Synthesis of new heterocycle-based selenoamides as potent cytotoxic agents

We report the synthesis of a new series of aryl-and heteroaryl (hydroxy)ethyl selenoamides, in a two-step, one-pot sequence based on the aminolysis/selenative demetalation of Fischer ethoxycarbene complexes, in good to excellent global yields, as small cytotoxic molecules. The molecular structure of a 2-thienyl based selenoamide was confirmed by single-crystal X-Ray diffraction analysis. In vitro analysis against different human cancer (HCT-15, U251 and PC-3) and human T-lymphocyte (MT2) cell lines revealed that the 2-thienyl based selenoamide can be considered a potent and selective compound against the human prostatic adenocarcinoma (PC-3) cell line with an IC 50 value of 14.5 µM.


Introduction
][10][11][12] A wide spectrum of organoselenium compounds has been tested against different human cancer cell lines, showing promising cell growth inhibition.In this context, our research group developed the synthesis of a ferrocene(hydroxy)alkylselenoamides family, 13 that act as cytotoxic agents against different human cancer cell lines, especially on MCF-7, HCT-15, and U251, exerting superior activities than those displayed by marketed drugs tamoxifen and cisplatin.This behavior has placed these compounds in an emerging and very promising class of ferrocene antitumor agents, [14][15][16] allowing the establishment of some structural key fragments for synthesizing new potentially cytotoxic selenoamides.Considering the precedent results obtained for leader ferrocenyl selenoamide, we decided to modify the structure at the aromatic ring position of this compound, based on the assumption that this substituent could enhance the bioactivity by promoting the selectivity or potency of these new compounds.For instance, we chose diverse aromatic rings, including p-methoxyphenyl and methylenedioxyphenyl, and different heterocycles like N-methylpyrrol-2-yl, N-methylindol-2-yl, thien-2-yl and furan-2-yl.These rings are frequently occurring motifs in drug-like molecules (Figure 1). 17he conversion of carbonyl groups into the corresponding selenocarbonyl compounds through treatment with various selenating reagents possessing reactive metal-selenium bonds [20][21][22][23][24] or Woolins reagent [25][26] remain as the most accessible approaches to obtain this kind of compound, despite the difficulty of preparing and using such selenating reagents.As an alternative, we recently developed a methodology that uses elemental selenium in combination with NaBH4 as the reagent to introduce selenium functionalities into organometallic compounds in mild reaction conditions. 13ntinuing with our studies related to the chemistry of selenocarbonyl compounds, we describe here the synthesis and study of the cytotoxic properties of new aryl and heteroaryl selenoamides, with the aim to obtain new potent and selective organoselenium compounds.We used as key starting material a family of

AUTHOR(S)
Fischer carbene complexes that allows the easy introduction of a selenocarbonyl functionality, in a two-step, one pot synthesis.

Results and Discussion
4][15][16][17][18][19] The complex 1c is new and exhibits the characteristic absorption bands around 2057 and 1905 cm -1 assigned to M-CO.From the 13 C NMR spectrum, we identified a signal at 339.8 ppm assigned to C=Cr.We also observed two signals around 223.8 and 216.7 ppm assigned to (CrCOax) and (CrCOeq), respectively.After preparing the ethoxycarbene complexes, these compounds were exposed to an aminolysis reaction using ethanolamine, affording the corresponding Fischer aminocarbene complexes (2a-h) in good yields (Table 1).The new carbene complexes showed in their infrared spectra bands around 2054, 1974 and 1911 cm -1 characteristic of Cr-CO.In all cases, a molecular ion was observed in the mass spectra (FAB+).In the 13 C NMR spectra, we observed a characteristic signal around δ 270 ppm assigned to carbenic carbon as well as signals around δ 210-225 ppm for Cr-CO.All the aminocarbene complexes, with the exception of 2e and 2h, were obtained as a mixture of non-separable E/Z geometric stereoisomers.The 1 H NMR spectra displayed two sets of signals, the most easily seen being those assigned to the NH group, with a shift around 9.4 ppm for the E stereoisomer and around 9.0 ppm for the Z stereoisomer.3] However, for 2h, the Z stereoisomer is the only species, 13 as result of the steric interaction between the ferrocenyl group and the hydroxyalkyl chain.With the aminocarbene complexes in hand, we carried out their selenative demetalation to obtain the arylselenoamides (3a-h), using the protocol earlier developed by our group for synthesizing ferrocenyl selenoamides. 13,31 his procedure provided the new arylselenoamides as yellow-red solids, in 15 to 45 min, depending on the ethoxy carbene complex used as precursor (Table S1, SI).To increase the global yields of these compounds, we attempted a two-step, one-pot synthesis based on the aminolysis-demetalation reaction as a key strategy.This improved the global yields of the selenoamides by up to 80% starting from the respective commercially available aromatic ring compound (Scheme 1).No selenols were detected in the reaction mixture and the persistence of the OH functional group in all the products obtained, confirmed the selectivity and tolerance of the reagent formed by the mixture Se/NaBH4, providing a useful method for obtaining selenocarbonyl derivatives in mild reaction conditions via Fischer carbene complexes.

Lead
Scheme 1. Synthesis of aryl selenoamides in one-pot, two-step conditions.a Global yield calculated from the corresponding aryl (or heteroaryl) substrate.
The arylselenoamides were characterized using conventional spectroscopic techniques.They exhibited in infrared spectra bands around 1440, 1090 and 650 cm -1 .These bands are characteristic of a mixed vibrational mode assigned to the fragment N-C=Se, where the stretching vibration (C=Se) is strongly coupled to (C-N). 34- 35In all cases, the mass spectrum (EI) of each selenocarbonyl displayed a molecular ion in concordance with the molecular formula for each compound and confirmed the complete removal of metallic fragment [Cr(CO)5].The 1 H NMR spectra of these compounds show the signal of selenoamide proton shifted to low-field around 8-9 ppm revealing a possible intramolecular interaction via a hydrogen bond between N-H … OH.This behavior is more evident in the case of compound 3c, which shows both signals more shifted to high frequencies compared to those displayed for the other members of this series of compounds.Likewise, the 13 C NMR spectra showed a signal around δ 203 ppm assigned to C=Se for benzene based selenoamides, and high-field shifts around δ 185 ppm for their heterocyclic analogues.The purity of selenoamides obtained was certified using different analytical techniques. 36 addition, we confirmed the structural arrangement of the thienyl based selenoamide 3f 37 through singlecrystal X-ray diffraction analysis (Figure 2).This study revealed that selenocarbonyl moiety is directly bonded to the α position of the thiophene ring; the sum of bond angles around C(6) (Σ = 359.98°)indicates the trigonal geometry of this group.The C=Se double bond distance [Se(1)-C(6) 1.836(2)Å] is quite similar to other reported selenoamides; nevertheless, the N(1)-C(6) 1.321(2) Å bond distance is relatively longer.2] Once formed, A reacts with the electrophilic Fischer carbene forming the intermediate B. This species loses the metallic fragment [Cr(CO)5] and (H2BSe -), affording the corresponding selenoamide.In an attempt to obtain insight on the plausible pathway of this transformation, we conducted two additional experiments.When this reaction as conducted in stochiometric conditions, we only recovered the selenoamide coordinated with [Cr(CO)5] in low yields, which implies that the 10-fold excess of selenating reagent (Se/NaBH4) favors the complete removal of chromium fragment.On the other hand, when we used benzyl bromide in the place of the carbene complex, we obtained quantitively the corresponding benzyl diselenide.This result confirms the identity and the nucleophilic character of adduct A. Likewise, after the work-up of the reaction, we observed the formation of a black powder, presumably due to the disproportion of C affording black selenium.] The initial screening data listed in Table S2 show a substantial difference in the bioactivity displayed by these new selenoamides in comparison with the ferrocenylselenoamide Lead (3h), which possesses a really good activity towards HCT15, U251 and specially against MCF-7. 13Compounds 3(a-g) display good activity towards HCT-15, U251 and particularly against PC-3 human cancer cell lines.Comparing the percentage of inhibition of selenoamides 3(a-g) in these cell lines, we observed good results for 3b, 3c, 3e and 3f, with values greater than 65% of cell growth inhibition (Table S3, entries 2, 3, 5 and 6).
To correlate the effect of the aromatic ring on the structure of these selenoamides with their biological activity, we chose the HCT-15, U251 and PC-3 cancer cell lines.Thus, the IC50 values against the selected human cancer cell lines were determined and compared with the ferrocenyl selenoamide Lead (3h) and two well-known commercial drugs, etoposide and cisplatin as references.Likewise, to know the cytotoxic selectivity, we also determined the IC50 values against MT2 cells (human T-lymphocytes).The results listed in Table 2 show that the aromatic fragments included in this series of compounds do not improve the inhibitory activity of ferrocenyl selenoamide (3h, Lead) in cell lines HCT-15 and U251.Only the selenoamide 3a exhibited activity close to cisplatin in HCT-15 (Table 2, entries 1 and 9).Comparing the IC50 obtained in HCT-15 for 3h and etoposide, the former exhibits a better biological response (entries 8 and 10).
On the other hand, when we compare the biological response obtained in the PC-3 cell line, we observed that selenoamides 3a, 3d, 3f and 3h exhibit a better cytotoxic activity than cisplatin and etoposide, with IC50 values between 14-17 M (Table 2, entries 1, 4, 6 and 8-10).These results are particularly interesting, showing that the incorporation of a 2-thienyl fragment, instead of the ferrocenyl motif, improves the cytotoxic activity of the selenoamide (Table 2, entries 6 and 8).Although the nature of these fragments -aromatic rings with electron-donor properties -we consider that it is not only electronic effects playing a role in the biological response.Both steric hindrance and −stacking interactions could facilitate a better recognition of the biological target responsible of the cytotoxic response.Finally, with the aim of knowing the cytotoxic behavior of compounds 3(a-h) in normal cells, the selenoamides were also tested against human T-lymphocytes (MT2) cells.In general, IC50 values determined for non-cancer MT2 cells are higher than those found in cancer cell lines (Table 2).On the contrary, cisplatin was shown to be more cytotoxic against MT2 cells.These results reveal a high selectivity towards cancer cell lines and allow us to consider this family of molecules as good candidates for further chemical variations, focused on improving their pharmacological behavior.

Conclusions
In conclusion, we have optimized the synthesis of novel aryl and heteroaryl selenoamides through a two-step sequence in one-pot based on the aminolysis/selenative demetalation reactions of Fischer ethoxycarbene complexes, in excellent global yields to obtain cytotoxic small molecules.The synthetic methodology is simple, efficient, and robust, allowing the introduction of several structural motifs.
The biological assays revealed that varying the aromatic ring directly attached to selenoamide moiety resulted in diverse cytotoxic activities IC50 towards HCT-15, U251 and PC-3 human cancer cell lines as compared with ferrocene selenoamide lead and the well-known reference marketed drugs, cisplatin and etoposide.This evaluation showed that modifications in the nature of the aromatic rings can improve the biological response of these organoselenium compounds.The IC50 values determined for MT2 cells were higher than those for HCT-15, U251 and PC-3 cell lines, which indicates a very good selectivity towards human cancer cells.
Likewise, these results along with the structural modifications, indicate that these selenocarbonyl compounds are good candidates for conducting a further QSAR model to reach an in-depth understanding of their pharmacological response behavior.

Experimental Section
General. Materials and Instruments.THF and diethyl ether were distilled from sodium/benzophenone under a nitrogen atmosphere.All reagents and solvents were obtained from commercial suppliers and used without further purification.All compounds were characterized by IR spectra, recorded on a Bruker Tensor 27 spectrophotometer, by KBr or film techniques, and all data are expressed in wave numbers (cm -1 ).Melting points were obtained on a Melt-Temp II apparatus and are uncorrected.NMR spectra were measured with a JEOL Eclipse +300 and Bruker Avance III 300, using CDCl3 and CD3CN as solvents.Chemical shifts are in ppm (δ), relative to TMS.The MS-EI were obtained on a JEOL JEM-AX505HA using 70 eV as ionization energy and for MS-FAB on a JEOL SX 102A.All tested compounds synthesized are more than 95% pure, analyzed using HPLC HP 1100 with diode-array detector.

Synthesis of Fischer type-carbene chromium(0) complexes 1(a-h).
4][15][16][17][18][19] To a solution of the corresponding aryl substrate (8 mmol) in anhydrous THF (10 mL) under argon atmosphere was added at 0 °C, a solution of nBuLi (8.2 mmol).The reaction mixture was stirred at rt for 20 to 60 min and then transferred by cannula to a suspension of Cr(CO)6 (1.74 g, 8 mmol) in THF (20 mL), the mixture was then stirred for the time specified in Table 1, at rt.The solvent was removed under vacuum and to the residue was added the mixture obtained from triethyloxonium tetrafluoroborate (2 g, 10 mmol) added to ice/water (ca.20 g).The organic phase was washed with a saturated solution of NaHCO3 and then with brine.The organic phase was dried with anhydrous Na2SO4 and the solvent was evaporated under vacuum.The mixture was purified by chromatography on silica gel or alumina using hexane as eluent.29][30][31] Synthesis of selenoamides (3a-h) in one-pot two-step conditions (a) Synthesis of aryl Fischer hydroxyethylamino carbene complexes 2. To a solution of the corresponding Fischer ethoxycarbene complex 1 (1 mmol) in 10 mL of anhydrous Et2O under N2 atmosphere was added ethanolamine (2.1 mmol).The reaction mixture was stirred at rt for 5 to 20 min and then diluted with water (10 mL).The organic phase was separated and dried with anhydrous Na2SO4, and the solvent was evaporated in vacuum.The crude product was used for the next reaction step.(b) Synthesis of selenoamides 3(a-h).Preparation of selenating agent.To a solution of of NaBH4 (0.01 mol) in EtOH (10 mL) was added of powdered selenium (0.01 mol), and the mixture was vigorously stirred at rt for 30 min under N2.The selenating agent was then added to a solution of the corresponding amino carbene complex 2, previously obtained, in EtOH (5 mL), under N2.The reaction was monitored by TLC on silica-gel.
After the reaction was completed, the solvent was evaporated under vacuum, the residual mixture was dissolved in distilled water and the product was extracted with CH2Cl2 and then dried with anhydrous Na2SO4.After the evaporation of the solvent, the resultant mixture was purified by silica-gel column using hexane/CH2Cl2 (1:1) as eluent.
Determination of purity.The purity of the final products is close to 95-99% and was determined using 60:40 and 40:60 (hexane/ammonium acetate) as the mobile phase with a flow rate of 0.2 mL/min on a Zorbax Bonus RP column (3.5 μm, 2.1 x 100 mm, Agilent).
X-ray crystallography.Suitable X-ray quality crystals of 3f were grown by slow evaporation of a CHCl3 solution at rt.A crystal of 3f was mounted on a glass fiber at room temperature, then placed on a Bruker Smart Apex CCD diffractometer, equipped with Mo KR radiation; decay was negligible in both cases.Systematic absences and intensity statistics were used in space group determination.The structure was solved using direct methods. 45Anisotropic structure refinements were achieved using full matrix, least-squares technique on all non-hydrogen atoms.All hydrogen atoms were placed in idealized positions, based on hybridization, with isotropic thermal parameters fixed at 1.2 times the value of the attached atom.Structure solution and refinement were per-formed using SHELXTL V6.10. 46

Figure 1 .
Figure 1.Design for the obtainment of potentially cytotoxic arylselenoamides.

Table 1 .
Synthesis of ethoxycarbene complexes (1a-h) and aminocarbene complexes (2a-h) a Yields obtained of pure product after flash column chromatography purification on SiO2.b The yield is given for the mixture of E/Z conformers.c Phenyllithium (available commercially) was used as starting substrate, going directly to step 2. d The formation of these lithium derivatives was conducted at -78 °C.e t-BuLi was used as base.f Unstable compound in solution.