Synthesis , Characterization and in vitro Anticancer Activity of Novel 8 , 4 ’-Oxyneolignan Analogues

Neolignans are a class of natural products with a wide range of biological effects. These substances are of great synthetic and biological interest, especially in searching for novel anticancer agents. In this paper, we report the synthesis of a new subclass of 8,4’-oxyneolignan analogues (β-ketoethers and β-ketoesters) and their cell viability assay on twenty four different cancer cells, among leukemias and carcinomas. Three compounds inhibited the growth of most human cancer cells. 2-Oxo-2-phenylethyl(2E)-3-[4-(2-oxo-2-phenylethoxy) phenyl]prop-2-enoate showed an antiproliferative activity superior to doxorubicin for U-87, U-138 MG and H1299 cell types and (E)-2-oxo-2-phenylethyl 3-(3-methoxy-4-(2-oxo-2-phenylethoxy)phenyl)acrylate was found to be very selective, demonstrating a growth inhibition of 92.0% against KG-1 cells. Furthermore, 1-oxo-1-phenylpropan-2-yl cinnamate exhibited significant inhibition activity in a range of 52.2 to 91.2% against twelve kinds of leukemia cell lines, revealing excellent results and very comparable to the reference drug.


Introduction
Cancer is a generic term for a large group of diseases that can affect any part of the body and remains a leading cause of death worldwide.It is considered a public health problem according to the World Health Organization, and many efforts have been made towards its prevention and cure.Treatment usually involves a series of interventions, and approximately 90% of tumors can be treated with antiblastic drugs, 1,2 which makes chemotherapy the most used treatment.3][4][5] As a consequence, there is an urgent need for novel and effective drugs that act against cancer.
2][33][34] Therefore, this type of natural product is of great synthetic and biological interest, especially in searching for novel anticancer agents.In this paper, we report the synthesis of new 8,4'-oxyneolignan analogues and the cell viability assays for different neoplasms, among leukemias and carcinomas.

In vitro cell viability assay -MTT assay
All steps in this assay were automated in Liquid Handling Workstation epMotion ® 5070 (Eppendorf, Vaudaux, Schonenbuch, Switzerland).Cells were distributed in 96 wells (100 μL cells well -1 ) and incubated for 48 h, before addition of test compounds.Cells were then exposed to the compounds at a concentration of 10 μM.After 24 h of exposure at 37 °C, cell viability was determined by colorimetric MTT 35 (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) based on the metabolic ability of active cells to convert the yellow MTT reagent into a blue insoluble salt (formazan), which is spectrophotometrically measured.Then, the amount of formazan produced was dissolved in solution containing 150 μL of isopropanol and optical density was read by a spectrophotometer at 570 nm (Bio-Tek Power Wave XS).Absorbance of wells containing the compounds and those with cells in control (cells treated with vehicle, 0.1% DMSO) were compared to estimate the cell viability.The results were expressed as inhibition percentage relative to control (considered as 100%) and doxorubicin was used as a reference drug.Compound 6c was also evaluated for cytotoxicity against human leukemia cells, using the MTT method.Doxorubicin was used as positive control.All assays were performed in triplicate and mean ± standard deviation (SD) values were used to estimate cell viability.

Chemistry
In this work, 25 oxyneolignan analogues (β-ketoethers or β-ketoesters) were synthesized and the synthetic strategies for their preparation are summarized in Scheme 1.The β-ketoethers and β-ketoesters analogues 3a-3j, 4a-4c, 5a-5b, 6a-6f, 7a-7b, 8 and 9 (Table 1) were obtained following a procedure described by Barata et al. 26 Initially, the α-bromoketone intermediates 2a-2b were prepared according to a known procedure, 26 then, without any purification due to their lacrimogenic property, were reacted with an in situ generated phenoxy and/or carboxylate ion.These condensation reactions were carried out in the presence of K 2 CO 3 and butanone as solvent furnishing the products in yields ranging from 40 to 100%.The phenolic/carboxylic compounds were used in an excess of 2% instead of using excess of α-bromoketone.During isolation, the volume of solvent was reduced to 1/3 of the initial volume before work-up, since the slight solubility of the solvents in water hampers the isolation procedure.
In order to obtain compounds 7a-7b and 8, previous esterification of the cinnamic acid derivatives with methanol and sulfuric acid was mandatory to avoid the competitive nucleophilic displacement of the bromine atom by the carboxylate anions.This was confirmed in the obtention of compounds 4a-4c and 5a-5b by using 2 equivalents of 2a-2b.As far as we know, this creates a new subclass of 8,4'-oxyneolignan analogues, which we called 8,4'-oxyneolignan cinnamic analogues.Compound 9 was synthesized in a similar fashion.The 1 H NMR spectra of the products show the presence of one peak (compounds 3, 6, 7 and 8) or two peaks (compounds 4, 5 and 9) in between 5.12-6.14ppm, assignable to the CH 2 /CH carbinolic protons.These signals confirm the formation of the C-O-C bond and, consequently, the expected products.
All synthesized substances were purified by crystallization and fully characterized by usual spectroscopic methods (melting points, HRMS, 1 H and 13 C NMR).The chemical structures are described in Table 1.

Biology
To evaluate the antiproliferative activity, the amount of surviving cells at the dose level of 10 μM was measured after 24 h of incubation by the MTT method colorimetric assay 35 and the results were expressed as percentage of inhibition relative to control and compared with the reference drug (doxorubicin).The tests were carried out in triplicate, using doxorubicin as positive control and these data are schematically listed in Table 2. Vol. 28, No. 11, 2017   The antiproliferative screening results show that five compounds presented a promising antiproliferative activity (≥ 50% of cell inhibition) against the leukemia cell lines.The other analogues were less active or completely inactive at the dose of 10 μM.Compounds 4a, 4b, 6d and 9 (entries 11, 12, 19 and 25, respectively) demonstrate similar inhibition of growth proliferation against HL-60, Ramos and Nalm-6 (48.6-66.7%).The same substances did not reveal a good inhibition level against K-562 cells.The best compound of the series was 6c (entry 18) inhibiting cell proliferation over 50% against four leukemia cell lines and very comparable to the positive control used in this test (entry 26).For Nalm-6 and Ramos cells, the percentage of inhibition was even superior to doxorubicin inhibition, presenting 73.2 and 84.6%, respectively.
Compounds which presented significant activity profile against leukemia cells (4a, 4b, 6c, 6d, 9) were selected to be evaluated for their activity on cell proliferation on other nineteen different kinds of human neoplasms, among tumors and leukemia cell lines.These tests were performed by using the MTT method, as previously mentioned.The cells used in this evaluation are listed in the Experimental section and the obtained results are shown on Tables 3-4 and Figures 1-2.
The evaluation of compounds 4a, 4b, 6c, 6d and 9 on leukemic cells are expressed in Table 4 and Figures 3-4.
Compound 6c was found to be the most promising of the series, presenting antileukemic activity higher than 50% for all cell lines used in this study and superior or comparable results when compared to the reference drug.In the case of KG-1 cells, the extent of inhibition levels of products 4b and 6c were even better than doxorubicin, indicating its possible efficacy against this leukemia type.
Considering the results obtained in the antiproliferative tests (Table 4, Figure 4), in vitro cytotoxicity assay was used to assess the activity of the most potent compound (6c).The analogue was tested against eleven human leukemic cell lines.The assays were carried out in triplicate, and doxorubicin was used as the positive control.The biological endpoint was determined according to the concentration,  which causes fifty percent of cell growth inhibition (IC 50 ).Compound 6c showed similar values of IC 50 against leukemia cells (Table 5), ranging from moderate to good activity, except for K-562 cells, for which the drug showed no activity.Compound 6c induced better cytotoxic effects on Ramos cells, which presented an IC 50 of 9.4 μM.
The correlation between the structures of the synthesized compounds and their antiproliferative activities leads to the conclusion that the compounds bearing a cinnamic moiety on its structure possess better potency in MTT assay.The presence of the cinnamic portion in the basic structure of the 8,4'-oxyneolignans and the variation on its position on the aromatic ring affect considerably the antiproliferative activities.When comparing compounds 4a-4b and 5a-5b, 7a-7b and 8 better inhibition showed by compounds 4a and 4b against cancer cells could be attributed to its para substitution, whereas meta-substituted compounds 5a-5b did not show an expressive percentage of inhibition.However, in compounds 7a-7b and 8, the cinnamate moiety and the absence of an aromatic ring may be responsible for the drastic decrease in activity.
In the 8,4'-oxyneolignans 3a-3j, the effects of substitution on the aromatic rings were not clearly observed.These compounds did not show enhanced activity when comparing substituent groups, positions and side chain length.
Among compounds 6a-6f, as shown in Tables 2 and 3, the effect of alkyl chain substitution in compound 6c greatly  decreased cell proliferation when compared to compound 6a.In the same aspect, the presence of a halogen substituent in compound 6d intensified the cytotoxicity level compared to 6a and also compared with 6b, which possesses an electron-withdrawing group at the same position.These results suggest that the electronic characteristics of the substituent groups and the alkyl chain affect the capability of the molecule to interact with the bioactive target increasing/ decreasing the antiproliferative activity.

Conclusions
From our study we were able to produce a new subclass of neolignan analogues, the 8,4'-oxyneolignan cinnamic analogues.These compounds were evaluated against a variety of cancer cell lines, among tumors and leukemias.In tumor cells assays we identified that compound 4a showed an antiproliferative activity superior to doxorubicin for U-87, U-138 MG and H1299 cell types.Compound 6c exhibited significant inhibition activity in a range of 52.2 to 91.2% against twelve kinds of leukemia cell lines, revealing excellent results and very comparable to the reference drug.In addition, compound 4b was found to be very selective, demonstrating a growth inhibition of 92.0% against KG-1 cells.These preliminary results suggest that further investigation is needed to elucidate the characteristics underlying the antiproliferative activities of these analogues.

Figure 2 .
Figure 2. MTT assay for human tumor cell lines of compounds 4a and 4b.

Figure 4 .
Figure 4. MTT assay for human leukemia cell lines of compounds 4b and 6c.

Table 1 .
Chemical structures, yields, and melting points (mp) of the synthesized compounds

Table 1 .
Chemical structures, yields, and melting points (mp) of the synthesized compounds (cont.)

Table 2 .
Evaluation of cytotoxicity towards leukemia cells (% inhibition) a for all synthesized compounds

Table 5 .
In vitro cytotoxicity of compound 6c against leukemia cell lines IC 50 >100 μM means not active; NT: not tested.