Carboxamide Appended Quinoline Moieties as Potential Antiproliferative Agents, Apoptotic Inducers and Pim-1 Kinase Inhibitors

The targeted approach of protein kinases (PKs), as PKs are the main regulators of cell survival and proliferation, has been a promising strategy for treatments for cancer. Here we analyze the potential of quinoline-carboximide derivatives for four cell lines: MCF-7, CACO, HepG-2 and HCT-116 as anticancer agents. 3e , 4b , 11b and 13d derivatives showed good anti-proliferative activities in comparison to the reference standard Doxorubicin, against the four cell lines tested. They have been chosen for further studies. First of all, the IC 50 value surveys were carried out to ensure the protection of our hits and demonstrate that the cytotoxic effect (IC 50 > 113 μM) is highly selective on normal human cells (WI-38). Secondly, apoptosis was accomplished by down-regulation of Bcl-2 and up-regulation of BAX and Caspase-3 by these active compounds. Also, the Pim-1 inhibitory activity of the active hybrids was done, which indicate that compound 3e was the most active with percentage of inhibition 82.27% and IC 50 equal 0.11 when compaired to SGI-1776 as a reference standered. In addition, the in silico assessment of ADME properties, all of the strongest compounds are orally bioavailable without blood brain barrier penetration. 13 13 3 3 129.54, 130.14, 131.38, 133.56, 134.15, 137.91, 140.21, 140.32, 141.30, 141.34, 142.90, 145.27, 147.92, 160.80, 161.91, 162.04; Anal. Calcd for C 29 H 18 ClN 5 O 2 (503.95): C, 69.12; H, 3.60; N, 13.90; Found: C, 69.23; H, 3.44; N, 13.85.

| P a g e Pim-1 is highly expressed in a wide variety of human tumours of both hematopoietic and epithelial origin, including myeloid and lymphoid acuteleukemia, large cell lymphoma (LCDL) diffuse, and neoplastic prostate cancer [15].
Various classes of heterocyclic and fused heterocyclic compounds have been identified in recent times in the search for anticancer agents through molecular biology, empirical screening and rational drug development [16,17]. In many alkaloids, for instance camtothecin, the quinolin nucleus occurs naturally. Therefore, quinoline ring plays a major role in the production of anticancer drugs, as its derivatives have been shown to yield excellent results with various mechanisms of action such as growth inhibitors though cell cycle arrest, apoptosis, angiogenesis inhibition, cell migration interference, and nuclear receptor response modulation [18][19][20][21][22]. The amide is a common backbone of many organic molecules and natural products that are characterized by a wide range of chemical and pharmacological characteristics [22][23][24]. Amides thus play an important part in essential life processes, because the amide bond is, for example, the key to the production of proteins. In addition, amide bond is commonly used in the structure of 25% of the well-known drugs, according to the Comprehensive Medicinal Chemistry database [25,26].
More essential for synthesizing polyhetero-arometic molecules are amides that typically are stable, neutrals and both have the hydrogen-bonds receiver/donator properties. It has now been documented to be effective for cancer therapy [27] and for reduction of undesirable side effects [17] by developing a single molecule with more than one pharmacopher with different action modes. Based on the above findings, and in continuation to our work on the synthesis of new compounds with biological activities [28][29][30][31][32][33][34], we have synthesized a new series of quinolone-3-carboxamide derivatives containing a series of biologically active moites that are expected to be strongly inhibitory for several human cell lines and PIM-1 inhibitors as a result of our commitment to search for new potential anticancer agents related to heterocyclic [35][36][37][38][39][40][41][42].

Chemistry
Schemes 1-4 outline the synthetic pathways used to obtain compounds 1-13. The starting materials 2-chloro or 2-keto 3-formylquinoline 1 were prepared according to the reported method [43]. The reactivity of 1 towards some active methylene compounds was discussed. Thus, condensation of 1 with some cyanoacetamide derivatives 2a-c in ethanol containing piperidine as catalyst afforded the corresponding amide derivatives 3a-e. The structure of 3 has been assigned as a reaction product on the basis of analytical and spectral data. The IR spectrum of 3c as example displayed absorption bands at 3370 cm -1 due to NH function, at 2211 cm -1 due to conjugated C≡N function, at 1694 cm -1 due to amidic C=O function. The 1 H NMR spectrum (DMSO-d6) exhibited one sharp singlet signal at δ 4.42 ppm assignable to CH2 protons, another two multiplet signals at δ 6.31-6.42 ppm due to furan protons and at δ 7.27-8.33 ppm specific for 4 protons of quinoline, in addition to three signals at δ 8.73, 9.10 and 12.29 owing to methine, 4H-quinoline and NH proton. Moreover, its 13  The IR spectrumof 4a as example showed the lack of absorption band corresponding to NH which confirm the cyclic structure and presence of bands at 2228 and 1664 cm -1 due to nitrile and carbonyl groups respectively. 1 H NMR spectrum exhibited singlet signal at δ 2.45 ppm due to CH3 in addition to the aromatic protons. In attempt to obtain pyranoquinoline derivative 5 by heating 3d,e in DMF containing trimethylamine but the attempt was failed which may be due to the presence of 2-hydroxyquinoline in keto form (So we should draw in the form of keton). were obtained through the reaction of the aldehyde 1a with cyanoacetamide and acetylacetone in presence of piperidine as catalyst. The analytical and spectral data are in agreement with the proposed structure. The IR spectrum of 6a showed the presence of two C=O groups stretching at 1730, 1664 cm -1 and CN group at 2228 cm -1 . 1 H NMR spectrum of the reaction product showed three singlet signals at δ 2.42, 2.74 and 2.89 ppm corresponding to three methyl groups. Also, its 13 C-NMR proved the presence of methyl groups as three signals at δ 11.55, 21.38 ppm and 31.24.
On the other hand, when 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one was used as active methylene to react with the aldehyde derivative 1 and cyanoacetanilide , the corresponding tetrahydropyrano[2,3-c]pyrazole (7) was obtained. The chemical structure of 7 was established on the basis of its elemental analysis and spectral data. The IR spectrum showed the presence of two C=O groups stretching at 1731, 1650 cm -1 . Its 1 H NMR spectrum displayed two doublet signals at 5. 16  with Doxorubicin (DOX) as a reference anticancer drug. The cytotoxic activities expressed as IC50 (µM/mL) value; the dose that reduces cell growth and survival to 50% of our tested compounds which were presented in Table 1.
These results revealed that the best activities were obtained when the 2-cyano-acrylamide moieties are directly attached to the quinoline rings (compounds 13d, 3e and 11b). Similarly, good anticancer activity was observed when the quinoline ring is attached to cyanoacetamide moiety through a linker (compound 9b), chromene ring through a spacer (compound 10) or fused with 2pyridone moiety (compound 4b).
These results revealed that the best activities were obtained when the 2-cyano-acrylamide moieties are directly attached to the quinoline rings (compounds 13d, 11a, 11b and 13c). Similarly, excellent anticancer activity was observed when the quinoline ring is attached to chromene moiety through a linker (compound 10) or cyanoacetamide moiety through a spacer (compound 9b). These results revealed that the best activities were obtained when the 2-cyano-acrylamide moieties are directly attached to the quinoline rings (compounds 3e, 13d, 13c, 13b, 11b and 11a). Similarly, excellent anticancer activity was observed when the quinoline ring is attached to chromene moiety through a linker (compound 10), cyanoacetamide moiety through a spacer (compound 9a) or fused with2-pyridone moiety (compound 4b).

Structure activity relationship (SAR)
The sensitivity of the screened cancer cells to the target compounds were in the following descending  The most active compounds were OQMAP tagged with oxoquinoline-3-yl-acrylamide moiety 11b and CQMAP tagged with chromene-3-carbocamide moiety 10.

Effect on the active Caspase-3 level
Stimulation of apoptosis is believed to be the main mechanism of action for most of the known anticancer agents [45]. Elevated level of Caspases is an indication of apoptosis. The effect of the targets quinoline derivatives 3e, 4b, 11b and 13d on the level of Caspase-3 was evaluated and the obtained results are summarized in table 4. It is clear that the level of Caspase-3 in cancer cells treated with the | P a g e target quinoline derivatives was 5 to 6 times more than the level of Caspase-3 in cancer cells, which are not treated with the target quinoline derivatives (Table 2) (Figure 4). This demonstrates that the antiproliferative activity of the quinoline target compounds is through the stimulation of the apoptotic pathway.

Effect on mitochondrial apoptosis pathway proteins BAX and Bcl-2
It was reported that the over expression of BAX (pro-apoptotic) stimulates apoptosis in cancer cells [46]. were evaluated for their effect on some apoptosis key markers, BAX and Bcl-2 ( Table 2). Pim-1's inhibition of apoptosis is by phosphorylication in target molecules such as BAD, ASK1 or PRAS 40 that inhibit or activate anti-apoptotic (Bcl-2) and pro-survival (mTOR) functions, for instance, the JNK / p38-Caspa-3 axis. In this way it is possible to minimize apoptosis.
This article aims to update Pim kinase inhibitors with a key role in developing anticancer compounds against Pim kinase targets.
To investigate their function in Pim-1 kinase inhibition with a variety of ten concentrations, compounds 3e, 4b, 11b, and 13d were chosen, and values for IC50 were measured. The most potent Pim-1 inhibitors | P a g e were compounds, 3e, 4b and 11b with IC50 of 0.11-0.92 μM, and 13d with less effective activity (IC50 = 3.24 μM). The positive control with the test SGI-1776 was used for IC50 0.048 μM. The profiles showed that Pim-1 is a potential target for those compounds in combination with cell-wide inhibition data for the 3a, 4b, 11b and 13d compounds described in Table 3.

In-silico computational analysis studies
The typical reference medication, Doxorubicin, was also subjected to in-silico physicochemical, pharmacokinetic/ADME, and drug likeliness attributes along with all the synthesized compounds. The These physicochemical properties are shown in Table 4. The absorption rate in the chosen compounds varies from 63.82% to 81.78%. Some compounds in water with a good molar refractiveness are moderately soluble and have been shown to be moderately to goodly absorbent with a median of 81.78% and 75.15% of reference and synthesized drugs respectively.   [52] had to analyze the molecule as an important candidate for drugs. The number of violations of the rules previously called along with their bioavailability scores are listed in (Table 6). The Lipinski

DOX.
No (3) No (2) No (1) No (1) No (3) 0.17 log S not greater than 6, saturation: fraction of carbons in the sp 3 hybridization not less than 0.25, and flexibility: no more than nine rotatable bonds. In this case, the compound is predicted not orally bioavailable, because too flexible and too polar.

Conclusion
In

Chemistry
Melting points were measured with a Gallenkamp apparatus and are uncorrected. IR spectra were recorded for KBr disc on a Mattson 5000 FTIR spectrophotometer. 1 H NMR and 13 C NMR spectra were measured on a Bruker AC 500 (500 MHz) in DMSO-d6 as solvent, using TMS as an internal standard, and chemical shifts are expressed as ppm. Mass spectra were determined on Finnigan Incos 500 (70 eV). Elemental analyses were carried out at the Microanalytical Center of Cairo University. Anticancer activity was carried out in local strain identified in Regional Center for genetic engineering, faculty of Science (Boys), Al-Azhar University and the results were within ± 0.4% of calculated value.

amide (13a-d)
The aldehyde derivatives 1 (0.01mol) was added to shiff bases cynoacetanilide 12a, b (0.01mol) in ethanol (20 mL) in the presence of pipridine. The mixture was heated under reflux for 3h and the solid formed was filtered, washed with ethanol, dried, and recrystallized from a mixture of EtOH/DMF.    Wash with acetic acid 1% to remove unbound dye (end point: colorless drainage). The plates will be air dried 24 h. The dye will be solubilized with 50 μl/well of 10 mM tris base (PH 7.4) for 5 min on a shaker at 1600 rpm. The optical density (OD) of each well will be measured at 570 nm with an ELISA microplate reader (EXL 800 USA). The relative cell viability in percentage was calculated as (A570 of treated samples/A570 of untreated sample) X 100 and The IC50 values will be calculated using sigmoidal concentration response curve fitting models (Sigmaplot software) [37].

Determination of Caspase-3
The PC-3 cells were planted into RPMI 1640 with a 10% fetal serum of bovine serum at 37 ° C, were stimulated by Caspase-3 compounds, and lysed with the cell extraction buffer. The lysate was diluted in the regular diluent tank over the test range and tested for human active Caspase-3 content (cells are plated in the density of 1.2-1.8 per 100 μL of cells per well in a 96 wave plate for 48 hours before the enzymes test) [36].

Determination of BAX and Bcl-2
Cells PC-3 were grown in RPMI 1640 containing a fetal 10% of fetal serum at a temperature of 37°C, stimulated by the compounds for BAX/Bcl-2 testing and lysed by a buffer for cell removal. This lysate was diluted across the test range and tested for human active BAX / Bcl-2 contents in a Regular Diluent Buffer. (Cells are placed at a density of 1.2 -1.8 μL per pool in a total growth medium of 10,000 cells / well volume + 100 uL of the compounds tested in a 96well platform 24 hours before human BAX / Bcl-2 measurement) [37].

PIM1-Kinase detection
ADP-Glo™ Kinase Assay is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is converted into light by Ultra-Glo™ Luciferase . The luminescent signal positively correlates with ADP amount and kinase activity.
The assay is well suited for measuring the effects chemical compounds have on the activity of a broad range of purified kinases-making it ideal for both primary screening as well as kinase selectivity profiling. The ADP-Glo™ Kinase Assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) using up to 1mM ATP.

In-silico study
In the present study, all the synthesized derivatives as well as the standard reference drug i.e.

Notes
The authors declare no competing financial interest.