Synthesis of Novel Palladacycles Inhibitors of the Cathepsin B Activity and Antitumoral Agents

The reaction of 2-bromo-3,4,5-trimethoxybenzaldehyde 1 with Pd(dba)2; dba=dibenzylideneacetone) in the presence of a stoichiometric amount of nitrogen donor ligands, such as N,N,N’,N’-tetramethyl-ethane-1,2-diamine (TMEDA), 2,2’-bipyridine (bpy) 4,4’-dimethyl-2,2’-bipyridine (dmbpy) and an 1,10-phenanthroline (Phen), should be added to with equimolar ratio in degassed acetone under nitrogen to give mononuclear σ-aryl palladium (II) complexes cis-[2Pd{C6H(CHO)-6-(OMe)3-3,4,5}BrL2] 3a-d, where L2=TMEDA (3a); L2=bpy (3b); L2=dmbpy (3c); L2=Phen (3d) in good yields 48-65%. The reaction of the synthesized five-membered C,N-palladacycle cis-[2-Pd{C6H(CHO)-6-(OMe)3-3,4,5} BrL2] 3a-d, where L2=TMEDA (3a); L=bpy (3b); L2=dmbpy (3c); L2=Phen (3d), with an1-naphthylisocyanates (C10H7NCO) and an 1-naphthylisothiocyanates (C10H7-NCS), leads to the formation of novel palladacycle 4a-d and 5a-d, which was characterized in solution by 1H NMR spectroscopy. The solid products were characterized by satisfactory elemental analysis and spectra studies. All the resulting complexes 3a-d, 4a-d and 5a-d were tested in vitro against a number of cell lines. For example, it inhibited K562 leukaemia cells with an IC50 value in the range of (3.00 -4.3) μM (1 h exposure) and displayed cathepsin B inhibitory action with an IC50 value in the range of (0.045-0.055 μM). . *Corresponding author: Abdel-Sattar S Hamad Elgazwy, Department of Chemistry, Faculty of Science, Ain Shams University, Abbassia 11566, Cairo, Egypt, Tel/Fax: 002024831836; E-mail: elgazwy@sci.asu.edu.eg Received July 22, 2013; Accepted August 29, 2013; Published August 30, 2013 Citation: Hamad Elgazwy ASS, Shehata MR, Zaki MY, Solima DHS, Elbakkry MM (2013) Synthesis of Novel Palladacycles Inhibitors of the Cathepsin B Activity and Antitumoral Agents. Med chem 3: 254-261. doi:10.4172/2161-0444.1000148 Copyright: © 2013 Hamad Elgazwy ASS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

A few examples of insertion of isocyanates into some other orthofunctionalized aryl palladium (II) complexes, leading to heterocyclic compounds have also been reported. The interest of this subject has prompted us to prepare aryl palladium complexes containing ortho--CHO functionalized. Herein, we report our results describing the preparation and full characterization of novel palladacycles 3a-d. In this context, the methodology of the synthesis of aryl palladium complexes 3a-d was studied. The reactivity of these aryl palladium complexes towards bulky of 1-naphthylisocyanate-(-N=C=O) and 1-naphthylisothiocyanate (-N=C=S), depend on the nature of substitution and the reaction conditions. The products of these reactions are mono-inserted complexes and prepared in single solvent, and no diffraction-quality crystals were grown by slow diffusion of Et 2 O into a CH 2 Cl 2 solution.
In addition to that, the electron releasing methoxyl groups (MeO) 3of the side chain could confer special properties to the formyl group, for example, facilitating its coordination to metallic centers to give cyclometalated species. Finally, this aryl moiety is present in organic molecules of pharmaceutical interest. For example, the antileukemic lactones steganacin, steganangin [45,46], the antibacterial agent trimethoprim [47], and the cytotoxic colchicines [48]. The cactus alkaloid, mescaline, beta-(3,4,5-trimethoxyphenyl)-ethylamine, has been studied for some years, because of its most interesting effects on the psychic states of human subjects. Since the elucidation of the chemical structure of the alkaloid through the synthesis by Spnth [49], a few other methods of preparation have been published [50,51].

Experimental Procedure General
Starting materials and commercial grade solvents were purchased from Sigma-Aldrich or Alfa Aesar and used without further purification. Reactions were carried out under nitrogen. Vero cells were from the American Tissue Culture Collection, The B16 cells were kindly donated by Dr. Nadia (Cancer Institute' Hospital). Reactions were carried out without precautions to exclude atmospheric moisture, unless otherwise stated. The IR and C, H, N Elemental analyses and melting point determinations were carried out as described elsewhere [69]. NMR spectra were recorded on Varian Unity 300 and a Bruker Unity 200 instruments. Chemical shifts were referred to TMS ( 1 H and 13 C{ 1 H})-NMR assignments were made with the help of DEPT techniques. Chromatographic separations were carried out by TLC on silica gel 60 ACC (70-230 mesh). Complex of "Pd-(dba) 2 " ([Pd 2 (dba) 3 ] dba), was prepared as previously reported [70,71].

Synthesis of Cis-[Pd{C6H(CHO)-6-OMe 3 -2,3,4}Br(L 2 )] (3ad)
General methods: 2-bromo-3,4,5-trimethoxybenzaldehyde 1 (1.19 mmol) was added to a suspension of "Pd(dba) 2 "(288 mg, 0.5 mmol) and N-donor ligands (0.5 mmol) in degassed acetone (15 ml) under nitrogen, and the resulting mixture was stirred at 0°C for 30 mins, and continue stirring at room temperature for 3 h. The solvent was evaporated in vacuo, the residue extracted with CH 2 Cl 2 (20 mL), and the resulting suspension filtered over anhydrous MgSO 4 . The solvent was concentrated to dryness and the resulting solid was separated by filtration, washed with Et 2 O (2×20 mL) and air-dried to give 3a-d as a yellow solid and air dried.   Reaction with bulky naphthylisocyanate and naphthylisothiocycnate 4.3.1) General methods reaction with naphthylisocyanate: Cis-[Pd{C 6 H(CHO)-6-OMe 3 -2,3,4}Br(L)2] 3a-d (0.1 mmol) was added to a suspension of naphthylisocyanate (14 µl, 0.1 mmol) in dry toluene (15 ml), and the resulting mixture was refluxing for 3 h. The solvent was concentrated to dryness and the resulting solid was separated by filtration, washed with Et 2 O (2×20 mL) and air-dried to give 4a-d as a yellow solid and air dried.  (15 ml), and the resulting mixture was refluxing for 3 h. The solvent was concentrated to dryness and the resulting solid was separated by filtration, washed with Et 2 O (2×20 mL) and air-dried to give a grey solid of 4a, yield; 41 mg, 70%

Cell culture
B16 and Vero cells were maintained in an atmosphere of 5% (v/v) CO 2 at 37°C. IC 50 values were obtained using published methodologies. Briefly, 5×10 3 cells/wellwere used to seed 96 well cell culture treated plates (Sigma). Compounds were dissolved at 5 mg ml-1 in sterile DMSO, and further diluted with the appropriate complete cell culture medium. After 72 h, cell viability was assessed using MTT(Sigma), also following published protocols [20,[72][73][74].

Growth inhibitory activity against the human K562 cell line
K562 human chronic myeloid leukemia cells were maintained in RPM1 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37°C in a humidified atmosphere containing 5% CO 2 , and were incubated with a specified dose of test agent for 1 h at 37°C in the dark. The incubation was terminated by centrifugation (5 min, 300 g), and the cells were washed once with drug-free medium. Following the appropriate drug treatment, the cells were transferred to 96-well microtitre plates. Plates were then kept in the dark at 37°C in a humidified atmosphere containing 5% CO 2 . The assay is based in the ability of viable cells to reduce a yellow soluble tetrazolium salt, 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma Chemical Co.) to an insoluble purple formazan precipitate. The optical density was then read at a wavelength of 550 nm on a plate reader, and a doseresponse curve was constructed. For each curve, an IC 50 value was read as the dose required reducing the final optical density to 50% of the control value.

Results and Discussion
In our present work, we describe the synthesis of several palladacycles in the form of TMEDA, bipyridyl and phenanthroline complexes, as well as the screening of these drugs for antitumor activity, depending on the cyclopalladated fine structure, different antitumor properties were observed involving inhibition of the respiratory activity.
Palladium Complexes 3a-d, 4a-d and 5a-d were tested for in vitro anticancer activity against a K562 human leukaemic cell line via a medium throughput screen. For comparison, a number of palladacycles 3a-d, 4a-d and 5a-d are presented in Table 1. However, complexes 3a-d, 4a-d and 5a-d display good in vitro activity, with an IC 50 of range (3.02-4.3 µM). Having established palladium complexes (3-5)a-d as a "hit" from this primary screen, we have evaluated it in other immortal cell lines namely B16 (Murine Melanoma) and Vero (African Green Monkey Kidney Epithelia) [52][53][54]. Preliminary data show 3a-d, 4a-d and 5a-d to have submicromolar activity (Figures 1  and 2). Furthermore, some related organo palladium complex has been tested for cathepsin B inhibitory activity [55] and registered an IC50 value of 2.98 µM, which was in the same range as that of the presented palladium complexes (3-5)a-d ( Figure 3).
It is better to present a generalized picture of mechanistic aspects of this processes begin with the oxidative addition of the organic halide to a Pd(0) substrate. These reaction seem to take place through a S N 2 mechanism which is consistent with other group [56][57][58], and the resulting cis-aryl Pd(II) complexes afforded as described in Scheme 2.
Another piece of evidence to support our configuration and structure determination of the resulting cis-aryl Palladium(II) complexes (3a-d) was presented by the reactions of (aryl) 2 mercury, and aryl mercury bromide with a variety of nitrogen ligands have been studied [59]. The presence of the electron-withdrawing heteroatoms results in these mercurials being stronger acceptors than the corresponding phenylmercury compounds. The most common routes to organomercury compounds involve the direct reaction of mercury with an alkyl bromide, to form the mercury analog of a Grignard Reagent (GR). The subsequent reaction of RHgBr with potassium cyanide yields the appropriate dialkyl mercury derivative. In order to obtain insight into the pathway of the reaction, we have prepared, these palladacycles (3a-d) [60][61][62][63][64]  . To our surprise, the reaction occurs without precipitation of any of the reaction products, and if acetone is removed and more water added, the byproduct RHgBr precipitates quantitatively leaving a yellow aqueous solution of some water-soluble arylpalladium(II) complexes. The dichloromethane solutions of 2,2'-bipyridine (bpy) or 4,4'-dimethyl-2,2'-bipyridine (dmbpy) or 1,l0-phenanthroline (phen) added and extraction of the water solution with more dichloromethane added, allow the isolation of complexes 3b-d in moderate yields (48,51,53) %, respectively. All of these compounds show in their IR spectra a strong band at ca. 1630-1660 cm -1 assignable to ν(CO) of the formyl gzroup. This frequency is similar to that observed in [HgR 2 ], HgRBr, or 2-bromo-3,4,5-trimethoxybenzaldehyde (1), indicating that there is no coordination of the formyl group to the metal atom.

Reaction with bulky 1-naphthyl isocyanate and isothiocyanate
Synthesis of cis-o-formylaryl palladium complexes 3a-d: As part of a systematic investigation to discover new organometallic approach, thus the insertion of 1-naphthylisocyanate (-N=C=O) and 1-naphthylisothiocyanate (-N=C=S) into the Pd-C bond, resulting in the formation of imidoyl palladium complexes, constitutes a key step in the palladium-catalyzed of organic substrates in laboratory synthesis and also in industrial processes. The palladacycles 3a-d was examined their reactivity towards naphthyl isocyanate in different molars in refluxing toluene afforded the corresponding spiro metalo-complexes of cis-arylpalladium complexes 4a-d and cis-arylpalladium complexes 5a-d in good yields. as described in Scheme 3. This is probably due to the result of the interchange between nitrogen donor of ligand and the bulky naphthyl isocyanate, which is a very well-unknown process. One might believe that palladium complexes as intermediates for the formation of complex 4a-d and/or 5a-d. This suggested that the presence of naphthylisocyanate or naphthylisothiocycanate as ligands, could be responsible for the interchange of the ligands and existence of complexes 4a-d or 5a-d in solution derived from two cis form.
These resulting compounds having 3,4,5-trimethoxyphenyl group similar to analogues of cactus alkaloid, mescaline [beta-(3,4,5trimethoxyphenyl)-arylmethylamine], that were used for induced changes of brain-cortex ribosomes [67]. In complexes 4a-d show in their IR spectra, a strong band at 1665 cm -1 assignable to ν(CO) group. This frequency is similar to that observed in iso-indenone, indicating that there is coordination of the formyl group to the isocyanate inserted. A piece of evidence to support our assumption that the reasons of inhibition the insertion of naphthyl isocyanate into bulky substrates, this caused by the steric hindrance of bulky reactants. The l H NMR spectrum at room temperature indicates that formation of the complexes seems to be favored by the potentially great steric hindrance and their ligands were in a square planar disposition. In this case, such steric hindrance should monitored by UV during the reaction process. All the reactions of aryl palladium complexes with 1-naphthylisocyanate (NCO) and 1-naphthylisothiocyanate (NCS) were investigated, and some of these results were consistent with the results reported recently [62][63][64]68]. The band assignable to ν(aryl), ν(bpy), ν(dmbpy) and ν(phen) in the IR spectra of the palladacycles are observed within the range 1515-1731 cm -1 and two bands at 1716 and 1615 cm -1 ; one of them may be due to the (C=C), and the other remaining band may be assignable to the ν(C=N) mode, corresponding to the ligands coordinated to the palladium atom. Complexes 3ad, 4a-d and 5a-d were tested for in vitro anticancer activity against a K562 human leukaemic cell line via a medium throughput screen. For comparison, a number of palladacycles 3a-d, 4a-d and 5a-d were synthesized and tested (Table 1). However, complexes of palladacycles 4a-d and 5a-d displays good in vitro activity, with an IC 50 of 4.3 µM.

Conclusion
Novel Palladacycles 3-5 give a good result of inhibitory activity against cathepsin B and leukimia cells in vitro over a wide concentration range. Palladacycles 3a-d is cytotoxic and inhibits cathepsin B with IC 50 values in the low µM range [52][53][54]. This study are aiming to address some complexes can be used as biological probes for proteins and biomolecules, e.g. cysteine, selenocysteine proteases [73][74][75]. All these exciting aspects of palladacycle chemistry will be divulged in due course.