Accepted Manuscript Molecular docking studies, biological and toxicity evaluation of dihydroisoquinoline derivatives as potential anticancer agents Joanna Ziemska, Adam Guśpiel, Joanna Jarosz, Anna Nasulewicz-Goldeman, Joanna Wietrzyk, Robert Kawęcki, Krzysztof Pypowski, Małgorzata Jarończyk, Jolanta Solecka PII: DOI: Reference:
S0968-0896(16)30671-X http://dx.doi.org/10.1016/j.bmc.2016.08.054 BMC 13237
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
30 May 2016 5 August 2016 27 August 2016
Please cite this article as: Ziemska, J., Guśpiel, A., Jarosz, J., Nasulewicz-Goldeman, A., Wietrzyk, J., Kawęcki, R., Pypowski, K., Jarończyk, M., Solecka, J., Molecular docking studies, biological and toxicity evaluation of dihydroisoquinoline derivatives as potential anticancer agents, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.08.054
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Molecular docking studies, biological and toxicity evaluation of dihydroisoquinoline derivatives as potential anticancer agents Joanna Ziemskaa, Adam Guśpiela, Joanna Jaroszb, Anna Nasulewicz-Goldemanb, Joanna Wietrzykb, Robert Kawęckic, Krzysztof Pypowskic, Małgorzata Jarończykd, Jolanta Soleckaa,* a
National Institute of Public Health - National Institute of Hygiene, Laboratory of
Biologically Active Compounds, 24 Chocimska Str., 00-791 Warsaw, Poland b
Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,
R. Weigla Str.12, 53-114 Wroclaw, Poland c
Institute of Chemistry, Siedlce University, 3.Maja 54, Siedlce 80-110, Poland
National Medicines Institute, 30/34 Chelmska Str., 00-725 Warsaw, Poland
*Corresponding author: Jolanta Solecka e-mail address: [email protected]
Abstract We report a study of a series of isoquinoline derivatives, including their synthesis, in vitro microsomal leucine aminopeptidase (LAP) inhibition and antiproliferative activity on cancer cell lines. Among fourteen tested compounds, one (compound 3b) was determined to have good activity against LAP and significant antiproliferative activity against HL-60 human promyelocytic leukemia, Burkitt′s lymphoma Raji, camptothecin resistant CEM/C2 leukemia cells with mutated catalytic site of topoisomerase I, its parental cell line CCRF/CEM and LoVo colon cancer. Its influence on the cell cycle was also observed. Moreover, we have confirmed that antiproliferative activity towards cancer cells is due to LAP inhibition. Docking simulation based on positioning compound 3b into the LAP active site was performed to explore the possible binding mode. The compound was able to form hydrogen bonds with Gly362 and coordinate zinc ions, which was previously suggested to be essential for inhibitory activity. Compound 3b was also characterized with a good selectivity index for cancer versus normal mammalian cells. Toxicological studies involving examination of skin sensitization, acute skin irritation/corrosion, acute dermal toxicity, acute oral toxicity and acute eye irritation/corrosion established that compound 3b is safe for use. Keywords: leucine aminopeptidase, antiproliferative activity, docking study, toxicity
1. Introduction According to statistics the number of people living beyond cancer diagnosis reached nearly 14.5 million in 2014 and is expected to rise to almost 19 million by 2024.1 Nevertheless, as cancer is still a leading cause of death and there is an urgent need to search for novel anticancer agents. Among anticancer drug targets there is membrane leucine aminopeptidase (EC 188.8.131.52) as well as cytosolic
leucine aminopeptidase (EC 184.108.40.206).2-5
metalloenzymes that catalyze hydrolysis of the N-terminal peptide bond in proteins and peptides. They are essential for protein maturation, activation and their stability. They play also an important role in degradation and regulation of hormonal and nonhormonal peptides.57
Altered aminopeptidase activity is associated with certain pathological disorders, such as
ageing, cancers, cataracts, cystic fibrosis and leukemia.6;8 Moreover, aminopeptidases seem to be necessary for the survival and proliferation of cancer cells by regulating the supply of free cellular amino acids. Many tumor cells depend on specific amino acids, which depletion has a greater impact on cancer cells than normal cells. Myeloid leukemia and multiple myeloma cells are highly reliant on the unfolded protein response in which aminopeptidases play a key part.4;9;10 Also, it was recently observed that leucine aminopeptidases (LAPs) promote G1/S transition in cancer cells.11;12 Therefore, at present the mechanism of action based on leucine aminopeptidase inhibition attracts attention of scientists and exploration for novel aminopeptidase inhibitors seems of utmost importance. Several LAPs inhibitors were described so far. They can be divided into amino acid analogues (e.g. L-leucinal) and peptide analogues (e.g. bestatin).6 Isoquinoline derivatives represent a well-known class of substances with potent biological activities, e.g. antioxidant13, antimicrobial14;15, anti-HIV, antimalarial16, antidiabetic17, cytotoxic18 as well as enzyme inhibitory activities - butyrylcholiesterase, acetylcholinesterase, D-amino
acid oxidase13, prolyl oligopeptidase.19 This work is the first report on substituted
dihydroisoquinoline derivatives with anticancer activity based on leucine aminopeptidase inhibition. In the study, we evaluated anticancer activities (in vitro microsomal LAP inhibition and antiproliferative activity against cancer cell lines) of a series of 14 dihydroisoquinoline derivatives. We also examined the influence of the most active compound - 3b, on cell cycle and cell death and its toxicology (skin sensitization study on guinea pigs, acute skin
irritation/corrosion study on rabbits, acute dermal toxicity study on rats, acute oral toxicity study on mice and acute eye irritation/corrosion on rabbits) was determined. Docking simulations were performed to investigate the interactions of compound 3b with a leucine aminopeptidase and explore its binding mode in the active site of the enzyme. For the study, leucine aminopeptidase from bovine lens (blLAP) was used, which X-ray structure was characterized and deposited in the Protein Data Bank (PDB).20 Its primary structure was described to show 81% homology to the human enzyme.21 Moreover, it is likely that both, the human and bovine enzyme share the same active site architecture.7 Enzymatic assays were done with the use of microsomal porcine kidney LAP (pkLAP), which is the only LAP commercially available.6;7 BlLAP and pkLAP reveal high first-order sequence homology and similar kinetics properties22. 2. Results and discussion 2.1. Chemistry CO2R
1a 1b 1c
R1 I H OBn
R R2 H OBn OBn
2a 2b 2c
R1 I H OBn
R2 R3 H Bn OBn Et OBn Bn
3a 3b 3c
R1 I H OBn
R2 H OBn OBn
R3 Bn Et Bn
4a 4b 4c
R1 R2 I H H OH OH OH
Scheme 1. Synthetic pathway of target compounds. Reagents and conditions: (a) K2CO3, dibenzyl or diethyl formamidomalonate, acetone; (b) POCl3, MeCN; (c) BBr3, CH2Cl2. Derivatives of 3,4-dihydroisoquinoline-3,3-dicarboxylic acid esters 3a-c have been prepared by Bischler-Napieralski cyclization of appropriate diethyl or dibenzyl formamidomalonates 2a-c. Most satisfactory results were obtained using POCl3 and protection of hydroxylic groups, such as benzyl ethers. Cleavage of benzyl ethers and esters with BBr3 gave 3,4-
dihydroisoquinoline-3-carboxylic acids 4a-c, which were purified on ion exchange and polyacrylamide column. Synthesis of compounds 4b, d-j, 5 and 6 a,b were previously described.13 R
Me CO2H O
Cl 4d 4e 4f 4g 4h 4i 4j
R1 H H H H OH H H
R2 OH OH H Br H OH OH
R3 H H OH OH Br Br I
R4 H Me H H H H H
R1 R2 R3 R4 OMe OMe CO2Bn Bn OH OH H H
Scheme 2. Chemical structures of tested compounds 2.2. Biological activity 2.2.1. Microsomal leucine aminopeptidase (LAP) inhibitory assay Microsomal LAP inhibitory activity was evaluated and obtained results are presented in Table 1. Compound 3b displayed good activity against LAP with IC50 value of 16.5±3.4 µM, which was comparable to the reference drug – bestatin (IC50 5.2±0.8 µM). Results of microsomal LAP inhibition obtained for compound 3b were consistent with its antiproliferative activity. Additionally, several other compounds tested, such as 4j, 4i, 4a and 4g, showed moderate LAP inhibitory activity, with four-fold higher IC50 in comparison to 3b. Compound 3b and bestatin were evaluated as microsomal LAP inhibitors in additional experiment including 10 h reaction. Ten hour reaction resulted in IC50 value of 33.6±2.2 µM for compound 3b (bestatin IC50 = 11.2±0.7 µM). 2.2.2. Structure-activity relationship Dihydroisoquinoline derivatives were previously identified as biologically active compounds. Taking into account a variety of activities, we were focused on the design of dihydroisoquinoline structures with different substituents: halogen atoms, alkyl groups as well as benzene rings. We designed a series of dihydroisoquinoline derivatives, however in the paper, we present just some of them (fourteen compounds) due to some difficulties with synthesis and biological evaluation (poor solubility in water). We consider structures such as diethyl
azeto[2,1-a]isoquinoline-4-carboxylic acid (5), dibenzyl 7,8-dimethoxy-4,5-dihydro-3H-2benzazepine-3,3-dicarboxylate (6a) and 7,8-dihydroxy-4,5-dihydro-3H-2-benzazepine-3carboxylic acid (6b). We were interested to see, which substituents will influence leucine aminopeptidase inhibitory activity. Structure-activity relationship (SAR) analysis indicated that among dihydroisoquinoline structures substituted with hydroxyl groups, disubstituted compounds (4d) are more active towards LAP than trisubstituted (4c). Ortho location of two hydroxyl groups (4d) caused slightly better inhibitory activity than meta substitution (4f). Replacement of hydroxyl group by iodine atom (4a) at 7-position caused a slight increase in biological activity. The presence of two hydroxyl groups at 6- and 7-position and halogen atom at 8-position (4i, 4j) resulted in better microsomal LAP inhibitory activity in comparison to compound 4d. Introduction of bromine atom at 7-position (4g) significantly enhanced LAP inhibitory activity among meta- dihydroxy substituted dihydroisoquinolines (4f, 4g). There is no difference in biological activity between compounds mono- and dihydroxy- substituted compounds with iodine atom (4a and 4j). Introduction of methyl group at 3-position (4e) did not enhance anti-LAP activity in comparison to compound 4d. Compound 6b, with a dihydrobenzoazepine ring, showed slightly worse microsomal LAP inhibitory activity in comparison to the similar compound, 4d, with a dihydropyridine ring. The introduction of methoxy- groups at 6- and 7-position and two benzyloxycarbonyl groups at 3-position (6a) did not enhance the activity towards LAP. Also, 2-oxo-1,4,5,9b-tetrahydro-2H-azeto[2,1-a]isoquinoline analogue (5) did not show biological activity. The presence of two carbonyl oxygen atoms and ethyl groups from ethyl carboxylate (3b) is necessary for potent interactions in the LAP binding site. We found that two benzyloxysubstituents in meta location and two ethoxycarbonyl groups at 3-position (3b) resulted in significant anti-LAP activity in comparison to dihydroxy- substituted derivative (4d). Moreover, it should be said that the presence of hydrophilic groups like hydroxyl moiety at 6and 7- (4d, 4e, 4i, 4j) as well as 6- and 8-position (4f, 4g) is not enough for potent anti-LAP activity. The presence of hydrophobic benzyl substituents improved binding potency of a dihydroisoquinoline derivative (3b).
All compounds were filtered based on the physico-chemical properties of known actives (206
Compounds 2b, 3b, 4a-j, 5, 6a-b fit Lipinski rule of five
and potentially will absorb in the
human body. 2.2.3. Antiproliferative activity Synthesized compounds were tested in vitro for their antiproliferative activity towards HL-60 human promyelocytic leukemia cells and its mitoxantrone resistant subline HL-60/MX2, human breast cancer cell line MCF-7 and three normal cell lines: normal breast epithelial MCF-10 cell line, murine fibroblasts BALB/3T3 as well as human endothelial HLMEC. HL60/MX2 cells were the most sensitive to the inhibitory effect of these compounds and antiproliferative activity of 3b was the highest among all tested compounds. The IC50 of compound 3b was 4-8 times lower towards cancer cell lines (both HL-60 and MCF-7 cells) than towards normal fibroblasts and breast epithelial cells. Similar antiproliferative activity of 3b as against cancer cells was observed against human lung endothelium HLMEC (Table 1). Table 1. Microsomal leucine aminopeptidase inhibitory activity (IC50) and antiproliferative activity (IC50) of all tested compounds
Antiproliferative activity (µM ± SD) Microsomal LAP (µM ± SD)
149.6±16.48 63.8±3.1 a
UN – not active in the highest concentration used NT – not tested a inhibition of proliferation (%) in the highest concentration used, 100 µg/ml (290 µM).
Compound 3b was determined to be the most active and has been examined against additional cell lines (Table 2). It was shown to demonstrate very strong antiproliferative activity towards Burkitt′s lymphoma cell line Raji. Similar activity of 3b was noticed against camptothecin resistant CEM/C2 human T cell leukemia cell line with mutated catalytic site of topoisomerase I25 and against its parental cell line CCRF/CEM. This result together with observations of antiproliferative activity of 3b towards HL-60/MX2, displaying crossresistance to agents which cytotoxicity results from interaction with the nuclear enzyme DNA topoisomerase II (topo II)26, suggest that the mechanism of antiproliferative activity of the compound is not dependent on both topoisomerases. The antiproliferative activity of 3b was also very high towards the LoVo colon cancer cell line, LoVo/Dx-variant resistant to doxorubicin with multidrug cross-resistance, LNCaP prostate cancer cell line, PC3 prostate cancer cell line and HCV29T urinary bladder cancer cell line. At the same time, the compound was inactive towards other colon cancer HT-29, Du145 prostate cancer cell line, MV4-11 biphenotypic B myelomonocytic leukemia, acute T cell leukemia Jurkat and K562 chronic myelogenous leukemia. Compound 3b showed moderate activity towards A549 nonsmall cell lung and A498 kidney cancer cell lines. In our study, we tested antiproliferative activity of a known drug, bestatin to compare it to the compound 3b. Bestatin showed inhibitory activity against MCF-7, LoVo and A549 cell lines with IC50 149.6±16.5 µM, 233.5±26.8 µM and 241.3 µM, respectively. Compound 3b was more active towards mentioned cell lines than bestatin (Table 1 and 2). The IC50 value of the compound 3b against MCF-7 cell line was approximately 15-fold lower than IC50 value of bestatin. Whereas, the IC50 values of 3b towards LoVo and A549 cells were 212-fold and 18fold lower, respectively than IC50 values of bestatin. Comparison of antiproliferative and LAP inhibitory activities values of compound 3b (Table 1, 2) may suggest additional mechanism of action. In the aim to search for another mode of action, we evaluated other potential activities of compound 3b, including free-radical scavenging activity against DPPH· and ABTS·+ radicals. However, compound 3b did not scavenge ABTS·+ radical and showed very low activity against DPPH· radical (IC50 = 1mM). This data suggested that free radical scavenging is not an additional mechanism of action. Possibly, electrophilic properties of compound 3b may contribute to antiproliferative activity. Table 2. Antiproliferative activity (IC50) and calculated selectivity index SI of compound 3b and reference anticancer drug, cisplatin
Antiproliferative activity (µM ± SD)
Calculated selectivity index SI
UN – not active in the highest concentration used. Bestatin in the concentration of 100 µg/ml inhibited proliferation of LoVo, LoVo/Dx and A549 cells in 54.8±3.7% 26.9±5.4% and 48±8.1%, respectively.
The selectivity index (SI), which is an important pharmaceutical parameter, representing the ratio of IC50 value calculated for normal cell line (BALB/3T3) to IC50 value for respective cancer cell line27, was estimated. SI for compound 3b towards HL-60, HL-60/MX2 and MCF7 was calculated to be 5.8, 7.0 and 3.7, respectively. SI for reference anticancer drug, cisplatin towards HL-60, HL-60/MX2 and MCF-7 was equal to 6.2, 7.3 and 0.7, respectively. SI values for tested compound 3b and cisplatin towards other cancer cell lines are shown in Table 2. SI values higher than 1 indicate greater efficacy against tumor cell than toxicity against normal
cells. SI values greater than 3 mean that the compound is highly selective. In our study, for most of the tested cancer cell lines, SI values of compound 3b were much higher than 3, implying that it can kill cancer cells selectively. It is worth mentioning, that for compound 3b, SI > 30 was determined towards Raji, CEM/C2, CCRF/CEM and LoVo cell lines, which is 6 – 8 times higher in comparison to SI for cisplatin. Compound 3b showed higher values of SI also towards PC-3, LNCaP, LoVo/Dx, HCV29T, A549, A498 cell lines. It should be concluded that compound 3b is highly selective towards these cancer cell lines and may be promising as the potential anticancer substance for more detailed in vitro and in vivo studies. Moreover, it could be considered as a lead compound for further development as anti-LAP drug for the treatment of various cancers. 2.2.4. The influence on cell cycle and cell death Compound 3b exhibited the highest antiproliferative activity and was subjected to analysis of its influence on the cell cycle and apoptosis of the HL-60 cell line. Studies of other authors show that LAP is frequently overproduced in various types of cancer cells. This enzyme was determined to promote G1/S transition in human esophageal squamous cell carcinoma and human hepatocellular carcinoma (HCC).11;12 G1/S phase transition is a major checkpoint for cell cycle progression. Knockdown of LAP in HepG2 HCC cells induced cell cycle arrest at G1 phase.11;12 Analysis of the cell cycle by flow cytometry showed that compound 3b demonstrates a tendency to stop the cell in G0/G1 cell cycle phase (Fig. 1A). It seems likely to be the result of LAP inhibition by 3b. Influence of compound 3b on the cell cycle was also evaluated by examining the changes in expression of cyclins using Western-blot technique. Previous studies have shown that LAP controls cell cycle progression through activation of cyclin-dependent kinases, CDK2, CDK4, CDK6 and cyclin A. CDK2, CDK4 and CDK6 are critical positive regulators during G1/S transition.12 In our study, the level of cyclin A was not affected by 3b (Fig. 1B). Based on literature data, the level of cyclin D increases in early steps of G1 and accumulates until the G1/S phase transition, then rapidly decreases.28 In our experiments, expression of cyclin D increased after treatment with 3b (Fig. 1B). Moreover, decreased expression of cyclin E was detected in cells treated with 3b (Fig. 1B). Cyclin E, overexpressed in cancer, is the regulatory cyclin for CDK2, and the complex cyclin E/CDK2 is considered a requisite regulator of G1 to S progression.29 Taken together, alteration in expression of these two cell
cycle regulators (cyclins D and E) in the presence of 3b confirmed its role in inhibition of cell cycle G1/S transition. Apoptosis is the programmed cell death and plays a crucial role in removal of unwanted cells. In the early stage of apoptosis, phosphatidylserine is translocated to the cell surface. Annexin V, which binds to exposed phosphatidylserine, can be used to identify apoptotic cells. To separate the early from the late apoptotic cells, annexin V staining and propidium iodide (PI) were used. Results are summarized in Figure 2A. Compound 3b increased the number of late apoptotic cells (p <0.05 as compared to control cells). The solvent (DMSO) did not impact apoptosis at the concentration used. We analyzed also the influence of compound 3b on the mitochondrial membrane potential (MMP) and activation of caspase cascades. In the first stage of apoptosis, MMP decreases. To measure MMP, we used staining with JC-1 dye, which accumulates in mitochondria in a potential-dependent manner. In normal cells with high MMP JC-1, Jaggregates exhibit red fluorescence and in cells with low MMP JC-1 - monomers are fluorescent green. The conducted study showed a tendency in decreasing MMP in cells treated with 3b relative to both controls (Fig. 2B). Anticancer therapies almost always result in activation of caspases, a family of cysteine proteases that act as common death effector molecules in various forms of cell death. 30
Compound 3b is able to increase the activation of caspase 3 as compared to cell and solvent
controls, which confirms its ability to induce cell death (Fig. 2C). Tian et al.11 and Zhang et al.12 also reported that LAP inhibition markedly promotes cell death in hepatocellular and squamous cancer cells treated with cisplatin. Our studies are in accordance with these observations, showing an increased number of apoptotic cells with a decrease in mitochondrial membrane potential and increased caspase-3 activity after treatment of HL-60 cells with 3b. These results, together with data showing LAP inhibitory properties of 3b, may underlie LAP inhibition as the main mechanism of action of the compound. Members of the Bcl-2 protein family are critical mediators of apoptotic cell death in health and disease. They are often deregulated in cancer and believed to lead to the survival of malignant cells.31 This family can be divided into pro-apoptotic effector proteins (e.g. BAX), and anti-apoptotic Bcl-2 proteins (e.g. Bcl-2).32 In our studies, compound 3b did not affect the expression of Bax or Bcl-2 proteins (Fig. 2D), suggesting that they are not involved in cell death induced by this compound.
Autophagy is a common pathway for degradation of cellular components and is related not only to physiological processes, but also to many pathologies, including cell survival and cell death. It is also involved in cancer and may influence effectiveness of anticancer therapies.33 However, compound 3b do not influence this process as assessed by LC3b-II detection (Fig. 2E).
Figure 1. Cell cycle parameters of HL-60 cells treated with 3b, cisplatin or solvent – DMSO. A. Flow cytometric analysis of cell cycle distribution. 1 - cell control, 2 - solvent control (DMSO), 3 - cisplatin 0.14 µg/mL, 4 - cisplatin 0.28 µg/mL, 5- 3b 1 µg/mL, 6- 3b 3 µg/mL. Three independent experiments are conducted and the mean value ± SD is presented. B. Expression of cell cycle regulating proteins: cyclin D, E and A. Densitometric analysis of the western blots was carried out using ImageJ 1.46r. The blots were normalized to actin and the fold-change protein level expression is reported in comparison to control. “C” means cell control.
Figure 2. Cell death analysis of HL-60 cells treated with 3b or solvent – DMSO. A. Detection of apoptosis using Annexin V. Representative dot-plots and graph summarizing mean ± SD values from three independent experiments. Q1- death cells (Annexin negative-PI
positive), Q2- end stage of apoptosis (Annexin positive- PI positive), Q3- viable cells (Annexin negative-PI negative), Q4- early stage of apoptosis. ∗ Indicates statistically significant values (p≤0.05 as compared to control). B. Mitochondrial membrane potential. Representative dot-plots of mitochondrial membrane potential of HL-60 cells treated with 3b or solvent (DMSO) and graph indicating mean ± SD from three independent experiments. C. Activity of caspase-3. Results are normalized to the protein content and reported as relative caspase-3/7 activity in comparison to the untreated control. Three independent experiments are conducted and the mean value ± SD is presented. D. Bax and Bcl-2 expression. E. LC3bII expression. The cells were exposed to the test compound at concentration of 1 µg/mL and DMSO as solvent for 3b. Following 72 h of incubation with compound and solvent, the cells were collected for cell death analyses. The densitometric analysis of the western blots was carried out using ImageJ 1.46r. The blots were normalized to actin and the fold-change protein level expression is reported in comparison to control (mean ± SD). 2.2.5. Toxicological studies Results of the LAP inhibitory assay determined compound 3b to be the most active, which led to its selection for further investigation of its toxicity. Several studies were performed to evaluate the safety of the compound, including skin sensitization of guinea pigs, acute skin irritation/corrosion of rabbits, acute dermal toxicity on rats, acute oral toxicity on mice and acute eye irritation/corrosion on rabbits. The skin sensitization study was performed to determine the potential allergic contact dermatitis caused by 3b. During the study, allergic skin reactions were determined in two out of eleven animals treated (18.2%). Taking these results into consideration, compound 3b can be classified as: agent causing mild sensitization – according to the Magnusson and Kligman classification34 and agent beyond categorization – according to the Regulation UE No. 286/201135 and (EC) No. 1272/2008.36 When trying to evaluate and assess toxic properties of a given test item, acute skin irritation/corrosion study is a significantly important step. The examination was performed on three albino rabbits. The treated skin of the rabbits did not exhibit any pathological changes 1, 24, 48, and 72 hours after the end of the treatment. Results of the study are presented based on the scoring system described in the OECD Guideline No. 404 / EU Method B.4.37 On the grounds of the study, compound 3b was characterized as follows: not irritative for the rabbit skin – according to the Annex to the Regulation38; beyond categorization – according to the
Regulation (EC) No. 1272/200836, amending and repealing Directives 67/548/EEC39 and 1999/45/EC40 and amending Regulation (EC) No. 1907/2006.41 An acute dermal toxicity study on ten rats was performed to provide initial information on health hazards resulting from short-term dermal exposure. After single application of the test item (2000 mg/kg body weight (bw)), the animals did not exhibit any general clinical signs. No pathological changes on the treated skin were noticed. On the grounds of the study, it can be determined that the median lethal dermal dose (LD50) for 3b is greater than 2000 mg/kg bw. According to the Regulation (EC) No. 1272/2008 36, amending and repealing Directives 67/548/EEC39 and 1999/45/EC40 and amending Regulation (EC) No. 1907/200641, it may be concluded that compound 3b is beyond categorization. The acute toxic class method, according to the OECD Guideline No. 423 / EU Method B.1. TRIS., was performed to evaluate acute oral toxicity of compound 3b on mice.37;42-45 Following single administration of the test item at a dose of 300 mg/kg bw to three females at the first stage of the experiment, temporary clinical signs were observed on the day of administration (day 0). During the 14-day observation period, animals did not exhibit any clinical signs. Following single administration of the test item at a dose of 2000 mg/kg bw to three females at the second stage of the experiment, the animals exhibited some clinical signs visible on the day of administration. These changes were temporary. During the 14-day observation period, no clinical signs were reported. Single administration of the test item at a dose of 2000 mg/kg bw to the next three females at the third stage of the experiment induced some clinical signs visible on the day of administration. These changes were temporary. On the grounds of the study, the test item (compound 3b) can be classified to category 5 (unclassified), according to the Globally Harmonized System (GHS). Acute eye irritation/corrosion study for compound 3b was performed on albino rabbits to obtain information on health hazards likely to arise from possible contact of the test item with the eye. All animals exhibited changes in the conjunctiva (erythema, swelling). Changes were temporary. The compound did not irritate the rabbit eye, according to the regulation of the Minister of Health of 08.2012 on the classification of chemical substances and their mixtures. Compound 3b is also beyond categorization, according to the regulation (EC) No. 1272/200836 and repealing Directives 67/548/EEC and 1999/45/EC and amending Regulation (EC) No. 1907/2006.37;43-45 2.3. Binding mode of compound 3b
Molecular docking of compound 3b into the binding site of bovine lens leucine aminopeptidase (PDB code: 1LAN46) was performed in order to understand the enzymeinhibitor key interactions. Interactions of compound 3b with the enzyme’s active site were afterwards compared to the binding of known inhibitors, L-leucinal, which is originally in crystal complex 1LAN (PDB), and bestatin. Residues determined to be involved in ligand binding with the leucine aminopeptidase enzyme were: Lys250, Lys262, Met270, Asn330, Ala333, Asp273, Arg336, Thr359, Leu360, Gly362, Ile421, Ala451, Met454 (Fig. 3). Previous studies showed that the LAP binding site can be divided into pockets named S1, S1’, S2’, etc.6;47 The S1 pocket of the leucine aminopeptidase has a hydrophobic character and is formed by Met270, Ala451, Thr359, Gly362 and Met454 residues.6 It seems that different residues in this pocket interact with the ligand through van der Waals and hydrophobic interactions. Both ethyl groups of compound 3b are located next to these amino acids. The enzyme S1’ pocket is also hydrophobic and is formed by Asn330, Ala333 and Ile421 side chains.48;49 One of the phenyl groups of compound 3b is located in this pocket and the second interacts with Ser260 and Ile261. Active site residues of LAP important for inhibitor binding, include Lys262, which side chain amino group is involved in the hydrogen bond with the oxygen atom of the inhibitors; Leu360, which carbonyl oxygen is involved in the hydrogen bond with the amide group of the scissile peptide bond of the substrate or peptide analogues as well as hydroxyl group of amino acid analogues; Asp273, which carboxyl oxygen forms a hydrogen bond with the N-terminal amine group of the substrate and inhibitors, and the amide group of Gly362, which interacts with the carboxyl group of the dipeptide substrate or its analogues. Besides the positively charged amino group of Lys262, the guanidinyl moiety of Arg336 is also present in the active site.6 The active site of the enzyme possesses also two zinc ions: Zn488 and Zn489, which participate in substrate binding and activation and are important also for inhibitor binding.6;20 The interaction with two zinc ions, as well as the positively charged amino group of Lys26.2 in the enzyme active site, appear to be the most important traits for tight binding with LAP inhibitors.6 Compound 3b binds to LAP via a hydrogen bond between the oxygen atom of one carbonyl group with the amino hydrogen atom of Gly362 (O···H-N: 1.67Å). The oxygen atom from the second carbonyl group coordinates Zn488 (Zn···O=C: 2.33Å). The Zn488 is coordinated
by the carboxylate oxygen atoms of Asp255, Glu334 and by carbonyl and carboxyl oxygen atoms of Asp332. The Zn489 is also coordinated by carboxylate oxygen atoms of Asp273 and Glu334 and liganded by Lys250. All the above mentioned interactions are depicted on Figure 3. In comparison to a known inhibitor - L-leucinal, crystallized in complex with LAP (in 1LAN, PDB), compound 3b shows similarity in the position of hydrophobic chains (ethyl group in compound 3b and isopropyl group in L-leucinal) and the carbonyl group which coordinates zinc ions.20 Results of the docking experiments show one carbonyl group of 3b to be close (C=O...H-N: 3.15Å) to Lys262. It seems probable that the oxygen atom from the carbonyl group may interact with Lys262 amino group via the water molecule. Moreover, in our study Asp273 and Leu360 were found within 3-4Å from the ligand 3b. The docking score for selected pose of 3b was -9.86.
Figure 3. A - Structure of blLAP in complex with 3b. B - interactions of the bILAP active site residues with compound 3b. Superposition of the crystallographically-determined binding pose of L-leucinal (in 1LAN crystal complex) with its model predicted by ICM Pro (Molsoft LLC, USA), show strong agreement. L-leucinal binds to several amino acids in the active site of blLAP. The nitrogen atom of its terminal α-amino group is coordinated to Zn489. Moreover, this α-amino group is involved in a hydrogen bond with the side chain amino group of Asp273. One oxygen atom of L-leucinal
forms a hydrogen bond with the amino group of Lys262 and coordinates Zn488.
Second oxygen atom coordinates Zn489 and is involved in hydrogen bonding with a water molecule present in the LAP active site.20 Another known LAP inhibitor, bestatin, was described to be stabilized in the active site by three hydrogen bonds, between the carboxyl oxygen and the side chain amino group of Lys262, between the NH group of bestatin and the backbone carbonyl oxygen atom of
Leu360, and between one of the carboxylate oxygen atoms of bestatin and the backbone NH group of Gly362.48;50;51 In summary, interactions of compound 3b in the LAP binding site seem to play crucial role in inhibition of the enzyme. Data from molecular docking studies and biological assays suggest that compound 3b is a potential inhibitor of LAP. 3.
In conclusion, a series of 3,4-dihydroisoquinoline derivatives were synthesized and their in vitro inhibitory activity towards microsomal leucine aminopeptidase as well as antiproliferative activity and toxicity were evaluated. Among the tested derivatives, compound 3b exhibited significant activity against microsomal LAP (IC50=16.5 µM) and promising antiproliferative activities on human cancer cell lines, including HL-60, MCF-7, Burkitt′s lymphoma Raji, CEM/C2, CCRF/CEM, LoVo, LoVo/Dx, LNCaP, PC3 and HCV29T. Most of these activities were comparable to or better than for cisplatin. Its antiproliferative activity towards MCF-7, LoVo and LoVo/Dx cell lines was stronger than bestatin, the known LAP inhibitor. It is worth mentioning that compound 3b showed very high values of SI towards these cancer cell lines, which were also from a few to several fold greater in comparison to SI of cisplatin. Additionally, the molecular docking simulations performed for the selected active compound - 3b, revealed that it could occupy the active site of blLAP through hydrophobic and H-bonding interactions. The compound displayed H-bond interactions with such residues as Gly362 or zinc ions, which were suggested to be essential for inhibitory activity. Altogether, the enzyme assay data and molecular docking study imply that compound 3b is an effective inhibitor of leucine aminopeptidase. Assays examining the influence of 3b on the cell cycle confirmed that LAP inhibition is one of its mechanisms of action. Results of the apoptosis study showed that compound 3b increased the number of late apoptopic cells and was able to increase caspase 3 activity, confirming its role in cell death induction. The safety profile of compound 3b was examined by toxicological studies. Taking into account the high SI values, promising antitumor activities and toxicity characteristics, compound 3b can be considered as a good candidate for further drug design studies. According to our knowledge, this is the first report of a 3,4-dihydroisoquinoline derivative acting as a LAP inhibitor. In the light of this study, molecules with 3,4-dihydroisoquinoline moiety might be regarded as a new class of anti-tumor agents and model compounds for designing and development of anticancer drugs.
All commercially available products were used without further purification. All chemicals used in the study were of analytical grade. Benzyl chlorides 1a-c have been prepared from appropriate alcohols using thionyl chloride. Compounds 4b,d-j, 5, 6a,b were described previously.13 4.2.
4.2.1. Dibenzyl 2-(3-(benzyloxy)-4-iodobenzyl)-2-formamidomalonate 2a To a solution of 3-benzyloxy-4-iodobenzyl chloride (1.17 g, 3.23 mmol) and dibenzyl formamidomalonate (1.08 g, 3.23 mmol) in acetone (30 mL), K2CO3 (6.0 g, 43.0 mmol) and KI (1.61 g, 9.69 mmol) were added. The mixture was stirred at 60 oC under a reflux condenser for 24 hrs. After cooling, the suspension was filtered through Celite and the residue washed with CH2Cl2. The solvent was evaporated and the residue was purified by chromatography on silica gel (CH2Cl2/AcOEt 50:1). The yield noted was 1.05 g (50%). 1H NMR (CDCl3) δ: 3.59 (s, 2H); 5.03 (s, 2H); 5.07 (1/2AB, J=12.1 Hz, 2H); 5.15 (1/2AB, J=12.1 Hz, 2H); 6.11 (dd, J=7.9 Hz, 1.7 Hz, 1H); 6.41 (d, J=1.7 Hz, 1H); 6.48 (bs, 1H), 7.22-7.66 (m, 4H); 7.30-7.40 (m, 11H); 7.50 (d, J=7.9 Hz, 1H); 7.85 (d, J=0.8 Hz, 1H).
C NMR (CDCl3) δ: 37.6; 66.5;
68.4;70.4; 85.5; 114.7; 128.4; 123.9; 128.5; 126.7; 128.6; 128.7; 127.8; 134.3; 136.2; 136.4; 139.2; 156.8; 159.8; 166.5. HR MS ESI calculated for (M+H) C32H29INO6 650.1034. Found: 650.1033. 4.2.2. Diethyl 2-(3,5-dibenzyloxybenzyl)-2-formamidomalonate 2b Compound 2b was prepared according to procedure 4.2.1, using 3,5-dibenzyloxybenzyl chloride (20 g, 59.0 mmol), diethyl formamidomalonate (12.24 g, 59.0 mmol), acetone (350 mL), K2CO3 (110 g, 0.79 mol) and KI (29.4 g, 0.177 mmol). The product was recrystalized from hexane. The yield noted was 27.1 g (91%). 1H NMR (CDCl3) δ: 1.27 (t, J=7.1 Hz, 6H); 3.56 (s, 2H); 4.14-4.32 (m, 4H); 5.0 (s, 4H); 6.24 (d, J=2.1 Hz, 2H); 6.53 (t, J=2.1 Hz, 1H); 6.56 (bs, 1H); 7.28-7.44 (m, 10H); 7.92 (d, J=0.8 Hz, 1H).
C NMR (CDCl3) δ: 13.9; 38.0;
62.8; 66.5; 69.9; 101.1; 109.2; 127.3; 127.9; 128.6; 136.8; 136.9; 159.7; 159.8; 166.9. HR MS ESI calculated for C29H31NO7 (M+H) 506.2173. Found 506.2164. 4.2.3. Dibenzyl 2-(3,4,5-tribenzyloxybenzyl)-2-formamidomalonate 2c
Compound 2c was prepared according to procedure 4.2.1, using 3,4,5-tribenzyloxybenzyl chloride (10 g, 22.02 mmol), dibenzyl formamidomalonate (7.4 g, 22.02 mmol), K2CO3 (41 g, 293 mmol) and KI (10 g, 22.02 mmol). The yield noted was 13.87 g (86%). 1H NMR (CDCl3) δ: 3.54 (s, 2H); 4.96 (s, 4H); 5.04 (s, 4H); 5.10 (s, 2H); 6.20 (s, 2H); 6.37 (bs, 1H); 7.16-7.46 (m, 25H); 7.72 (d, J=1.2 Hz, 1H).
C NMR (CDCl3) δ: 38.0; 66.7; 68.1; 70.9; 75.0; 109.8;
127.1; 127.2; 127.71; 127.73; 128.1; 128.4; 128.56; 128.57; 128.6; 129.9; 134.5; 137.1; 137.71; 137.73; 152.4; 159.8; 166.5. HR MS ESI calculated for C46H42NO8 (M+H) 736.2905. Found 736.2845. 4.2.4. Dibenzyl 6-benzyloxy-7-iodo-3,4-dihydroisoquinoline-3,3-dicarboxylate 3a POCl3 (0.22 mL, 2.42 mmol) was added to a stirred solution of dibenzyl 2-(3-(benzyloxy)-4iodobenzyl)-2-formamidomalonate (500 mg, 0.77 mmol) in acetonitrile (15 mL) and the mixture was heated on an oil bath at 85 oC for 24 hrs. The solvent was evaporated. Residue was dissolved in CH2Cl2 (15 mL) and neutralized with aqueous NaHCO3 (20 mL). The mixture was extracted with CH2Cl2 (3 × 15 mL). The combined organic layers were dried with MgSO4 and concentrated. The product was purified by chromatography on silica gel. The yield noted was 405 mg (82%). 1H NMR (CDCl3) δ: 3.29 (s, 2H); 5.08 (1/2AB, J=12.5 Hz, 2H); 5.09 (s, 2H); 5.17 (1/2AB, J=12.5 Hz, 2H); 6.57 (s, 1H); 7.13-7.48 (m, 15H); 7.75 (s, 1H); 8.37 (s, 1H).
C NMR (CDCl3) δ: 31,2; 67,8; 70,3; 71,0; 84,5; 111,4; 122,9; 126,7;
126,7; 126,9; 128,1; 128,2; 128,3; 128,4; 128,6; 135,0; 135,4; 135,5; 139,0; 159,9; 160,8;168,4. HR MS ESI calculated for (M+H) C32H27INO5 632.0928. Found: 632.0924. 4.2.5. Diethyl 6,8-dibenzyloxy-3,4-dihydroisoquinoline-3,3-dicarboxylate 3b Compound 3b was prepared according to procedure 4.2.4, using POCl3 (0.45 mL, 4.9 mmol), diethyl 2-(3,5-dibenzyloxybenzyl)-2-formamidomalonate (0.8 g, 1.58 mmol) and acetonitrile (40 mL). The mixture was heated on an oil bath at 75oC for 48 hrs. The product was purified by crystallization from hexane. The yield noted was 0.639 mg (83%). 1H NMR (CDCl3) δ: 1.24 (t, J=7.1 Hz, 6H); 3.3 (s, 2H); 4.13-4.32 (m, 4H); 5.0 (s, 2H); 5.1 (s, 2H); 6.41 (d, J=2.1 Hz, 1H); 6.44 (d, J=2.1 Hz, 1H); 7.29-7.46 (m, 10H); 8.86 (d, J=0.5 Hz, 1H).
(CDCl3) δ: 13.9; 31.5; 62.1; 69.8; 70.2; 99.1; 105.8; 111.4; 127.0; 127,5; 128,1; 128,3; 128,6; 128.7; 136.1; 136.2; 136.9; 157.4; 158.1; 162.8; 169.3. HR MS ESI calculated for C29H30NO6 (M+H) 488.2068. Found 488.2060. 4.2.6. Dibenzyl 6,7,8-tribenzyloxy-3,4-dihydroisoquinoline-3,3-dicarboxylate 3c
Compound 3c was prepared according to procedure 4.2.4, using dibenzyl 2-(3,4,5tribenzyloxybenzyl)-2-formamidomalonate (6.48 g, 8.81 mmol) and POCl3 (2.52 mL). The reaction has been carried out for 24 hrs at 75oC. The product was crystallized from ethyl acetate–hexane solution. The yield noted was 5.6 g (89%). 1H NMR (CDCl3) δ: 3.27 (s, 2H); 5.01 (s, 2H); 5.03 (s, 2H); 5.08 (s, 2H); 5.10 and 5.19 (AB, J=12 Hz, 4H); 6.53 (s, 1H); 7.177.44 (m, 25H); 8.73 (s, 1H).
C NMR (CDCl3) δ: 29.7; 31.1; 67.6; 70.4; 70.9; 75.6; 108.3;
115.5; 127.5; 127.98; 128.10; 128.18; 128.24; 128.28; 128.30; 128.39; 128.41; 128.51; 128.58; 128.61; 130.1; 135.2; 136.0; 136.6; 137.1; 140.3; 151.7; 156.3; 158.2; 168.9. HR MS ESI calculated for C46H40NO7 (M+H) 718.2799. Found 718.2793. 4.2.7. 6-Hydroxy-7-iodo-3,4-dihydroisoquinoline-3-carboxylic acid 4a BBr3 (237 mg, 0.94 mmol) was added dropwise under argon to a solution of dibenzyl 6benzyloxy-7-iodo-3,4-dihydroisoquinoline-3,3-dicarboxylate (148 mg, 0.234 mmol) in CH2Cl2 (25 mL) at -20°C. The reaction mixture was stirred for 0.5 h at the same temperature and quenched with H2O (10 mL). The resultant mixture was then stirred at room temperature (rt) for 0.5 h. An additional portion of water (80 mL) was added. The water layer was separated and washed 3 times with CH2Cl2. The aqueous solution was loaded on a column packed with ion exchange resin Dowex 50W X4 (30 mL, pretreated subsequently with water, 2 M HCl, and water to pH 6–7). The column was washed with water (200 mL) and the product was eluted with 2 M aqueous NH3. Brown to yellow colored fractions were collected. Ammonia was removed under water pump vacuum at rt and the solution was evaporated (water bath at 35°C). The residue was acidified with 2 M aqueous HCl. White precipitate was collected, washed with water and dried. The yield noted was 35 mg (47%). 1H NMR (D2O+DCl) δ: 3.15 and 3.26 (ABX, J= 8.1 Hz, 8.8 Hz, 16.9 Hz, 2H); 4.39 (dd, J=8.1 Hz, 8.8 Hz, 1H); 6.45 (s, 1H); 8.07 (s, 1H); 8.23 (s, 1H). HR MS ESI calculated for (M+H) C10H9INO3 317.9622. Found: 317.9619. 4.2.8. 6,7,8-trihydroxy-3,4-dihydroisoquinoline-3-carboxylic acid 4c BBr3 (1.75 mL, 17.97 mmol) was added dropwise under argon to a solution of dibenzyl 6,7,8-tribenzyloxy-3,4-dihydroisoquinoline-3,3-dicarboxylate (2.15 g, 3.0 mmol) in CH2Cl2 (60 mL) at -20°C. The reaction mixture was stirred for 0.5 h at the same temperature and quenched with H2O (25 mL). The resultant mixture was then stirred at rt for 0.5 h. The water layer was separated and washed 3 times with CH2Cl2. The residue was acidified with 2 M aqueous HCl to pH 2 and purified on polyacrylamide gel (Biogel P-2) using 0.1% aqueous TFA as an eluent. The yield noted was 0.6 g (90%). 1H NMR (D2O+DCl) δ: 3.00 and 3.10
(ABX, J=0.7 Hz, 8.6 Hz, 17.2 Hz, 2H); 4.59 (ddd, J=8.6 Hz, 7.3 Hz, 1.2 Hz, 1H); 6.21 (s, 1H); 8.6 (s, 1H).
C NMR (D2O) δ: 27.3; 53.2; 105.8; 108.7; 129.8; 130.5; 151.6; 156.8;
159.5; 171.0. HR MS ESI calculated for (M+H) C10H10NO5 224.0559. Found: 224.0594. 4.3.
Aminopeptidase inhibitory activity
4.3.1. Microsomal leucine aminopeptidase inhibitory activity The microsomal leucine aminopeptidase (LAP; Sigma-Aldrich, Germany) inhibition assay was based on detection of 7-amido-4-methylcoumarin that is obtained when L-Leu is cleaved from the substrate (L-leucine-7-amido-4-methylcoumarin), according to the previously described method52 with modification. The method was adapted for microtitre plates (Thermo Scientific, USA) and a total volume of 200 µL per well. An amount of 185 µL of 0.02 M TrisHCl with 0.5 mM CaCl2 (pH 8.5), 5 µL of inhibitor solution in DMSO, 5 µL of 0.1225 U/mL leucine aminopeptidase solution in Tris-HCl 0.02M + 0.5mM CaCl2 pH 8.5 and 5 µL of 4 mM L-leucine-7-amido-4-methylcoumarin in DMSO were mixed and incubated at 37°C for 60 min. During incubation in the FLUOstar Omega (BMG LABTECH, GmbH, Germany) microplate reader (ex. 380 nm, em. 440 nm), the fluorescence was read every 3 min. The positive control of the enzyme activity consisted of DMSO (5 µL) instead of inhibitor solution. Three blank samples were tested: inhibitor solution; inhibitor solution with substrate; 7-amido-4-methylcoumarin solution. All samples were triplicated and carried out under the same conditions. Different concentrations of the inhibitor were examined (final concentration: 2 – 250 µM). Additional experiment was done for compound 3b and for bestatin. It was based on the same reaction conditions (as described above) but 10h reaction period. The fluorescence was read every 6 min. The percentage of inhibition was calculated for each inhibitor concentration using formula:
where Fc stands for the change of fluorescence for the control and Fi represents the change of fluorescence in the presence of the inhibitor. The amount of the tested compound needed to inhibit the enzyme by 50%, IC50 (inhibitory concentration), was calculated by linear regression between inhibitor concentration and percentage of inhibition and expressed as µM. 4.4.
Free radical scavenging activity
Compound 3b was tested for the ability to scavenge DPPH· and ABTS·+ radicals according to the methods described previously 13. 4.5. Antiproliferative activity 4.5.1. Compounds Prior to usage, the compounds were dissolved in DMSO to the concentration of 20 mg/mL, and subsequently diluted in the test medium to reach the required concentrations (ranging from 0.01 to 1000 µg/mL). 4.5.2. In vitro anti-proliferative assay Twenty four hours prior to the addition of the tested compounds, cells were plated in 96-well plates (Sarstedt, Germany) at a density of 1 × 104 cells per well. The assay was performed after 72 h of exposure to varying concentrations of the tested agents. The in vitro cytotoxic effect of all agents was examined using the SRB assay for adherent cells (MCF-10, MCF-7, BALB/3T3 and HLMEC) or MTT assay for leukemia cells (HL-60 and HL-60/MX2) as described previously.53 Results were presented as IC50 – the concentration of tested agent which inhibits proliferation of 50% of the cancer cell population.54 IC50 values were calculated for each experiment separately and mean values ± SD are presented in Table 1 and 2. Each compound at the given concentrations was tested in triplicate in a single experiment. Each experiment was repeated 3-5 times. 220.127.116.11. SRB cytotoxic test Cells were added to each well of the microtitre plate on top of the culture medium and fixed with cold 50% trichloroacetic acid (TCA, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Plates were incubated at 4°C for 1 h and then washed five times with tap water. The cellular material was then stained with 0.4% sulphorhodamine B (SRB, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and dissolved in 1% acetic acid (Avantor Performance Materials Poland S.A., Poland) for 30 min. Unbound dye was removed by rinsing (4X) in 1% acetic acid. The protein-bound dye was extracted with 10 mM unbuffered Tris base (SigmaAldrich Chemie GmbH, Steinheim, Germany) and the optical density (λ = 540 nm) was determined in a computer-interfaced BioTek Synerg H4 Hybrid Microplate Reader (BioTek Instruments USA).
18.104.22.168. MTT cytotoxic test Twenty microliters of MTT solution (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, stock solution: 5 mg/mL) was added to each well and incubated for 4 h. After incubation, 80 µL of the lysis mixture was added to each well (lysis mixture: 225 mL dimethylformamide, 67.5 g sodium dodecyl sulphate and 275 mL of distilled water). Optical densities of the samples were read after 24 h on a BioTek Synerg H4 Hybrid Microplate Reader (BioTek Instruments USA) at 570 nm. All chemicals were purchased from SigmaAldrich Chemie GmbH, Steinheim, Germany. 4.5.3. Influence on the cell cycle and cell death by the tested compound Twenty four hours prior to the addition of the tested compounds, HL-60 cells were 5
seeded at the density of 2 × 10 cells/mL in 24-well plates (Sarstedt, Germany) to the final volume of 2 ml. Cells were exposed to the test compound 3b at a concentration of 1 µg/mL and 3 µg/mL. Cisplatin was used as a positive control. DMSO as solvent for 3b, in dilution corresponding to its highest concentration applied for the compound, induced no toxicity. After 72 h of incubation, cells were collected, washed in phosphate-buffered saline (PBS) and counted in a Burker haemocytometer. Cells (1 × 106) were washed twice in cold PBS and fixed in 70% ethanol at – 20°C. Then, cells were washed twice in PBS and incubated with RNAse (8 µg/mL, Fermentas, Germany) at 37°C for 1 h. Cells were stained 30 min with propidium iodide (50 µg/mL, Sigma-Aldrich Chemie GmbH, Germany) at 4°C and the cellular DNA content was analyzed by flow cytometry. 22.214.171.124. Determination of apoptotic cells by Annexin V staining Twenty four hours prior to the addition of the tested compounds, HL-60 cells were 5
seeded at the density of 2 × 10 cells/mL of the culture medium on 24-well plates (Sarstedt, Germany) to the final volume of 2 mL. Cells were exposed to the test compound at a concentration of 3 µg/mL. DMSO as solvent for 3b, in dilution corresponding to its concentration applied for the compound, induced no toxicity. After 72 h of incubation, cells were collected, washed in phosphate-buffered saline (PBS) and counted in a Burker haemocytometer. Following 72 h of incubation with compound and solvent, cells were collected, washed in PBS and counted in a haemocytometer. APC-Annexin V (Alexis Biochemicals) was diluted to a concentration of 1 mg/mL in binding buffer (Hepes buffer: 10
mM HEPES/NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) (IIET, Wroclaw, Poland) and cells were suspended in 200 µl of this solution (freshly prepared each time). After 15 min of incubation in the dark at rt, propidium iodide (PI) solution (1 mg/mL) was added to a final concentration of 0.1 mg/mL prior to the analysis. Data acquisition was performed by flow cytometry. Results were displayed as two-color dot plots with APCAnnexin V (Y axis) vs. PI (X axis). Double-negative cells were live cells, PI+/Annexin V+ corresponded to late apoptotic or necrotic cells, and PI-/Annexin V+ to early apoptotic cells. 126.96.36.199. Determination of mitochondrial membrane potential and caspase-3 activity 188.8.131.52.1. Mitochondrial membrane potential Twenty four hours prior to the addition of the tested compounds, HL-60 cells were seeded at 5
the density of 2 × 10 cells/mL of the culture medium on 24-well plates (Sarstedt, Germany) to the final volume of 2 mL. Cells were exposed to the test compound at a concentration of 3 µg/mL. As a positive control for cells with low potential, a 24-h incubation with 1 mM of Valinomycin (Sigma-Aldrich, Germany) was used. DMSO as solvent for 3b, in dilution corresponding to its concentration applied for the compound, induced no toxicity. Following 24 h of incubation with compound and solvent, cells were collected, washed in PBS and counted in a haemocytometer. Pelleted cells were resuspended in 100 µL of warm cultured medium with addition of 10 µL of JC-1 (Life Technologies, Scotland) (final concentration of JC-1 was 10 µg/mL) and incubated for 30 min at 37ºC in the dark. Next, cells were washed with 1 mL of PBS + 2% FBS and resuspended in 300 µl of PBS. The mitochondrial membrane potential was analyzed by flow cytometry using the CellQuest program. Data were analyzed using the BD FACSDiva program. 184.108.40.206.2. Enzymatic caspase-3/7 activity assay Twenty four hours prior to the addition of the tested compounds, HL-60 cells were seeded at 5
the density of 2 × 10 cells/mL of the culture medium on 24-well plates (Sarstedt, Germany) to the final volume of 2 mL. Cells were exposed to the test compound at a concentration of 3 µg/mL. DMSO as solvent for 3b, in dilution corresponding to its concentration applied for the compound, induced no toxicity. Following 24 h of incubation with the compound and solvent, cells were collected, washed in PBS, counted in a haemocytometer and resuspended at 1.2 x 106 in one milliliter of lysis buffer (50 mM HEPES, 10% (w/v) sucrose, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-
100, pH 7.3) (IIET, Wroclaw, Poland). After a 30-min incubation in 4oC, 40 µL of each sample was transferred to a white, 96-well plate (Corning, NY, USA), containing 160 µl of reaction buffer (20 mM HEPES, 10 % (w/v) sucrose, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 0.02% (v/v) Triton X-100, pH 7.3) (IIET, Wroclaw, Poland) with Ac-DEVD-ACC (final concentration on plate was 10 µM) (λex = 360 nm, λem = 460 nm) as a fluorogenic substrate. Increase of fluorescence intensity correlated with active caspase-3/7 level was continuously recorded at 37 oC for 90 min using BioTek Synerg H4 Hybrid Microplate Reader (BioTek Instruments, USA). Results were normalized to the protein content determined using the Lowry method (BioRad, Warsaw, Poland) and are reported as relative caspase-3/7 activity in comparison to the untreated control. 220.127.116.11. Western-Blot Analysis of Cyclin-dependent kinases, Bcl-2, Bax and LC3b Expression Twenty four hours prior to the addition of the tested compounds, HL-60 cells were seeded at 5
the density of 2 × 10 cells/mL of the culture medium on 24-well plates (Sarstedt, Germany) to the final volume of 2 mL. Cells were exposed to the test compound at a concentration of 1 µg/mL and DMSO as solvent for 3b. Following 72 h of incubation with the compound and solvent, cells were collected, washed twice in phosphate-buffered saline (PBS) and lysed in RIPA buffer with protease and phosphatase inhibitor cocktail and stored at -20°C for future use. Protein concentrations for each cell lysate were determined using a FOLIN protein assay (DC Protein Assay, Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins from cell lysates (100 µg) were separated by gel electrophoresis in 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (BioRad) and transferred to a nitrocellulose membrane (0.45 Micron, NitroBind; GE Water & Process Technologies, Osmonics, Hopkins, MN, USA). Membrane was blocked overnight in a 1% blocking reagent (Membrane blocking agent, Amersham, GE Healthcare, Little Chalfont, Buckinghamshire HP7 9NA, UK) at 4oC, and then rinsed 3 times for 10 min with 0.1% PBST (PBS with Tween-20). Next, the membrane was incubated for 1 h at rt with one of the primary antibody solution: rabbit anti- cyclin E cyclin A - cyclin D1, - Bcl-2, - Bax (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or rabbit anti-actin antibody (Sigma-Aldrich, Poznan, Poland). After incubation, blots were washed three times with 0.1% PBST and incubated for 1 h with the anti-rabbit immunoglobulins fluorescein-linked secondary antibody (Amersham, GE Healthcare). After
incubation, the blot was washed three times with 0.1% PBST and incubated for 1 h with the third antibody: anti-fluorescein with conjugated alkaline phosphatase (Amersham, GE Healthcare). Then, the membrane was washed three times with 0.1% PBST and incubated for 30 min with the ECF substrate (Amersham, GE Healthcare). Fluorescence was detected using a scanner (Typhoon scanner GH Healthcare). Western blot densitometry analysis was carried out using ImageJ 1.46r (National Institutes of Health, Bethesda, MD, USA). 4.6. Toxicological studies Toxicological examinations were performed in 2013 in the Department of Toxicological Studies at the Institute of Industrial Organic Chemistry, Branch Pszczyna, which holds the Statement of GLP Compliance No. 8/2012/DPL of June 28, 2012 issued by the Bureau for Chemical Substances. All experimental protocols were approved by the Ethic Committee of the Medical University of Silesia. Skin sensitization study was performed to examine the possibility of allergic contact dermatitis caused by skin contact with the test item. The experiment was conducted on the Dunkin-Hartley guinea pigs obtained from the Charles River Laboratories (Kissleg, Germany). It commenced with a pilot study which allowed determining the concentrations of the test item to be used in the main study. In order to determine the proper concentration of compound 3b to be used in stage I of the main study, six intradermal injections (compound concentrations: 0.5, 1 and 2%) were given in the shoulder region of two guinea pigs. Similarly, to establish proper concentrations for stages II and III of the main study, compound 3b was applied at concentrations 0% and 50% to the shaved flanks of two guinea pigs. As the test item was in solid form, the maximum concentration that could be applied to the skin was 50%. A 1% concentration of the test item causing mild skin changes was chosen for stage I (induction - intradermal injections) and a 50% concentration causing no skin changes was selected for stage II (induction – topical application) of the main study. A 50% concentration causing no skin changes was used for stage III (challenge – topical application) of the main study. The main study included 11 guinea pigs in the treated group and 6 guinea pigs in the control group. The examination was composed of three parts: a two-stage induction phase (stage I and II) and a challenge phase (stage III). At stage I of the main study, treated animals were given intradermal injections containing a 1% suspension in peanut oil of the test item with Freund’s Complete Adjuvant (FCA). At stage II, a 50% suspension in peanut oil of the test item was applied to the skin in the sites of the intradermal injections. Due to no weak skin
irritation after application of the 50% suspension, the day before stage II of the experiment 10% sodium lauryl sulfate in vaseline was applied at the place of intradermal injections, in order to create local skin irritation. During the induction period, control animals were subjected to sham treatment and were applied peanut oil (medium) instead of the test item. Sensitization challenge was performed by applying a 50% suspension of the test item in peanut oil to the right flank of the treated and control animals, while the medium alone was applied to the left flank. Following the challenge, the treated and control animals were observed for skin changes 24, 48 and 72 hours after exposure. General clinical observations were made during the main study. Detailed skin observations were conducted 24, 48, and 72 hours after exposure. In order to detect allergic contact dermatitis, animals were initially exposed to the test substance by intradermal injection and epidermal application (induction exposure). Following a rest period (induction period), during which an immune response is expected to develop, animals were exposed to a challenge dose. The extent and degree of skin reaction to the challenge exposure in the test animals is compared with that demonstrated by control animals which underwent sham treatment during induction and received the challenge exposure. After the observation period, the animals were euthanized. Acute skin irritation/corrosion study was performed on albino rabbits of the new Zealand strain obtained from the Institute of Occupational Medicine in Lodz (Poland). The study commenced with a sighting study on one animal. The test item (ground into a powder) in the amount of 0.5 g was applied to the shaved skin (area about 6cm2) of one animal (rabbit no. 1) and covered with a protecting band. The exposure lasted 4 hours. After the evaluation of the treated skin, the test item was applied to the skin of the next two animals (rabbit no. 2 and 3) for 4 hours in order to confirm/exclude its irritating properties. The procedure was the same as in case of rabbit no. 1. General clinical observations of all animals for morbidity and mortality were performed daily during the entire experiment. Detailed clinical observations of the treated skin were performed 1, 24, 48, and 72 hours after exposure. After the observation period, the animals were euthanized. Acute dermal toxicity study was performed on the Wistar rats obtained from the Experimental Medicine Centre at the Medical University of Bialystok (Poland). The test item at a single dose of 2000 mg/kg bw was applied to the shaved dorsal skin of 5 males (area about 41cm2/male) and 5 females (area about 31cm2/female) for 24 hours. After application of the test item, animals were observed for 14 days. General and detailed clinical observations of all
animals were performed daily during the entire experiment. After the observation period, the animals were euthanized. Acute oral toxicity study was performed according to the OECD Guideline No. 423 / EU Method B.1. TRIS. The study was composed of three stages. Three Balbc/cmdb female mice from the Experimental Medicine Centre at the Medical University of Bialystok (Poland) were used at each stage. At the first stage of the experiment, the test item was administered at a single dose of 300 mg/kg bw to three females. At the second stage of the experiment, the test item at a single dose of 2000 mg/kg bw was administered to three females. At the third stage of the experiment, the test item at a single dose of 2000 mg/kg bw was administered to three females. The test item in a volume of 0.5 mL/100 g bw in DMSO was given to animals through a metal stomach food. Animals were fasted previously for about 3 hours. After administration, animal observations were conducted for 14 days. General and detailed clinical observations of all animals were conducted daily during the entire experiment. Body weights of the animals were determined on days 0 (directly before the administration of the test item), 7 and 14 following application. After the 14-day observation period, all animals were euthanized and subjected to necropsy and detailed gross examination. Acute eye irritation/corrosion study by compound 3b was performed on 3 albino rabbits of the New Zealand strain obtained from the Institute of Occupational Medicine in Lodz (Poland). The main study was preceded by a preliminary examination on one animal. The test item was ground into a powder and applied to the conjunctival sac of one eye of the animal (rabbit no. 1) at a volume of 0.1 mL (0.068 g). The second eye served as a control. After evaluation of the treated eye, the test item was applied in the same manner to the eyes of the next two animals (rabbit no. 2 and 3) in order to confirm/exclude its irritating properties. General clinical observations of the animals for morbidity and mortality were performed daily during the entire experiment. Detailed clinical observations for changes in the cornea, iris and conjunctiva were performed after 1, 24, 48 and 72 hours of application. After the observation period, the animals were euthanized. 4.7. Molecular docking Molecular docking was performed using ICM Pro-Chemistry (Molsoft LLC, USA).55;56 We employed the crystal structure of bovine lens leucine aminopeptidase (blLAP) complexed with
(PDB code: 1LAN).20 Three dimensional structures of the examined
compounds were constructed with the use of ChemDraw Ultra 12.057 (CambridgeSoft, USA)
and optimized using ICM Pro–Chemistry. All water molecules and co-crystallized ligands were removed from PDB structure and hydrogen atoms were added. The binding site of the leucine aminopeptidase was identified using the ICM Pocket Finder module. Ligands were docked using a regular rigid receptor-flexible ligand docking approach that uses five potential energy maps combining hydrophobicity, electrostatics, hydrogen bond formation and two van-der-Waals parameters. Acknowledgements Financial support by the European Union within the European Regional Development Fund (grant number UDA-POIG.01.03.01-14-136/09/08) is gratefully acknowledged. Supplementary Data The following is the supplementary data related to this article: Copies of 1H and 13C NMR spectra. References 1. National Cancer Institute website, http://www.cancer.gov/about-cancer/what-iscancer/statistics, 2016 (accessed 25.05.16). 2. Müller, W.E.G.; Schuster, D.K.; Leyhausen, G.; Sobel, C.; Umezawa, H., Cell surface bound leucine aminopeptidase: target of the immunomodulator bestatin, in: G.W. Kreutzberg, M. Reddington, H. Zimmermann (Eds.), Cellular Biology of Ectoenzymes, Springer-Verlag Berlin Heidelberg, 1986, pp. 285-293. 3. Gluza, K.; Kafarski, P., Inhibitors of Proteinases as Potential Anti-Cancer Agents, in: Ch. Rundfeldt (Ed.), Drug Development - A Case Study Based Insight into Modern Strategies, InTech, 2011, pp. 39-74. 4. Hitzerd, S. M.; Verbrugge, S. E.; Ossenkoppele, G.; Jansen, G.; Peters, G. J. Amino Acids 2014, 46, 793. 5. Matsui, M.; Fowler, J. H.; Walling, L. L. Biol. Chem. 2006, 387,1535. 6. Grembecka, J.; Kafarski, P. Mini Rev. Med. Chem. 2001, 1, 133. 7. Taylor, A. FASEB J. 1993, 7, 290. 8. Mucha, A.; Drag, M.; Dalton, J. P.; Kafarski, P. Biochimie 2010, 92, 1509. 9. Moore, H. E.; Davenport, E. L.; Smith, E. M.; Muralikrishnan, S.; Dunlop, A. S.; Walker, B. A.; Krige, D.; Drummond, A. H.; Hooftman, L.; Morgan, G. J.; Davies, F. E. Mol. Cancer Ther. 2009, 8, 762. 10. Scott, L.; Lamb, J.; Smith, S.; Wheatley, D. N. Br. J. Cancer 2000, 83, 800. 11. Tian, S. Y.; Chen, S. H.; Shao, B. F.; Cai, H. Y.; Zhou, Y.; Zhou, Y. L.; Xu, A. B. Int. J. Clin. Exp. Pathol. 2014, 7, 3752. 12. Zhang, S.; Yang, X.; Shi, H.; Li, M.; Xue, Q.; Ren, H.; Yao, L.; Chen, X.; Zhang, J.; Wang, H. J. Mol. Histol. 2014, 45, 283. 13. Solecka, J.; Guspiel, A.; Postek, M.; Ziemska, J.; Kawecki, R.; Leczycka, K.; Osior, A.; Pietrzak, B.; Pypowski, K.; Wyrzykowska, A. Molecules 2014, 19, 15866 14. Solecka, J.; Rajnisz, A.; Laudy, A. E. J. Antibiot. (Tokyo) 2009, 62, 575.
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• A set of isoquinolines as microsomal leucine aminopeptidase (LAP) inhibitors was found • Compound 3b exhibited potent in vitro antitumor activity against cancer cell lines • Compound 3b showed good selectivity index and promising toxicological profile • Its influence on cell cycle was determined • Docking study of 3b was performed and confirmed significant LAP inhibition