Synthesis of bioisosteric 5-sulfa-rutaecarpine derivatives

Rutaecarpine ( Evodia rutaecarpa ) derivatives were synthesized by bioisosteric replacements of the quinazolone moiety of the pentacyclic system with benzothiadiazine 1,1-dioxide. Syntheses were performed efficiently by formation of phenylhydrazones via active methylene group transformations of pyrrolo-and pyrido[1,2 -b ]1,2,4-benzothiadiazine 5,5-dioxides, and subsequent Fischer-indolization. Preliminary testing of compound 3 showed cytotoxic activity against HeLa cells and apoptosis inducing effect.


Introduction
Fused heterocyclic compounds have traditionally been a starting-point for the design of novel anticancer agents, and current interest is in the development of bioisosteric approaches to new ring systems. 1 Traditional Chinese medicine has a unique position among traditional medicines as it provides new and safer bioactive lead structures with antitumour activity. 2utaecarpine-type alkaloids form an important class of indolopyridoquinazolinone heterocycles and belong to the subgroup of quinazoline-type alkaloids isolated from the heartwood and the fruit of numerous plants and trees of the Rutaceae family. 3,4,5Their extracts have long been used as important remedies in Chinese traditional medicine. 6"Wu-Chu-Yu", the dried unripe fruit of Evodia rutaecarpa, a Chinese herbal drug showed remarkable efficacy in headache, cholera, dysentery, worm infestations and post partum disturbances. 7Rutaecarpine 1 and evodiamine 2 (Figure 1), the major quinazolinocarboline alkaloids isolated from E. rutaecarpa showed various pharmacological activities in earlier studies 8,9 and notable examples include antimetastatic, antiproliferative, and apoptotic effect. 10,11Recent pharmacological researches proved that evodiamine stabilizes the topoisomerase-I-DNA complex and induces caspase-3 activity. 12Quinazoline and 1,2,4-benzothiadiazine 1,1-dioxide are considered as bioisosteric ring systems, and numerous attempts to vary the size of the ring or change their functional groups have been made in order to modify biological activity and selectivity. 13,2,4-Benzothiadiazine 1,1-dioxides are used as antihypertensive, diuretic, antidiabetic, glutaminergic neuromodulators 14,15 and K-channel inhibitors, 16 yet the ring system has also been known for its anti-microbial and anti-tubercular activity.17,18 Additionally, this class of compounds has been proved to inhibit hepatitis C virus (HCV) replication effectively in cell based replication systems with no apparent cytotoxicity.19 Anticancer agents containing the 1,2,4-benzothiadiazine 1,1-dioxide ring system also exhibit potent antiviral activity.20 Several hybrid compounds where quinazoline moiety or its bioisosteric analogue was built in to structures of well-known antitumour agents have been designed, synthesized and evaluated for biological activity in the past few years.21 Recently, we have been involved in the development of new synthetic strategies 22 for the preparation of new pentacyclic ring systems and also in the design of structurally modified quinazolinocarbolines and their hybrids for the development of more potent anticancer agents.23,24 The active methylene reactivity of tricyclic compound 8b was characterised by the kinetic constant (k = 1.78•10 -4 1/s) of acid catalysed hydrogen-deuterium exchange rate.The experimental data indicate decreased reactivity of the sulfa compounds compared to that of analogous tetrahdropyridoquinazolone (k = 4.29•10 -3 1/s). 28 Schem 2. Synthesis of 5-sulfa-rutaecarpine 3.
Reaction of the active methylene group at position 4 of compound 8a with phenyldiazonium chloride resulted in 6-phenylhydrazono derivative 12 (Scheme 2.).This type of reaction has been applied to similarly positioned active methylene groups earlier. 22,26Direct azocoupling reaction failed in the case of the five-membered tricyclic compound 8b which had decreased methylene group reactivity at the position 3.In order to increase reactivity on the C-3 carbon atom, formylation was accomplished to introduce an activating substituent (Scheme 3.).Vilsmeier-Haack formylation of 8b using two equivalents of phosphorous oxychloride in DMF at 60 °C for 3 hours gave the 3-dimethylaminomethylene derivative 13 in 94% yield.Only the sterically more favourable E geometric isomer is present in the DMSO-d6 solution, as proved by the NOE between the N-methyl and 2-methylene groups.Japp-Klingemann reaction 27 of 13 with phenyldiazonium chloride in acetic acid solution (0 °C, 3 h) resulted in the phenylhydrazone derivative 14 in 89% yield.
The phenylhydrazono derivatives 12, 14 exhibit a solvent dependent E/Z geometric isomerism, and indicate low activation energy of the rotation around the exo cyclic C(6)=N double bond.In CDCl3 the sterically more crowded Z form predominates (~100%), which is stabilized by an internal hydrogen bond between the amino group and ring nitrogen atom.The sterically more favourable E isomer is more abundant in DMSO-d6,, as the solvent forms a stronger hydrogen bridge with the amino group than in CDCl3.

Experimental Section
General.Melting points were measured with a digital melting point apparatus and are uncorrected.NMR experiments of all compounds (except compounds 7a,b) were carried out on a 600 MHz NMR spectrometer equipped with a dual 5-mm inverse-detection gradient (IDPFG) probehead.In case of structure identifications, standard pulse sequences and processing routines were used. 1 H and 13 C chemical shifts were referenced to internal TMS (= 0.000 ppm) or to the residual solvent signal unless otherwise stated.The probe temperature was maintained at 298 K and standard 5 mm NMR tubes were used in all experiments.NMR spectra of 7a,b in D2O were acquired at 25 C on NMR spectrometers operating at 500 and 800 MHz proton frequencies, both equipped with cryogenic HCN probeheads. 1 H and 13 C chemical shifts were referenced to internal sodium 3-trimethylsilyl-propanesulfonate (DSS).Standard pulse sequences and processing routines were applied.Single-tube 1 H NMR-pH titrations were conducted at 2 mM concentration, 25 C and 0.15 M ionic strength (NaCl) in H2O/D2O 9/1 solvent.For molecules 7a,b, in situ pH monitoring was accomplished by using trimethylamine, TRIS and imidazole (1 mM each).For compounds 8a,b protonating at pH < 7, acetic, chloroacetic and dichloroacetic acids (1 mM each) were applied as pH indicators.Degradation products were observed during the titration of 8b, studies on its pHdependent degradation kinetics are currently in progress.LC-MS conditions: For mass spectrometric analysis of compounds 3 and 4 a triple quadrupole equipped with an electrospray ionization source (ESI) was used coupled to a HPLC system.Full mass scan spectra were recorded in positive ion mode over an m/z range of 50-400 dalton (500 msec cycletime).ESI conditions were as follows: temperature: 350 °C, nebulizer pressure: 40 psi N2, drying gas flow: 9 L/min N2, fragmentor voltage: 100 V, capillary voltage: 3500 V. High purity nitrogen was used as collision gas during CID experiments and the collision energy was 30, 40 and 45 eV.Chromatographic conditions were as follows: a dead zero volume was used, with 100% acetonitrile as eluent.Injection volume was 3 µL.High-resolution mass spectra with electrospray ionization (HRMS-ESI) of all the other new compounds were recorded on a hydrid mass spectrometer consisting of a Linear Ion Trap Mass Spectrometer, combined with a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.
The reaction progress was monitored by thin layer chromatography (TLC) on silica gel plates, visualized with UV light and the plates were developed using Dragendorff reagent stains.To assess nucleosomal DNA fragmentation, HeLa cells were treated with 10 -6 M of each compound for 72 hours.Cells were then trypsinized, counted and 2•10 5 cells were fixed with 1 ml of -20 °C, 70% ethanol.Samples were kept at room temperature for 30 minutes, and afterwards they were stored at -20 °C, or processed.To extract fragmented DNA, samples were centrifuged at 450 g for 5 minutes, supernatants were discarded and the cell pellets were resuspended in 300 μl extraction buffer (200 mM Na2HPO4 containing 0.1 mg/ml RNAse A, adjusted to pH 7.8 with 200 mM citric acid).Cells were incubated with the extraction buffer for 15 minutes at room temperature, and then 3 μl propidium iodide solution (1 mg/ml) was added to each sample.After another 15 minutes of incubation at room temperature, samples were analyzed by flow cytometry.Cell counts were plotted against the FL2 channel fluorescence intensity, and cells gated in the sub-G1 region were considered to be positive for apoptotic DNA fragmentation.

7,8,9,10-Tetrahydropyrido[1,2-b][1,2,4]benzothiadiazine-5,5-dioxide (8a
). 1.00 g (3.932 mmol) of 7a was dissolved in 20 ml of phosphoryl chloride (POCl3) and a catalytic amount (2 drops) of dimethylformamide (DMF) was added to the solution.The mixture was heated on a steam bath at 90 °C for 5 minutes.Excess phosphoryl chloride was evaporated in vacuo and the residue was neutralized with 50 % potassium carbonate solution (10 ml).The aqueous phase was extracted with chloroform (3x20 ml), the organic extracts were combined, washed once with brine (30 ml), dried over anhydrous sodium sulphate and the solvent was removed in vacuo.The solid product was purified by recrystallization from isopropanol to obtain 0.57 g of 7,8,9,10tetrahydropyrido [ Phenyldiazonium chloride was produced from aniline (0.9 mL, 10 mmol) in a conventional way: it was put into 1:1 diluted hydrochloric acid (10 mL) and 5 mL sodium nitrite (0.69 g, 10 mmol) dissolved in water was added to it at 0 ºC.Consequently, this compound 8a (2.36 g, 10 mmol) was dissolved in 20 mL acetic acid.The solution was added gradually to the tincture of phenyldiazonium chloride and the sodium acetate trihydrate (6 g) at 0 ºC.The reaction mixture was stirred for 24 hours at 0 ºC and then it was diluted with water.The precipitated crystals were filtered and leached with water profoundly, dried and cleansed through boiling with isopropanol.Yield: 91%, mp: 166-168 °C (dec.