions of anilines or iminoquinones, Fischer reactions of the corresponding phenylhydrazones, benzannulation reactions of indoles.The pyranone fragment is introduced starting from 1-hydroxycarbazoles by Friedel-Crafts reactions and subsequent cyclization, 19,20 by Pechmann cyclization, 21 by reactions 21 with DMAD and Ph 3 P or from 2-formyl-1-hydroxycarbazoles 22 by Wittig or Knoevenagel reactions.
Recently we have prepared coumarins by the reactions of phenols [23][24][25][26] and especially hydroxyindoles 9 with DMAD and Ph 3 P and by Pechmann reactions of phenols with malic acid and H 2 SO 4 under microwave (MW) irradiation or with propiolates in the presence of ZnCl 2 . 27In the course of our interest on the synthesis of coumarin hybrid derivatives, to study further their biological activity, we report herein the reactions of 2-and 4-hydroxycarbazoles with ethyl propiolate or with DMAD and Ph 3 P and the biological study of new hybrid compounds as antioxidant agents.Free radicals are highly implicated in the induction of several significant pathophysiological disorders. 28Inflammation, cancer, arthritis, myocardial and central nervous system (CNS) ischemia among them are under intensive study.Consequently, compounds with antioxidative character can be expected to offer protection in several diseases and to lead to potentially effective drugs.

Results and Discussion
The reactions studied and the products obtained are depicted in Schemes 1-2.The treatment of the mixture of 4-hydroxycarbazole (1) and ethyl propiolate (2) with catalytic amount (11 mol%) of InCl 3 under stirring at 90 °C for 24 h gave pyrano[3,2-c]carbazol-2(7H)-one (3) (Scheme 1) in 77% yield (Method A, Table 1, entry 1).This reaction is a tandem Michael addition followed by cyclization for the formation of pyranone ring. 18In the literature, it is referred also that this process with InCl 3 failed to give amino substituted coumarin. 18The above procedure gave much better results than the attempts for the formation of derivative 3 from compounds 1 and 2 in the presence of catalytic amount of Pd(OAc) 2 and TFA 16 (Method B, Table 1, entry 2) or ZnCl 2 in dioxane 27 (Method C, Table 1, entry 3).The reaction also of 1 with malic acid in the presence of concentrated H 2 SO 4 under microwave irradiation 27 led to 8% of product 3 (Method D, Table 1, entry 4).
Taking into account the multifactorial character of oxidative stress and inflammation, and the role of indoles and coumarins in them, we decided to evaluate in the present investigation the new pyranocarbazolones (Table 2) as antioxidative agents.Nordihydroguaiaretic acid (NDGA) and Trolox, well known antioxidant agents, are used as reference compounds.Several assays should be used to assess in vitro antioxidant activity because the antioxidant ability of a compound must be evaluated in a variety of milieus.Most of them require a spectrophotometric measurement and a certain reaction time to obtain reproducible results.The interaction/reducing activity (RA) of the examined compounds with the stable free radical DPPH is shown in Table 2.This interaction, which indicates their radical scavenging ability in an iron-free system, was measured at 100 µM for 20/60 min.In the DPPH assay, the dominant chemical reaction involved is the reduction of the DPPH radical by an electron transfer from the antioxidant.Particularly effective such antioxidants are the phenoxide anions from phenolic compounds like catechol and derivatives, such as NDGA.Under our experimental conditions 25 very low interaction values (11-43%) were observed with the exception of compounds 3 and 5 which both contain an indolyl and a coumarin group.The results are similar, although compound 3 is a pyrano[3,2-c]carbazol-2(7H)-one and 5 is a pyrano[3,2-a]carbazole-3(11H)-one.An increase in reducing abilities is observed after 60 min of interaction for: 3, 5, 10a and 10b.
In our studies, AAPH was used as a free radical initiator to follow oxidative changes of linoleic acid to conjugated diene hydroperoxide.The water-soluble azo compound AAPH has been extensively used as a clean and controllable source of thermally produced alkylperoxyl free radicals. 27In the AAPH assay the highly reactive alkylperoxyl radicals are intercepted mainly by hydrogen atom transfer (HAT) from the antioxidant.Particularly effective HAT agents 31 are compounds with high hydrogen atom donating ability, that is compounds with low heteroatom-H bond dissociation energies and/or compounds from which hydrogen abstraction leads to sterically hindered radicals as well as compounds from which abstraction of hydrogen leads to C-centered radicals stabilized by resonance.Only pyranocarbazolones 9b, 11a-b, shown is Table 2 exhibit interesting anti-lipid peroxidation activity compared to the reference compound Trolox.Furanone 8b exhibits lower activity compared to the corresponding pyranone 9b.The same results are taken from furanones 10a-b and pyranones 9a-b and 11a-b.It seems that pyranocarbazolones are more potent and this is correlated to the presence of the coumarin ring in 11a-b.The rest carbazolones present low or no activity.
We evaluated compounds from both series for their ability to inhibit soybean lipoxygenase (LOX) by the UV absorbance based enzyme assay. 32The lipoxygenase (LOX) catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes.LTB4 generation is considered to be important in the pathogenesis of neutrophil-mediated inflammatory diseases with a marked relation to the severity of cardiovascular diseases, asthma and cancer.The most potent inhibitor is furanone 10a with an IC 50 value of 10 µM followed by pyranocarbazolones 9a and 11b (100 µM).Judging the special structural characteristics within the two groups of derivatives, compounds of the furanone group seem to be more potent.

Conclusions
Pyranocarbazolones were synthesized from ethyl propiolate and hydroxycarbazoles in the presence of catalytic amount of InCl 3 .Carbalkoxypyranocarbazolones and methylenefurocarbazolones were obtained from the reactions of hydroxycarbazoles with dialkyl acetylene dicarboxylates in the presence of Ph 3 P in toluene heated at reflux.Furanone 10a (IC 50 = 10 µM) followed by pyranocarbazolones 9a and 11b are interesting lipoxygenase inhibitors.Pyranocarbazolones 9b, 11a-b are potent anti-lipid peroxidation agents and this is correlated to the presence of the coumarin ring.Thus, the combination of carbazole and coumarin or furanone leads to interesting hybrid compounds in terms of biological activity.

Experimental Section
General.Melting points were determined on a Kofler hot-stage apparatus and are uncorrected.IR spectra were obtained with a Perkin-Elmer 1310 spectrophotometer as KBr disks. 1 H and 13 C NMR spectra were recorded at 300 and 75 MHz, respectively on a Bruker AM 300 with CDCl 3 as solvent and TMS as internal standard.Mass spectra were determined on a LCMS-2010 EV Instrument (Shimadzu) under Electrospray Ionization (ESI) conditions.Elemental analyses for C, H and N were obtained using a Perkin-Elmer 2400-II Element analyzer.The MW experiments were performed in a Biotage (Initiator 2.0) scientific MW oven.Silica gel N o 60, Merck A.G. was used for column chromatography.Ethyl propiolate, DMAD, diethyl acetylene dicarboxylate, InCl 3 , Ph 3 P were purchased from Fluka, (Buchs, Switzerland) and used without further purification.1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH), nordihydroguaiaretic acid (NDGA), trolox, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH), soybean lipoxygenase, linoleic acid sodium salt were purchased from Aldrich Chemical Co.(Milwaukee, WI, USA).

Biological evaluation
Determination of the reducing activity of the stable radical 1,1-diphenyl-picrylhydrazyl (DPPH). 25To a solution of DPPH (final concentration 50 µΜ) in absolute ethanol was added an equal volume of the compounds dissolved in DMSO.As a control solution ethanol was used.The final concentration of the test compounds was 100 µM.After 20 min/60 min at room temperature, the absorbance was recorded at 517 nm (Table 1).Inhibition of linoleic acid peroxidation. 25,27Production of conjugated diene hydroperoxide by oxidation of linoleic acid in an aqueous dispersion was monitored at 234 nm.Azobis(2amidinopropane) dihydrochloride (AAPH) is used as a free radical initiator.Ten microliters of the 16 mM linoleic acid dispersion were added to the UV cuvette containing 0.93 mL of 0.05 M phosphate buffer, pH 7.4, thermostated at 37 o C under air by the addition of 50 µL of 40 Mm AAPH solution.Oxidation was carried out in the presence of aliquots (10 µL) of the tested compounds.In the assay without antioxidant, lipid oxidation was measured in the presence of the same level of DMSO.The rate of oxidation at 37 o C was monitored by recording the increase in absorption at 234 nm caused by conjugated diene hydroperoxide formation (Table 1).Soybean lipoxygenase inhibition study in vitro. 25,27The in vitro study was evaluated as reported previously.The test compounds dissolved in ethanol were incubated at room temperature with sodium linoleate (100 µL) and 0.2 mL of enzyme solution (1/9 × 10 -4 w/v in saline).The conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded and compared with the appropriate standard inhibitor (NDGA) (Table 1).

Table 2 .
Biological evaluation of pyranocarbazolones and furocarbazolones as antioxidants a Interaction with DPPH; b In vitro % inhibition of soybean lipoxygenase (LOX); c % Inhibition of lipid peroxidation (AAPH); d na = no activity under the experimental conditions.