Morita-Baylis-Hillman Reaction with 7-Chloroquinoline Derivatives-New Compounds with Potential Anticancer Activity

Morita-Baylis-Hillman adducts (MBHA) is a class of polyfunctional molecules that has been standing out due to their versatility and expressive biological activities. Therefore, this paper describes the synthesis and antiproliferative activity of some new MBHA/7-choroquinoline hybrids. The Michael acceptors were obtained starting from 4,7-dichloroquinoline which were submitted to the Morita-Baylis-Hillman reaction with ortho, meta and para-nitrobenzaldehyde. The in vitro screening of the synthetized MBHA against NCI-H292, HCT-116 and MCF-7 cancer cells suggests the influence of the spacer chain in its inhibition potential. The 50% inhibitory concentration (IC50) obtained in the antiproliferative assay using MCF-7, HCT-116, HL-60 and NCI-H292 cancer cells indicate expressive cytotoxic potential of the adducts containing nitro group in the ortho position, with IC50 of 4.60 μmol L. MBHA/7-choroquinoline hybrids were more active than MBHA described in literature, indicating the improvement of the cytotoxic effect due to 7-chloroquinoline moiety in the molecular structure, with maximum selectivity index values of 11.89.


Introduction
Cancer is a generic word for a group of diseases that cause disordered growth of cells. It is the second cause of death in the world, causing about 10 million deaths in 2018. Some examples of the most common cancer are colorectal, breast and lung cancer. 1 Cancer can be initiated by intrinsic factors or factors external to the body that lead to genetic changes. Tumor development can occur anywhere in the body and spread beyond its place of origin through the process known as metastasis, making it difficult to treat the disease. 1,2 The increase in the incidence of various types of cancer creates a need for new anticancer drugs. For example, 1.7 million of new cancer cases and 606,880 cancer deaths were projected to occur in 2019 in the United States. 3 Quinolines belong to an important class of heterocycles, and have derivatives with interesting biological activities. 4 Quinoline core containing compounds are studied because of their demonstrated inhibition activity of protein kinases and inhibition of cell growth and interaction with the growth factor receptor. 5 Derivatives of the 7-chloroquinoline nucleus have been studied in recent years as candidates for various activities such as antitumor, leishmanicidal, antinociceptive and anti-inflammatory activities, antituberculosis and antizika virus. [6][7][8][9][10][11] Hybridization of this nucleus with other groups may increase its activity, since hybrid molecules may have double or multiple actions, depending on the different pharmacophoric units linked to a single chemical entity. Moreover, the potential advantages of this strategy include lower effective therapeutic doses and the reduction of drug resistance. 12 One way to hybridize chemical groups is through the Morita-Baylis-Hillman reaction (MBHR). This reaction consists in the formation of a C-C bond between two groups, efficiently and with total economy of atoms. 13, 14 Morita-Baylis-Hillman adducts (MBHA) are considered a class of molecules with good cytotoxic potential for various diseases, being efficiently produced by the MBHR. [15][16][17][18][19] The nitro benzaldehyde group was used in MBHR with 7-chloroquinoline derivatives as Michael acceptors aiming to enhance its activity according to Figure 1. This proposal is based on previous studies 15 performed by our research group that showed excellent results of several biological activities of the MBHA based on nitrobenzaldehyde derivatives.
Inspired by the biological profile of 7-chloroquinoline and its importance in pharmaceutical and biological fields, it was thought worthwhile the development of new molecules based on 7-chloroquinoline with a view to obtain new chemical entities with enhanced activity. Due to this context, this paper describes the synthesis and antiproliferative activity evaluation of new MBHA based on 7-chloroquinoline moiety.

General
All commercially available reagents were used without further purification. Reactions were monitored by thin-layer chromatography (TLC) using Silica gel 60 UV254 pre-coated silica gel plates (Macherey-Nagel, Düren, Germany). Flash column chromatography was performed on a silica gel (300-400 mesh) using an ethyl acetate (Vetec, Rio de Janeiro, Brazil) and hexane (Vetec, Rio de Janeiro, Brazil) mixture as eluent. Fourier transform infrared (FTIR) spectra were recorded on a Shimadzu IR Prestige-21 spectrophotometer. Hydrogen-nuclear magnetic resonance ( 1 H NMR) spectra were recorded in dimethyl sulfoxide (DMSO-d 6 )  Typical procedure for the synthesis of 7-chloroquinoline 4-substituted (2-4) 4,7-Dichloroquinoline (1) (3 mmol, 1 equiv.; Sigma-Aldrich, São Paulo, Brazil) and respective diol (36 mmol, 12 equiv.) were mixed at room temperature. It was added potassium t-butoxide (4.5 mmol, 1.5 equiv.; Sigma-Aldrich, São Paulo, Brazil) and t-butanol (15 mL; Sigma-Aldrich, São Paulo, Brazil) and the flask was connected to a reflux condenser. The mixture was stirred at 80 °C in oil bath for 18 h. 20 After reaction completion as evidenced by TLC, the reaction mixture was quenched and it was added 15 mL of saturated solution of NaHCO 3 (Sigma-Aldrich, São Paulo, Brazil). Then, it was extracted with dichloromethane (3 × 20 mL; Vetec, Rio de Janeiro, Brazil) and the resulting organic phases were dried with anhydrous Na 2 SO 4 (Sigma-Aldrich, São Paulo, Brazil). After filtration, the solvent was removed under reduced pressure to obtain the respective product (2-4) without further purification.  1 mmol of each derivative of 7-dichloroquinoline (2-4) was solubilized in 10 mL of dichloromethane and 5 mmol of thionyl chloride (SOCl 2 ; Sigma-Aldrich, São Paulo, Brazil). The resulting mixture was stirred at reflux 45 °C in oil bath for 16 h. After reaction completion as evidenced by TLC, the reaction mixture was quenched with 20 mL of a saturated solution of NaHCO 3 . Then, it was extracted with dichloromethane (3 × 20 mL) and the resulting organic phases were dried with anhydrous Na 2 SO 4 . After filtration, the solvent was removed under reduced pressure to obtain the respective product (2a-4a) without further purification.
Typical procedure for the synthesis of acrylates (5)(6)(7) A solution of 1 mmol of correspondent intermediate (2a-4a) and 1 mmol of potassium acrylate (Sigma-Aldrich, São Paulo, Brazil) in N,N-dimethylformamide (DMF, 5 mL; Sigma-Aldrich, São Paulo, Brazil) was stirred at reflux 160 °C in oil bath for 15 h. After total conversion, evidenced by TLC, the reaction mixture was extracted with dichloromethane (3 × 20 mL) and the resulting organic phases were dried with anhydrous Na 2 SO 4 . After filtration, the solvent was removed under reduced pressure to obtain the respective product (5-7) without further purification.
The synthesized compounds were evaluated against tumor cells (0.06-50 µmol L -1 ) in order to obtain the concentration that causes 50% inhibition of cell growth (IC 50 ). Negative control received the same amount of DMSO. Doxorubicin (Sigma-Aldrich, St. Louis, USA) (0.01-5 µg mL -1 ) was used as the positive control. After 69 h of treatment, 20 µL of MTT (5 mg mL -1 ) was added. At the end of the incubation, the MTT formazan product was dissolved in 100 µL of DMSO and the absorbance was measured at 595 nm in a plate spectrophotometer (Varioskan Flash; Thermo Fisher Scientific, Vantaa, Finland). After the preliminary study with a single concentration of 50 µmol L -1 , the percentage of cell growth inhibition (mean and standard deviation) was calculated and only compounds with at least 75% inhibition in a cell line were considered active for the IC 50 assay. IC 50 values were calculated by non-linear regression with a 95% confidence interval.

Results and Discussion
Chemistry 7-Chloroquinoline alcohols derivatives were synthesized as shown in Figure 2. 4,7-Dichloroquinoline was subjected to a nucleophilic aromatic substitution reaction (i) with the corresponding diol under basic catalysis according to described in literature. 20 The obtained products (2-4) were then subjected to a two steps reaction: (ii) the obtaining of the alkyl halides from alcohols and (iii) their esterification with potassium acrylate.
The conversion of alcohols to alkyl halides (2a-4a) was indicated by the absence of hydroxyl band in their respective FTIR spectra (see Supplementary Information section). The formation of acrylate was confirmed by 1 H NMR by the appearance of three double doublets for three vinyl hydrogen at 5.7-6.5 ppm.
Finally, the acrylates 5-7 were used as Michael acceptors in MBHR with ortho, meta or para-nitrobenzaldehyde, that are commercially available, according to the synthetic conditions given in Figure 3. DABCO was used as catalyst in the synthesis of the hybrids 8-16 whose yields are given in Table 1.
Although some of the synthesized molecules have been previously reported by our research group, 12 most of the molecules reported in this research are novel to the best of our knowledge. These structures were elucidated by 1 H NMR, 13 C NMR and mass spectrometry. The 13 C NMR reveals the formation of the desired MBHA by the appearance of a carbinolic carbon at range 65-72 ppm in 13 C NMR. Moreover, the absence of one of three vinyl hydrogen in 1 H NMR reinforces the MBHA formation.
All spectral data were in accordance with the assumed structures.

In silico study
The evaluation of pharmacokinetic properties via theoretical studies is an important tool in the search for   new drug candidates, minimizing both the cost and the time required in the study. These attributes allow the use of in silico ADME (administration, distribution, metabolism and excretion) models in order to discern promising molecules from those with an unfavorable pharmacokinetic profile. ADME parameters provide important information about these properties, particularly about the absorption, which reflects directly on the bioavailability of the drug. 21 One of the main studies for determining pharmacokinetic parameters based on physical-chemical properties is the Rule of Five, proposed by Lipinski et al. 22 Also called Lipinski's Rule, this rule establishes that the potential of a drug candidate is greater when the molecule meets at least three of the following conditions: molecular weight ≤ 500 g mol −1 , number of hydrogen bond acceptors ≤ 10 (N or O atoms), number of hydrogen bond donors ≤ 5 (NH or OH groups) and log P ≤ 5.
In addition to Lipinski's parameters, other physicochemical properties obtained via in silico studies have been explored by several research groups to predict the oral bioavailability of candidates to drugs. Veber et al. 23 proposes a simple approach based on the number of rotatable bonds and the topological polar surface area (TPSA) of the molecule to provide an effective rule for predicting drug absorption.
OSIRIS Property Explorer 24 and SwissADME 25 software were used to obtain the physicochemical properties of the MBHAs proposed in this study. The calculated values are given in Table 2. As expected, the data show that the compound's lipophilicity is directly influenced by the size of the spacer chain and the functional group/portion anchored to it. A brief comparison of cLog P between the classes of synthesized compounds allows us to verify that the alcohols (2-4) have a greater polarity in relation to the respective acrylated derivatives (5-7).
Variations in the size of the spacer chain in the synthesized MBHA has a direct influence on its lipophilicity, evidenced by the increase in the value of cLog P proportionally to the number of carbons in the chain. Taking the homologous MBHA 8 and 11 as an example, a variation of about 20% in the value of cLog P can be observed. On the other hand, it is observed that the position of the substituent -NO 2 in aromatics has practically no influence on the values of this parameter within the same series of molecules.
Regarding to the Rule of Five, it is possible to verify that all the evaluated molecules were within the limits established by Lipinski et al., 22 and none of these parameters were exceeded, reinforcing the possible oral bioavailability of the compounds in question.
TPSA refers to the molecular surface area resulting from the summation of the tabulated surface contributions of the 2D polar fragments (O or N atoms and H atoms attached to them). 26,27 It was found that the synthesized MBHA 8 to 16 showed a fixed TPSA value (114.47 Å 2 ), since their structural differences reside in the size of the spacer chain and in the position of the -NO 2 group. As described in the literature, 28  closely linked to the value of TPSA, therefore all MBHA synthesized had the same value for this parameter (69.51%). Veber et al. 23 highlighted that molecules with TPSA values ≤ 140 Å 2 and rotatable bonds (RB) ≤ 10 tend to have significant oral bioavailability. Among the proposed molecules, only adducts with a four-carbon spacer chain (14-16) do not simultaneously meet these conditions. As proposed by Zhao et al., 28 in addition to Lipinski's parameters, these results indicate considerable capacity to penetrate the cell membrane.

the percentage of absorption (ABS) is
Drug score (DS) indicates the overall potential of the compound to act as a drug and its calculation is based on the values of Log P, Log S, druglikeness, molecular mass and risk of toxicity. It was possible to verify that the DS values calculated in this study were very close to each other. We highlight 8-10 adducts, considering that the probability of a compound to be bioactive is greater the closer to the unit is the DS value. 24,29 In vitro study A single concentration screening of the molecules was performed aiming to identify the compounds with the highest inhibition potential against three cancer cells (NCI-H292, HCT-116 and MCF-7). After the preliminary assay, compounds with a percentage of inhibition greater than 75% were tested to determine their respective IC 50 . 30 The results presented in Table 3 are expressed as the mean of the inhibitory growth percentages (%) followed by their respective standard deviations (SD). These data demonstrate that the synthetized alcohols 2-4 were not promising for the cancer cells evaluated, whereas one of the synthesized acrylates (5) showed inhibition superior to the reference drug, leading us to study its IC 50 .
Regarding the MBHA obtained, the derivatives showed better results as the spacer chain increased, demonstrating a possible influence of the lipophilicity on their inhibitory activity. We draw attention to the most lipophilic compounds (14)(15)(16), with a four-carbon spacer chain, as active as doxorubicin.
The antiproliferative activity of 7-chloroquinoline derivatives was evaluated investigating their effects on four cancer cell lines: MCF-7 (breast cancer), HCT-116 (colorectal cancer), HL-60 (promyelocytic leukemia) and NCI-H292 (lung cancer). Doxorubicin was used as a positive control. The results available in Table 4 are given as the mean of the IC 50 values followed by their respective SD.
The IC 50 results demonstrate that nitro group at ortho position has a strong influence on the activity of the synthesized molecules, which was already expected compared to other results presented in the literature. 16 Expressive results can be observed in the activity of compounds 11 and 14 against HL-60, when compared to the MBHA previously published by our group. These values indicate a positive effect of the 7-chloroquinoline moiety, 15 increasing their cytotoxic activity.
Furthermore, comparing compounds with different spacers we can observe that 15 (with four carbons) had a better activity than 9 (with two carbons), possibly due its major conformational freedom. The tests performed showed that compound 14 showed better IC 50 values for MCF-7 and NCI-H292 cells. In contrast, compound 16 had the best result for HCT-116 cells and compound 11 had the best result for HL-60 cells.
After the tests against cancer cells, the molecules with the best IC 50 values had their selectivity evaluated in healthy cells, since one of the major obstacles of the chemotherapy treatment is related to the attack of the drug to these cells, causing complications in the treatment.
Selectivity index (SI) corresponds to the ratio between the respective IC 50 values of the pure compound against healthy cells (peripheral blood mononuclear cells, PBMC) and the pure compound against the cancer cell line, so that an SI value less than 2.0 indicates possible toxicity of the drug candidate. 31 Table 5 shows the obtained results, where the majority of the structures presented satisfactory results (SI ≥ 2.0). In general, the best selectivity results were observed in relation to the HL-60 cell line, whose SI values were greater than 5.0 for practically all tested molecules. It is also noteworthy that the acrylate 5 showed promising results against three of the four cell lines tested, creating perspectives for further studies of molecules endowed with Michael's accepting portion as anti-cancer agents.
All compounds evaluated here against the HCT-116 cell line were more selective than doxorubicin, which presented SI = 1.16, whereas the minimum selectivity obtained for the synthesized compounds was 2.29 (compound 11).
The results of cytotoxic activity against human healthy cells reinforces the antiproliferative potential of the compound 14, which has the best calculated SI (11.89) in comparison to the others MBHA tested. Furthermore, adduct 14 was about three times more selective than doxorubicin.

Conclusions
In this paper, we present the convenient synthesis of a series of novel 7-chloroquinoline derivatives that were designed, synthesized in acceptable to good yields and well confirmed by FTIR, 1 H NMR, 13 C NMR and HRMS. The drug-likeness study showed promising scores for the new compounds, which were active against the cancer cell lines studied, with low levels of toxicity and considerable selectivity in comparison to the reference drug. We can highlight compound 14 that showed the best results for the four cancer cell lines and for SI, thus demonstrating the relevance of the application of 7-chloroquinoline in MBHR to obtain new anticancer compounds.