Synthesis and Evaluation of Cytotoxic Effects of Amino-ester Derivatives of Natural α , β-Amyrin Mixture

Natural α,β-amyrins were isolated from endemic Brazilian Esenbeckia grandiflora Mart., and eight synthetic derivatives were obtained by esterification reactions with bromo acetate, followed by amine treatment. The structures of the all compounds were confirmed by H and C nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and high-resolution mass spectrometry (HRMS) data analysis. The derivatives were screened for cytotoxic activity against human tumor cell-lines PC3 (prostate carcinoma), HCT-116 (colon carcinoma) and HL60 (leukemia). HCT-116 and PC3 cell-lines showed weak tumor growth inhibition (range of 13.9-25.4 and 10.3-28.8%, respectively), but the derivatives presented moderate activity against HL60 (range of 13.6-59.0%). Diethyl, aniline, morpholine and imidazole moieties presented higher activities (range of 45.9-59.0%).


Introduction
Cancer is a generic term for a large group of diseases that can affect any part of the body, being also known by other terms such as malignant tumors and neoplasms.According to WHO (World Health Organization), it accounted 8.2 million deaths worldwide in 2012 (13% of all deaths) with 14.1 million new cases. 1Main risk in cancer treatment is multidrug resistance, when cells lose their sensitivity to chemotherapeutics, leading to intensify the action against cancer by developing and reinforcing of control programmers and to search alternative treatments.

Results and Discussion
The mixture of triterpenoid α,β-amyrin 1a,b was isolated from leaves of Esenbeckia grandiflora Mart., a Brazilian endemic plant from Rutaceae family (see Experimental section).Detailed 1 H nuclear magnetic resonance (NMR) analysis permitted to determinate the mixture at the 1:1 ratio of αand β-amyrin.The spectra displayed two triplets to H-12 at d 5.14 of the α-amyrin and d 5.19 of the β-amyrin, and its integrations showed 0.49:0.51proportion areas, respectively.All other analytical data were according to literature. 2 The α,β-amyrin derivatives 3-9a,b were synthesized by a modified literature procedure previously described (Scheme 1). 22Regarding this procedure, acylation of hydroxyl group of C-3 position was performed by treatment of mixture 1a,b with bromoacetyl bromide in CHCl 3 in the presence of anhydrous K 2 CO 3 at room temperature for 24 h, affording α-bromoacetyl mixture 2a,b derivative as a white solid with 85% yield.The Fourier transform infrared spectra (FTIR) showed absorption band at 1751 cm -1 , characteristic of C=O stretching of ester, corroborating with the structure of the synthetic product.The comparison of the 1 H NMR spectra of the product with the amyrins showed a new signal at d 3.84 due to the presence of bromoacetyl moiety, and the upshielding of H-3 signal (from d 3.24 to 4.58) confirms the esterification at C-3.The two new signals at d 167.1 and 28.0 registered in the 13 C NMR spectra also assigned the synthesis of 2a,b.Sequentially, the α-bromoester derivatives (2a,b) were submitted to a synthesis of amino-acetyl derivatives by simple treatment of these compounds with amine excess.To our delight, the reaction of α,β-amyrin derivatives 2a,b with diethylamine 3 in MeCN afforded a 3a,b mixture in 77% yield after 24 h as a white solid.The synthesis was corroborated by the 1 H NMR spectra analysis: the two signals at d 1.09 (6H, J 5 Hz) and 2.70 (4H, J 5 Hz), related to H 2a,b' and H 1a,b' , respectively, assure the presence of a new diethyl moiety.This same evidence was found in 13 C NMR spectra analysis by the two new intense signals at d 47.8 (C 1a,b' ) and 12.4 (C 2a,b' ).In the same way, the aminoacetyl structure was also assured by absorption band of C=O stretching at 1732 cm -1 observed in FTIR spectra.The same procedure was successfully applied to other amine derivatives (piperidine 4, morpholine 5, pyrrolidine 6, aniline 7 and benzylamine 8), it furnished aminoacetyl derivatives 4-8a,b with yields ranging 71-99%.The FTIR spectra of the prepared compounds showed C=O stretching due to the aminoacetyl moiety ranging 1724-1746 cm -1 .The 1 H NMR spectra of piperidine derivatives (4a,b) presented new signals at d 2.54 (4H, H 2" and H 6" ), 1.55 (4H, H 3" and H 5" ) and 1.44 (2H, H 4" ) when compared with the starting material, while 13 C NMR spectra revealed signals at d 60.4 (C 2" and C 6" ), 25.8 (C 3" and C 5" ) and 24.0 (C 4" ).These resonances are in accordance with the expected ones for the structure.The NMR spectra of the morpholine derivative 5a,b permitted to observe the presence of the oxy and aminomethylene signals at d 2.61 (4H, H 2" and H 6" ) and 3.76 (4H, H 3" and H 5" ), respectively, in the 1 H NMR spectrum, and at d 66.8 (C 3" and C 5" ) and 53.3 (C 2" and C 6" ), respectively, in the 13 C NMR spectra as well.For derivative 6a,b, d 2.86 (4H, H 2" and H 5" ) and 1.91 (4H, H 3" and H 4" ) in 1 H NMR and d 53.6 (C 2" and C 5" ) and 24.0 (C 3" and C 4" ) in the 13 C NMR assured the pyrrolidine moiety.For derivative 7a,b, new signals appeared at d 6.64 (2H, H 2" and H 6" ), 7.20 (2H, H 3" and H 5" ) and 6.77 (1H, H 4" ) in the 1 H NMR spectra, while in the 13 C NMR signals appeared at d 113.0 (C 2" and C 6" ), 129.3 (C 3" and C 5" ) and 118.2 (C 4" ) due to the aniline moiety.Benzylamine derivative 8a,b showed signals in the 1 H NMR spectra at d 3.87 (2H, H 4' ) due to the benzylmethylene hydrogens and the hydrogens of the aromatic ring at d 7.36 (5H).These findings were corroborated by the 13 C NMR data at d 53.1 (C 4' ), 138.6 (C 1" ), 128.5 (C 2" , C 3" , C 5" and C 6" ) and 127.4 (C 4" ).For the imidazole derivative, a slight modification in the synthesis was performed, which required DMF (dimethylformamide) as solvent 23 to lead 9a,b with 54% of yield.Their syntheses were corroborated by the appearance in the 1 H NMR spectra of signals at d 7.53 (1H, H 2" ), 7.11 (1H, H 4" ) and 6.96 (1H, H 5" ), while 13 C NMR spectra showed new signals at d 137.9 (C 2" ), 129.7 (C 4" ) and 199.9 (C 5" ).
The cytotoxic effects of aminoacetyl derivatives 3-9a,b were evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 24The compounds were incubated with three human tumor cell-lines (HCT-116, colon; HL-60, leukemia; and PC-3, prostate) at 5 µg mL -1 and the percentage reduction in cell viability (RCV) was determined after 72 h.The results presented in Table 1 are useful to carry out a preliminary structure-activity relationship study.Compounds 5a,b, 6a,b and 9a,b (morpholine, pyrrolidine and imidazole derivatives, respectively) presented medium activity against HCT-116.Compounds 5a,b and 7a,b (aniline derivative) showed medium activity against HL-60, while diethylamine and imidazole derivatives (compounds 3a,b and 9a,b, respectively) exhibited high activity against this cell-line.In addition, compounds 2a,b (bromoacetyl precursor) and 5a,b showed low activity against PC-3.Doxorubicin showed high cytotoxic effect against all tested cell-lines being used in this study as positive control of assay and not for the new compounds.
These results agree with previous reports of Barros et al., 21 in which ester derivatives of α,β-amyrin mixture demonstrated higher activity against tumor lines of leukemia cells HL-60 than other tested ones (MDAMB-435, melanoma; SF-295, glioblastoma; and HCT-8, colon cancer).However, in that previous study, Scheme 1. Reagents and conditions: (a) BrCH 2 COBr, K 2 CO 3 , CHCl 3 , room temperature, 24 h, 85%, and (b) amine (3 equiv.),MeCN or DMF*, room temperature, 24 h, 54-99%.the higher activity of the maleinate derivative attached to carbon 3 was assigned due to the presence of the unsaturation in the lateral chain.In the present study, higher activities were exhibited by diethylamine 3a,b and imidazole 9a,b derivatives.While the latter exhibits double bond in the structure, the first does not display.The presence of the nitrogen atom in the side chain generates an electron-rich center of coordination to promote chemical interactions with center deficient in electrons.Nevertheless, all other derivatives with lower activity contain nitrogen in its structure.Also, comparisons on ring sizes (five or six membered) and/or aromaticity in side chain are not conclusive.Free alkyl chains in diethylamine moiety and a second coordination atom in the imidazole ring seem to be the most prominent features of the most active derivatives, although more studies are needed for more conclusive structure-activity relationship.
In order, we tested the compounds in different concentrations (0.05-25 µg mL -1 ) against HCT-116 and PC-3 cells by MTT assay in order to determine the concentration able to reduce the number of viable cells by 50% (RC 50 ) after 72 h of incubation.The results show that none tested compound was active against the human cancer cell-lines used (RC 50 > 25 µg mL -1 ).Doxorrubicin (Dox) (positive control) showed RC 50 values from 0.01 (0.08-0.12) and 0.15 (0.10-0.21) µg mL -1 against HCT-116 and PC-3, respectively.

Conclusions
In conclusion, a series of novel aminoacetyl α,β-amyrin derivatives were synthesized and their cytotoxic activities were evaluated against human colon carcinoma HCT-116, human acute promyelocytic leukemia HL-60 and human prostate adenocarcinoma PC-3 tumor cell-line using an in vitro cytotoxicity assay.Among these compounds, diethylamine and imidazole derivatives (compounds 3a,b and 9a,b, respectively) displayed the most potent cytotoxic activity and were found to be more effective against HL-60.These preliminary results showed that the synthesis of new derivatives is required to improve anticancer activity in vitro of the tested compounds.

General experimental procedures
All reagents were purchased from Sigma-Aldrich and Acros Chemicals and used without further purification.α,β-Amyrin mixture was previously isolated from Eriope blanchetti (Lamiaceae).Chloroform was refluxed with CaH 2 and distilled prior to use.Acetonitrile was used in HPLC grade.N,N-Dimethylformamide (DMF) was treated with CaH 2 , distilled and stored on 4A molecular sieves.Aniline, benzylamine, morpholine, piperidine and pyrrolidine were distilled priori to use.All reactions were performed under argon atmosphere.Analytical thin layer chromatography (TLC) was performed on E. Merck TLC plates pre-coated with silica gel 60 F 254 (250 µm thickness).The visualization was accomplished using UV light and potassium permanganate solution.Column chromatography (CC) was performed on silica gel (60-120 Å pore size).The melting points were uncorrected and determined on a MQAPF-302 apparatus.FTIR spectra were measured using a Shimadzu IRAffinity-1S spectrophotometer.NMR spectra were recorded on Varian spectrometer Inova-500 in deuterated solvents.Electrospray high-resolution mass spectra (ESI-HRMS) were obtained using Agilent 6550 Q-TOF MS instrument in the positive mode.

Plant material: collection and identification of Esenbeckia grandiflora
Leaves of Esenbeckia grandiflora were collected at the surroundings of the city of Barra dos Coqueiros, Sergipe State, Brazil (10 o 48'20"S; 36 o 58'10"W).The specimen was identified by Dr Milton Groppo, and a voucher (No. 06137) was deposited in the Herbarium of the Departamento de Biologia of Universidade Federal do Sergipe (UFS), city of São Cristovão (Sergipe State, Brazil).

Isolation and identification of α,β-amyrin
The leaves (1.30 kg) were dried on shadow, and then were powdered and extracted with methanol for 36 h.The obtained filtrate was concentrated under reduced pressure in a rotary evaporator at 40 °C, which yielded 7.35 g of extract.The methanolic extract was partitioned with hexane/MeOH:H 2 O (9:1), CHCl 3 /MeOH:H 2 O (6:4) and EtOAc/H 2 O.The hexanic extract (5.35 g) was submitted to a filtration CC in silica-gel 60 using hexane/EtOAc in a growing order of polarity as mobile phase.The fractions were grouped by comparative TLC and submitted to new chromatographic partition.The mixture of α,β-amyrin (835.0 mg) was isolated from the fraction eluted with hexane/EtOAc (8:2).These compounds had their structures elucidated by analysis of the NMR data ( 1 H, 13 C, polarization transfers (DEPT 90 o and DEPT 135 o ), heteronuclear multiple-quantum correlation spectroscopy (HMQC) and heteronuclear multiple bond correlation (HMBC)) and by comparison with literature data. 2 Vol.28, No. 11, 2017

Synthesis of 3-bromoacetyl-α,β-amyrin esters (2a,b)
The mixture of α,β-amyrin (1a,b) (100 mg, 0.24 mmol) was dissolved in 5 mL of CHCl 3 in the presence of anhydrous K 2 CO 3 .After completely dissolving, bromoacetyl bromide (73.5 mg, 0.36 mmol) was added at room temperature and the mixture was stirred for 24 h in inert atmosphere.Then, the reaction mixture was evaporated and the resulting solid was chromatographed on a silica gel column eluted with CHCl 3 :MeOH (95:5) to afford compound 2a,b in 85% yield; FTIR (KBr) n / cm  General procedure to synthesis of α-aminoacetyl-α,β-amyrin esters (3-9a,b) 0.10 mmol of α-bromoesters 2a,b was dissolved in 5 mL of MeCN and mixed until complete dissolution.After, 0.30 mmol of amine was added to solution.The reaction proceeded for 24 h at room temperature under inert atmosphere.The reaction product was concentrated under vacuum and the resulting white solid was chromatographed on a silica gel column eluted with CHCl 3 :MeOH (9:1).For imidazole derivative, DMF was employed as solvent to lead 9a,b.