Synthesis , in vitro Antiproliferative and Anti-Mycobacterium tuberculosis Activities of Novel β-Carboline Derivatives

A series of β-carboline derivatives with amino or guanidinium were synthesized and evaluated in vitro against anti-Mycobacterium tuberculosis and for antiproliferative activities against nine human cancer cell lines. The compounds 1-(4-hydroxyphenyl)-3-carboxamide(ethylamine) β-carboline (24.9 μg mL) and 1-(4-methoxyphenyl)-3-carboxamide(ethylamine) β-carboline (26.9 μg mL) were the most active against M. Tuberculosis (MTB). Compounds 1-(4-hydroxyphenyl)-3carboxamide(ethylamine) β-carboline and 1-(4-methoxyphenyl)-3-carboxamide(propylamine) β-carboline, which had the same substituted groups, inhibited the growth of all human tumor cell lines with growth inhibitory activity (GI50) values from 1.37 to 9.20 mmol L. Also in this series, compounds 1-(4-hydroxyphenyl)-3-carboxamide(propylamine) β-carboline and 1-(3-nitrophenyl)3-carboxamide(propylamine) β-carboline demonstrated significant activity against NCI/ADR cells. Among compounds with a terminal guanidine group, compounds 1-(4-hydroxyphenyl)-3carboxamide(ethyl)guanidine β-carboline (27.8 μg mL) and 1-(3-nitrophenyl)-3-carboxamide(ethyl) guanidine β-carboline (37.4 μg mL) demonstrated the greatest activity against MTB. Additionally, compounds 1-(4-methoxyphenyl)-3-carboxamide(ethyl)guanidine β-carboline (GI50 = 0.45 mmol L) effectively inhibited growth and was highly selective against NCI/ADR. The in silico study revealed that 1-(4-hydroxyphenyl)-3-carboxamide(ethylamine) β-carboline, 1-(4-methoxyphenyl)3-carboxamide(ethylamine) β-carboline, 1-(4-hydroxyphenyl)-3-carboxamide(propylamine) β-carboline, 1-(4-methoxyphenyl)-3-carboxamide(propylamine) β-carboline and 1-(3-nitrophenyl)3-carboxamide(propylamine) β-carboline compounds follow the rules established by Lipinski, suggesting that this compound has no problems with oral bioavailability.


Introduction
Tuberculosis (TB) exhibits high morbidity and mortality.The long-term treatment regimen can cause patients to be non-compliant in completing the treatment, thus leading to emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) TB strains.Infections caused by MDR-TB and XDR-TB do not respond to first-line drugs that are used to treat TB, and alternative treatment regimens include mostly injected drugs and prolonged treatments. 1,2Due to the appearance of resistant strains and given high toxicity of anti-tuberculosis drugs, to the need to develop new drugs that are more effective and less toxic than current drugs, which would reduce time and complexity of treatment, is urgent. 3,4The discovery of new drugs is also necessary for the treatment of cancer, because most chemotherapeutic agents exhibit severe toxicity and cause many undesirable side effects; additionally, current agents are very expensive, mutagenic, carcinogenic and teratogenic. 5Tuberculosis, caused by Mycobacterium tuberculosis (MTB), 1 and cancer 6 have affected human health for thousands of years and remain a major cause of diseases affecting public health around the world, and the possible interaction of mycobacterial Vol. 27, No. 8, 2016   pathogens with cancer cells may be influenced by genetic alterations in the tumor cells.
6][17][18][19][20][21][22][23] Other studies have shown that β-carboline derivatives with a methyl-substituted group at position-1 and a guanidinium group-terminated side chain at C-3 exhibited anti-HIV-1 activity in MT4 cells by hindering the essential interaction of the regulatory protein Tat with trans-activation response region (TAR). 24,25Some results cited above indicated that β-carboline derivatives containing 4-hydroxyphenyl, 4-methoxyphenyl or 3-nitrophenyl group at C-1 showed potent anticancer activity for some of the human cancer cell lines tested.This led us to study novel β-carboline analogs that might serve as antitumoral and antituberculosis agents as part of our ongoing research program.
Based on the idea that the addition of appropriate substituents at positions-1 and -3 might result in more potent compounds, we synthesized novel 1-substitutedphenyl-β-carboline with an amino or guanidinium groupterminated side chain at C-3 and evaluated the in vitro antituberculosis, antiproliferative properties and in silico study.

Experimental
General procedure 1 H and 13 C nuclear magnetic ressonance (NMR) spectra were recorded on a Varian Mercury Plus (Palo Alto, EUA) spectrometer operating at 300 and 75.5 MHz, respectively, using deuterated dimethyl sulfoxide (DMSO-d 6 ), chloroform (CDCl 3 ) and methanol (CD 3 OD) as solvent, and tetramethylsilane (TMS) as internal reference.Infrared (IR) spectra were recorded as potassium bromide pellets on a BOMEM spectrometer model MB-100 (Houston, USA).Melting points were determined in a Micro-Química apparatus MQAPF-301 model (Palhoça, Brazil) and are uncorrected.The reactions were monitored by thin layer chromatography (TLC) conducted on Merck TLC plates (Silica Gel 60 F254, Darmstadt, Germany).All reagents were purchased from commercial suppliers.
Stock solutions of the test compounds were solubilized in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, USA) and diluted in Middlebrook 7H9 broth (Difco, Sparks, USA) supplemented with OADC enrichment BBL/Becton Dickinson.Rifampicin and isoniazid were solubilized according to the manufacturer's recommendations (Sigma-Aldrich, St. Louis, USA) and used as positive controls.
The change in absorbance, at 492 nm wavelength, was measured using a microplate reader TP-Reader (Thermo Plate ® , Männedorf, Switzerland).Each compound was analyzed in triplicate on alternate days.The minimum inhibitory concentration (MIC) was defined as the lowest concentration that resulted in 90% inhibition of the growth of M. tuberculosis. 28MIC values were used to classify a compound's activity as follows: inactive, > 150 μg mL -1 ; moderate, between > 10 and < 100 μg mL -1 ; and active, < 10 μg mL -1 .
The tests were performed using the colorimetric method with sulforhodamine B according to the National Cancer Institute (NCI) standard protocol; doxorubicin was used as a positive control. 29Assays were performed in a 96-well plate using four serial 10-fold dilutions (0.25-250 μg mL -1 ) for each test compound.The anticancer activity was determined based on concentration-response curves, and three concentration response parameters, growth inhibitory activity (GI 50 ), growth inhibition (TGI) and cytotoxic activity (LC 50 ) were calculated.The response parameter GI 50 refers to the drug concentration that produces 50% reduction of cell growth when compared to untreated control cells.The TGI and LC 50 parameters refer to the drug concentrations required for total growth inhibition and for 50% cell mortality, respectively.Compounds with GI 50 values < 100 μmol L -1 were considered active.

In silico study
The in silico computational study of compounds were performed to determine Lipinski's rules of five 30 (hydrogen bond donors ≤ 5; hydrogen bond acceptors ≤ 10; molecular weight ≤ 500; the Log P is ≤ 5), topological polar surface area (TPSA) and percentage of absorption (%ABS).Calculations were performed using Molinspiration online property calculation toolkit software 31 and OSIRIS property explorer software. 32The percentage of absorption was estimated using the following equation: %ABS = 10 -[0.345 × TPSA].

Chemistry
The synthetic pathway for the preparation of 1,3-disubstituted β-carbolines is presented in Scheme 1.The methyl esters in 1a-c were prepared by a Pictet-Spengler condensation of L-tryptophan with the appropriate aromatic aldehydes in acidic media, subsequent esterification of the resulting carboxylic acids with methanol and sulfuric acid, and oxidation with sulfur in refluxing xylene. 22ompounds 2a-c and 3a-c were obtained by the reaction of β-carboline methyl ester with 1,2-ethylenediamine and 1,3-propanediamine, respectively, and resulted in an amino group-terminated side chain at C-3.Finally, the coupling of β-carboline carboxamide derivatives 2a-c with S-methylisothiourea yielded compounds 4a-c, which include a terminal guanidinium group.
The novel compounds 2a-c, 3a-c and 4a-c were characterized using 1 H and 13

Anti-Mycobacterium tuberculosis activity (MTB)
Derivatives 2a-c, 3a-c and 4a-c were evaluated in vitro for their antimycobacterial activity against M. tuberculosis H37Rv (ATCC 27294) using the REMA method. 27The MIC values (μg mL -1 and μmol L -1 ) were measured with respect to two standard antitubercular drugs, isoniazid (INH) and rifampicin (RFP), and the screening results are presented in Table 1.Among the nine compounds evaluated against MTB, seven presented moderate activity, with MIC values ranging from 58.3-24.9μg mL -1 ; in particular, compounds 2a (24.9 μg mL -1 ), 2b (26.9 μg mL -1 ), 4a (27.8 μg mL -1 ) and 4c (37.4 μg mL -1 ) presented interesting activity.Compounds 2a and 2b, which had p-hidroxyphenyl and p-methoxyphenyl substituents, respectively, at position-1 and ethylenediamine moieties at C-3 were the most active derivatives in this series.The length of the terminated side chains affected the activities of these compounds.Substituting the guanidinium group led to reduced activity (compare compounds 2a and 4a).The effect of the guanidinium group was particularly significant when comparing compounds 2c (57.9 μg mL -1 ) and 4c (37.4 μg mL -1 ).The substituents at positions-1 and 3 strongly affected anti-MTB activities of these compounds.
The compounds with terminal guanidinium groups, including compound 4b (GI 50 = 0.45 mmol L -1 ; TGI = 72.09mmol L -1 ) effectively inhibited growth and was highly selective against adriamycin drug-resistant ovarian cancer cell lines (NCI/ADR) when compared with compounds 2a-c, which are amino group-terminated.However, the guanidinium-terminated compounds did not demonstrate any important interaction that could account for the cell growth inhibition.Substituting the phenyl group with electron-donating substituents at position-1 influenced each series differently.

Lipinski's rule of five
The drug-likeness concept helps optimize the pharmacokinetic properties of a compound, such as absorption, distribution, metabolism and excretion (ADME) in the human body. 36Lipinski's rule of five is a refinement of drug-likeness and is used to predict whether a chemical compound will have pharmacological or biological activity as an orally active drug in humans.This rule was formulated based on the observation that most medication drugs are relatively small and lipophilic molecules. 33The results of the analysis are shown in Table 4 and indicate that the compounds are in agreement with the values determined by Lipinski, except 4a-c derivatives, which showed the number of hydrogen bond donors (nHBD) > 5, in violation of the Lipinski rules.The calculated percent absorption (%ABS) of all compounds ranged from 51.90-76.91%,indicating good cell membrane permeability.Another important factor is obtained by the volume analysis and TPSA by the compounds showed lower than 140 Å² indicating that these derivatives have good absorption in the intestine, except the compound 4c (TPSA = 165.51).The compounds 2a-c, 3a-b and 4a-c exhibited good solubility (Log S = -3.87 to -4.98), except the compound 3c which showed a value of Log S less than -5.Compounds with high solubility are easily metabolized and eliminated from the body, thus resulting in a lower probability of adverse effects and bioaccumulation.

Conclusions
Thus, our results showed for the first time the synthesis and antitumor and anti-MTB activity of compounds with an amino or guanidinium group-terminated side chain at C-3 of a 1-substituted-phenyl-β-carboline nucleus.Compounds 2a, 2b and 4a were the most active against M. Tuberculosis H 37 Rv (ATCC27294).Compound 2a and 3b demonstrated promising antiproliferative activity for all cancer cell lines.Eight compounds inhibited the cell growth of adriamycin drug-resistant ovarian (NCI/ADR), showed activity and high selectivity for the 3a, 3c and 4b.Compound 2a demonstrated promising antiproliferative and anti-MTB activity, in addition to follow as established Lipinski's rule of five, suggesting that this compound has no problems with oral bioavailability, and indicates good permeability a in the plasma membrane of the cell, which may represent a precursor to development of new molecules.Further studies are required to explore the mechanism of action of these compounds in detail.
C NMR spectroscopy, as detailed in the Experimental section.The 1 H NMR spectra of carboxamides 2a-c and 3a-c showed additional signals at d H 1.64-3.95(reflecting the integration of two protons) and at d H 7.01-8.94(corresponding to aromatic hydrogens).The presence of the ethylamine or propilamine carboxamide in position C-3 group was confirmed by 13 C NMR, which showed signals at d c 32.8-41.9(CH 2 ) and d c 162.5-167.1 (C=ON).Derivatives 4a-c was characterized by the presence of an additional signal at d c 159.01-159.90, corresponding to the guanidinium group.

Table 1 .
Anti-Mycobacterium tuberculosis H 37 RV activity of compounds 2a

Table 2 .
In vitro cell growth inhibition (GI 50 ) of compounds 2a-c, 3a-c and 4a-c against neoplastic cells

Table 3 .
Total growth inhibition (TGI) and lethal concentration (LC 50 -in parentheses) of compounds