Moreover, the enzymatic assessment of the synthesised compounds against VEGFR-2 tyrosine kinase showed the significant inhibitory activities of the biarylureas  with IC50s of 4.4, 2.7 and 2.5 μM, respectively, and with 79.83, 72.58 and 71.6% inhibition of HUVEC at 10 μM, respectively. Additionally, compounds  were found to induce cell cycle arrest at S phase boundary. Compound 7b triggered a concurrent increase in cleaved caspase-3 expression level, indicating the apoptotic-induced cell death.In the designed compounds, either a biarylamide or biarylurea moiety or an N-substituted piperazine motif was linked to position 1 of the phthalazine core. The anti-proliferative activity of the synthesised compounds revealed that eight compounds ( However, malignant cells are generally less sensitive to these stresses and tend to evade apoptosis. Genetic regulation, initiation and effector mechanisms can be regarded as stages of apoptosis in its simplest model. Gamma and ultraviolet irradiation, anticancer drugs, deprivation of survival factors such as interleukin-1 (IL-1), or other cytokines that activate “death receptors” are considered as initiators of apoptosisFrom another point of view, angiogenesis, the process of sprouting of new blood vessel formations from pre-existing ones, is also regarded as an important hallmark of cancer development. Vascular endothelial growth factor family (VEGFs) are known to be one of the key regulators of angiogenesis. Their biologic effects is exerted by binding to extracellular domain.Multiple reports have detailed several small-molecule inhibitors of VEGFR-2 acting by binding to the ATP-binding site in the intracellular kinase domain resulting in diminished VEGF signal transduction, these inhibitors may be broadly categorised into two main types: type I; inhibitors represent ATP competitors that generally bind in or around the catalytic site of the kinase in its active conformation, in the region originally occupied by the adenine moiety of ATP(I) (IC50 VEGFR-2 = 43 nM)  = 43 nM) 6.(II) (IC50-VEGFR-2 = 90 nM) The biarylurea-based inhibitor, sorafenib = 90 nM) , has bee(IV), a hybrid-design type-II inhibitor derived from the type-I inhibitor vatalanib, was reported as a potent inhibitor of VEGFR family with VEGFR-2 IC50 of 23 nMMoreover, the biarylamide, (AAL993) V), the furopyridazine derivative, emerged as a potent and orally available inhibitor of VEGFR-2, VEGFR-3 with IC50s 6 and 4 nM, respectively. It is currently in clinical trials for gastric and colorectal cancerAlso, telatinib ((VI)   was repo2.(series A) or biarylurea tail  at position 1 of the phthalazine core via an amino or ether linkage were designed and synthesised as potent anti-proliferative derivatives as well as apoptosis inducers in tumour cells. Different substitution patterns were introduced to the terminal aromatic ring aiming to better occupy the hydrophobic pocket revealed by the kinase in its DFG-out conformation ((1) a flat heteroaromatic ring system to better occupy the ATP-binding region of the kinase, (2) a hydrogen bond donor–acceptor pair represented by the urea or amide moieties that usually form hydrogen bonds with Glu 885 and Asp 1046 residues, (3) a substituted terminal aryl moiety to occupy the hydrophobic pocket revealed by the movement of the DFG motif of the kinase in its inactive conformationBased on the above findings, three series of phthalazine-based derivatives linked to a biarylamide  were designed having a 4-chloroaniline moiety at position 1 of the phthalazine and an anilino or phenoxy motif linked to the position 4 via a methylene spacer.Furthermore, two other series of vatalanib analogs series F) was prepared as well, via linking the two major fragments: the phthalazine core recognised as a kinase-privileged fragment, to the piperazine moiety aiming to offer more rigidity and to facilitate deriving spatial–activity relationshipsIn addition, a sixth series of substituted piperazine-based derivatives (3.(1a–c), were prepared following the literature methods as illustrated in . Chlorination of 2a,b with phosphorous oxychloride afforded the corresponding 1-chlorophthalazines , which were used to prepare the target series of 1-substituted phthalazines via reaction with various nucleophiles. Thus, the reaction of 3a,b with p-phenylenediamine in butanol via the displacement of the chloro atom of phthalazine with the suitable nitrogen nucleophile such as p-phenylenediamine afforded the N1-benzene-1,4-diamines . The latter compounds  were further reacted with the respective benzoyl chloride in acetonitrile, in the presence of triethylamine to afford the target phthalazines bearing the biarylamide tail (5a–d) (series A). Moreover, the reaction of  with different arylisocyanates in DMF yielded the biarylureas (6a–f) (series B). On the other hand, 1-aryl-3–phenyl)ureas (7a–f) (series C) were obtained via reacting the respective 1-chlorophthalazines  with the appropriate intermediates (1a–c) in refluxing acetonitrile. This latter reaction proceeds via ipso addition by the nucleophile that gives an anion with a highly delocalised charge then followed by leaving of the chlorine atom of the respective 1-chlorophthalazines  to afford the alkoxyphthalazines (7a–f). Refluxing the 1-chlorophthalazines  with the appropriate piperazine derivative in ethanol yielded the target phthalazines bearing the substituted piperazinyl tail (8a–j) (series F) in good yields  having a chloro substituent at 4 position of the phthalazine . Thereafter, the target compounds (12a–c and 13a–c) were obtained in a manner similar to that adopted in As for the synthesis of the biarylureas   were synthesised via initial bromination of the key intermediate (14) with NBS in presence of catalytic amount of dibenzoyl peroxide. The reaction of the bromo derivative (15) with the respective aniline or phenol in acetone then afforded the titled compounds (Compounds ompounds .4.4.1.4.1.1.www.dtp.nci.nih.gov), Bethesda, Maryland, USA. In vitro NCI anticancer screening involves two-stage process, the first stage started with the evaluation of the selected candidates against the full NCI 60 cell lines panel representing leukaemia, non-small cell lung cancer, melanoma, colon, CNS, breast, ovarian, renal and prostate cancers at a single dose of 10 µM. NCI selection is based on degree of structure variation and application of computer modelling techniques to prioritise compounds based on their ability to add diversity to the NCI small molecule compound collection.Aiming to evaluate the general antitumour activity of the synthesised phthalazines, the structures of the final compounds were submitted to National Cancer Institute “NCI”  were selected by NCI under drug discovery programme for screening against the full NCI 60 cell panel at single dose 10 µM. Among the investigated 18 compounds, eight compounds  were selected by NCI for further screening at 5-log dose molar range due to their prominent cell growth inhibition against various cell lines.Eighteen of the submitted phthalazines  showed a distinctive pattern of sensitivity against various NCI cell lines . Thus, the biarylurea-based derivatives having a terminal 4-chloro substituent  exhibited remarkable broad spectrum cell growth inhibition above 90% against various cell lines including leukaemia, non-small cell lung cancer, melanoma, prostate cancer and breast cancer cell lines. Compound 6e even exhibited lethal effect (above 100% inhibition) against several leukaemia, melanoma, renal cancer cell lines. Interestingly, it showed highly potent inhibitory or lethal effects against most of the tested leukaemia and melanoma cell lines.Primary 7b) linked to the phthalazine nucleus via an ether linkage also showed excellent broad spectrum inhibitory or lethal activity against most of the tested NCI cell lines representing all the nine tumour subpanels specially those of leukaemia, colon, melanoma and breast cancer cell lines, with mean growth inhibition of 106.99%. However, its analogue (7e) having a methyl substituent at 4-position of phthalazine core only showed moderate cell growth inhibition against leukaemia and breast cell lines .Furthermore, their analogue ((13a) displayed broad spectrum cell growth inhibition above 90% against various cell lines including non-small cell lung cancer, melanoma, CNS cancer, melanoma and breast cancer cell lines. Interestingly, its disubstituted analogue (13c) exhibited an excellent broad spectrum lethal activity against most of the tested NCI cell lines representing all the nine tumour subpanels specially those of non-small lung cancer, colon, CNS and melanoma cell lines, with mean growth inhibition of 145.09%. Surprisingly, the 4-chloro substituted derivative (13b) did not show any significant cell line growth inhibition .Thus, the unsubstituted biarylurea derivative  or 4-phenoxy substituent (17a) linked to the phthalazine via methylene spacer; It was found that all the three tested derivatives displayed remarkable broad spectrum cell growth inhibition above 90% against various cell lines including leukaemia, melanoma and breast cancer cell lines, with the meta methoxy analogue (16d) showing the most prominent inhibition against most of the tested cell lines with mean growth inhibition of 116.00%.For the vatalanib analogues having a 4-anilino 5b, and 5d) nor the piperazines  showed any significant cell line growth inhibition under the same test conditions .Neither of the biarylamides  were selected by NCI for further 5 log dose screening against full NCI 60 cell panel. All the 60 cell lines, representing nine tumour subpanels  which were incubated at five different concentrations  of each of the test compounds. The outcomes were used to create log concentration vs % growth inhibition curves, then three response parameters  were calculated for each cell line, .Eight compounds  and vatalanib analogues  revealed potent antiproliferative activity against most of the tested cell lines representing the nine different subpanels with GI50 values between 0.15–8.41 µM, except for 16a which was insensitive to prostate and MCF-7 breast cancer cell lines.All investigated biarylureas 7b and 13c) exhibited the highest broad spectrum submicromolar inhibitory activity against most of the tested cell lines specially those of leukaemia, colon, melanoma, breast cancer and renal (13c only) cell panels with GI50 values of 0.15–2.81 µM and 0.2–2.66 µM for 7b and 13c, respectively. It is worth noting that 13c showed submicromolar GI50s against all tested leukaemia and melanoma cell lines.With regard to the sensitivity against the tested cell lines, compound  by their individual subpanel MID 4.1.2. was also evaluated against two specific cell lines, namely MCF-7 and HCT-116 using doxorubicin as a reference drug. In addition, compounds  were evaluated against a third cell line (HepG-2) These first two cell lines were selected based on the sensitivity of the previously NCI-tested derivatives towards them amongst the 60 NCI panel. The growth inhibition is expressed as the median growth inhibitory concentration (IC50) which corresponds to the concentration required for 50% inhibition of cell viability and are provided in .The growth inhibition of the rest of synthesised compounds (not selected by NCI) (series B and C) generally exhibited moderate to significant cytotoxicity on both cell lines. This was relatively in accordance with the NCI findings for the same series of compounds.Investigation of the results of the growth inhibition of the tested molecules revealed that the biarylurea-based phthalazines (series B) revealed that, the 4-chloro-3-trifluoromethyl derivatives (6c and 6f), bearing an electron withdrawing substitution pattern similar to that of sorafenib, displayed enhanced growth inhibition on both cell lines compared to their unsubstituted analogues (6a and 6d) .Further analysis of the inhibitory results within the biarylurea-based derivatives linked to phthalazine core via an NH-linker (series 7), the 4-chloro-3-trifluoromethyl derivative (7c) similarly displayed significant inhibiton on both cell lines, (IC50s = 2.5 and 2.6 µM). Surprisingly, its methyl analogue (7f) demonstrated weaker activity (IC50s = 31 and 49 µM) compared to its unsubstituted analogue . However, the amide-based derivatives (series A) and the piperazine-based derivatives have not shown any interesting cell line inhibition which was also consistent our previous results.As for the biarylureas bearing an ether linker 12a–c) nor the vatalanib analogues (series D and E) showed any significant cell line inhibition .Unfortunately, neither the 4-chlorophthalazines (4.1.3.4.1.3.1www.bpsbioscience.com). All the synthesised compounds representing the six series were evaluated for their ability to inhibit VEGFR-2 tyrosine kinase at single dose of 10 µM.The VEGFR-2 tyrosine kinase assay was performed in an attempt to investigate the mechanism of potent antiproliferative activity exerted by the synthesised compounds. The VEGFR tyrosine kinase assays were performed at BPS Bioscience : derivatives having a 4-chloro-3-trifluoromethyl substitution pattern on the terminal phenyl ring similar to that of sorafenib  tended to exhibit enhanced VEGFR-2 inhibitory activity as excepted from cell line results compared to their monosubstituted or unsubstituted analogues.Investigation of the results of VEGFR-2 inhibitory activities among the synthesised phthalazines revealed that: Within the biaryl ureas 3 or unsubstituted derivatives, resulted in favourable increase in VEGFR-2 inhibitory activity , which might be attributed to enhanced lipophilicity of the chloro group. Thus, compounds 6c, 12b, 12c and 13c showed 70–100% inhibition at 10 µM concentration  , they generally exhibited weak to moderate activity, with the derivatives having unsubstituted anilino or phenoxy moiety  showing relatively better activity than their meta substituted analogues  (series A) nor the piperazines (8a–j)(series F) showed any significant VEGFR-2 inhibition in accordance with cell lines results.As expected from the cell lines results, neither of the amide-based derivatives 4.1.3.2., which exhibited VEGFR-2 inhibition percent above 70% at 10 µM concentration, were evaluated at five different concentrations  to calculate their IC50 values  or a terminal 4-chloro-3-CF3 substitution  with IC50s of 4.4, 2.7 and 2.5 µM respectively.Results confirmed the significant inhibition of 4-chlorophthalazines bearing a biarylurea tail with a terminal 4-chloro substitution 4.1.4. showing best VEGFR-2 IC50s were selected to be tested for their ability to in vitro inhibit VEGF-induced HUVEC cell line proliferation at single dose of 10 µM concentration.In order to further assess the antiangiogenic activity of the promising candidates, three compounds www.bpsbioscience.com).The HUVEC anti-proliferative assay was also carried out in BPS Bioscience Corporation, San Diego, CA, USA  outgrowth from the parent vessel, followed by proliferation, migration, tube formation, alignment, and anastomosis to other vessels. Several  manifested significant inhibition of HUVEC cell lines of 79.83%, 72.58% and 71.60%, respectively, which may support their potential antiangiogenic activity (The test compounds 4.1.5.(7b and 16a) were selected to investigate their effect on cell-cycle progression, and induction of apoptosis using breast cancer adenocarcinoma cell line (MCF-7) and colon cancer cell line (HCT-116), respectively.In an attempt to elucidate the potential mechanism of action of the synthesised phthalazines that exhibited potent anti-proliferative activity against the NCI cell panel but weak or moderate VEGFR-2 inhibitory activity, compounds 13c) (VEGFR-2 IC50 = 2.5 µM) was selected to test its effect on cell cycle analysis and induction of apoptosis using breast cancer adenocarcinoma cell line (MCF-7).From another point of view, since the inhibition of VEGF pathway has a greater effect on the induction of endothelial cell apoptosis and the decrease of vascular tubulogenesis, as the repair of endothelial lining defects caused by endothelial apoptosis is coordinated by growth factors, mainly by vascular endothelial growth factor (VEGF), which conveys survival signals to endothelial cells and acts as a potent antiapoptotic, proliferation stimulating factorCell cycle is a chain of events that takes place in a cell leading to its division and replication. G1 phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis) are considered as the four main distinct phases of the cell cycle. The G1-phase, also called post-mitotic pre-synthesis phase, directly follows cell division. The S- or synthesis phase is characterised by the process of DNA replication. The G2-, premitotic or post-synthetic phase is the time when the cell prepares to split off in two cells, the actual division. Finally, in the M- or mitosis-phase division, the doubled DNA organised in chromosomes is separated7b and 13c) on the normal cell-cycle progression was characterised using flow cytometric analysis of the DNA ploidy in MCF-7 cells using BD FACSDIVA TM software. Exposure of MCF-7 cells to either 7b or 13c at their 50GI concentration  for 24 h and 48 h was shown to induce a remarkable disruption in cell cycle profile and cell-cycle arrest at S-phase boundary with concurrent time-dependent increase in pre-G cell population  for 24 h and 48 h also seemed to induce a significant action at both S phase and sub-G1 cell population  to induce apoptosis, an Annexin V (conjugated to FITC) apoptosis detection kit was employed. This assay detects phosphatidylserine (PS) expressed on the surface of apoptotic cells and fluoresces green after interacting with the labelled Annexin V. During early apoptosis, membrane asymmetry is lost, and PS translocates from the cytoplasmic side of the membrane to the external leaflet. Propidium iodide (PI), the counter stain used in this assay, has the ability to cross only compromised membranes to intercalate into the DNA. Therefore, PI is used to detect the late stages of apoptosis by the presence of red fluorescence. Exposure of MCF-7 cells to 7b at its GI50 (0.32 µM) for 24 h and 48 h increased the percentage of Annexin-V positive cells indicating an early (lower right quadrant) or late (upper right quadrant) apoptosis in a time-dependent manner compared to DMSO treated cells  were studied following Dapi staining. The effect of 7b on cell morphology was initially analysed with light microscopy. While control cells treated with DMSO exhibited normal morphological features, cells treated with 7b for 24 and 48 h showed deteriorated morphological changes including cell rounding, detachment and cellular fragmentation , blubbing with the appearance of nuclear fragmentation (arrow heads indicate apoptotic nucleus) (7b) to induce cellular apoptosis in MCF-7 cells as a mechanism of cancer cell death.To further examine the apoptosis inducing effect of entation . Similarnucleus) . These r4.1.8.7b to increase the expression of cleaved caspase-3 was studied as a driving force in 7b-induced apoptotic cell death. Exposure of MCF-7 cells to compound (7b) (0.32 µM) for 24 h and 48 h resulted in time-dependent increase in cleaved caspase-3 protein expression, indicating a pivotal involvement of caspases in the induced cancer cell death (Caspase-3 is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases is considered as one of the important keys in cellular apoptosis process. Actually, caspases exists as inactive proenzymes that changed to the active form by undergoing proteolytic processing. Cleaved caspase-3 further activates a cascade of caspases that induce cell death. Thus, the ability of compound 4.2.Molecular docking study was performed using AutoDock VinaFigure S7 in Supplementary material), we excluded investigating halogen-bond contacts in our docking-based assessment.Owing to the fact that the chloro group of the co-crystallised ligand (sorafenib) is in the far proximity to any halogen-bond6c) reproduced the key interactions of the co-crystallised ligand (sorafenib) which includes H-bonding interactions of Glu885 and Asp1046 with the urea group of 6c. In addition, other hydrophobic interactions can be observed for the hydrophobic side chains of Ile892, Leu1019 and Val898 with the halogen groups (chloro and trifluoromethyl) of the phenyl moiety of 6c. Nevertheless, losing the H-bonding interaction of the Cys 919 with the terminal amide group of sorafenib can explain the lower activity of 6c compared to sorafenib  on the phenyl moiety which contributes to an additional hydrophobic interaction, this also agrees with the predicted score of the best poses by AutoDock Vina6d, 6e and 6f can be found in .The relative higher inhibitory activity of 6c, the predicted best scored poses of both biarylurea derivatives 12b and 12c showed a flip of 180 degrees compared to sorafenib and biarylurea compound (6c) . The flipped pose showed interaction pattern in the binding site that maintained the H-bonding interaction of Glu885, however, in this case with the amino group of 12b and 12c. Also, chlorophthalazine ring was packed in the hydrophobic region of Ile892, Val898 and Leu1019. Similarly, the chlorophenyl moiety of 12b and the 4-chloro-3-trifluoromethylphenyl moiety of 12c showed hydrophobic contacts with the side chains of Phe918 and Leu840  compared to 12a and 12b, the predicted scores showed relative improvement .Unlike und (6c) . This imd Leu840 . Owing trovement . This al12c, the predicted pose of 13c showed the same flip and relatively the same interaction pattern. Only exception is that the isosteric ether link in 13c showed H-bonding interaction with Asp1046 instead of Glu885 with 12c as shown in (12c and 13c.Like shown in . This ag5.5a–d), biarylurea  or a substituted piperazine moiety at position 1 of the phathalazine nucleus (8a–j), in addition to two other series of vatalanib analogs  were designed, synthesised as anticancer agents exerting potent anti-proliferative activity as well as apoptosis inducers in tumour cells. Eight of the phthalazines derivatives; namely the biarylureas  and the vatalanib analogues  exhibited excellent broad spectrum anti-proliferative activity in NCI 5-log dose screening against 60 cell panel with GI50 values ranging from 0.15 to 8.41 µM, especially on leukaemia, renal, melanoma and breast cancer cell lines. Accordingly, the three compounds  were selected to further investigate the potential underlying mechanisms that induced cancer cell death. The latter three compounds were found to induce cell-cycle arrest with concurrent increase in pre-G cell population and increase the apoptotic cell population in time-dependent manner. Moreover, compound (7b) was found to disrupt the normal cellular and nuclear morphology and increase the expression of cleaved caspase-3, the pro-apoptotic protein, in MCF-7 cell lines which collectively indicate the involvement of these compounds in apoptotic-induced cell death. Moreover, all the synthesised phthalazines were evaluated for their VEGFR-2 inhibitory activity, which revealed the significant inhibition of the 4-chlorophthalazines having a biarylurea tail with either a terminal 4-chloro (12b) or a terminal 4-chloro-3-trifhuoromethyl substituents (12c and 13c) with IC50 of 4.4, 2.7 and 2.5 µM, respectively. 4-chloropthalazines were found to exhibit better VEGFR-2 activity than their corresponding 4-methyl or 4-unsubstituted analogues. 12b, 12c and 13c also displayed significant inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVEC) with 79.83, 72.58 and 71.6% inhibition, respectively, at 10 µM concentration. Finally, molecular docking study of the active biarylurea compounds  in VEGFR-2 kinase active site revealed their ability to form the essential H bond interactions with Glu 885 or Asp1046 key residues in the VEGFR-2 active site.Four series of phthalazine-based derivatives bearing biarylamides . Elemental analyses were performed at the Microanalytical Center, Al-Azhar University. Compounds 1a–c2a,band 3a,b9 and 10Starting materials and reagents were purchased from Sigma – Aldrich or Acros Organics. Melting points were recorded on Gallen Kamp apparatus and were uncorrected. FT-IR spectra were recorded on a Shimadzu IR 435 spectrophotometer. 6.1.1.General procedure:p-Phenylenediamine  and the respective chlorophthalazine  were treated with 2-BuOH (7.5 ml) in a tube and heated to 110 °C. The reaction quickly became a solid, yellow mass. After four hours, the reaction was cooled and diluted with water. The resultant slurry was then partitioned between equal volumes of DCM and 1 N NaOH (15 ml). The aqueous layer was extracted into DCM (2x15 ml). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting orange solid was crystallised from (EtOAc/pet ether 1:1) to afford the title compounds  as an orange crystals.6.1.1.1.1HNMR : δ 9.15 , 9.04 , δ 8.95–8.92 , δ 8.18–8.15 , δ 7.97 , δ 7.65–7.62 , δ 6.97–6.83 ; FT-IR : 3400 (NH2), 3329 (NH); MS (Mwt.: 236.11): m/z 236.00 , 212.00 (100.00%), 118.00 (54.00%), 56.00 (70.00%); Anal. Calcd for C14H12N4: C, 71.17; H, 5.12; N, 23.71; Found: C, 71.34; H, 5.17; N, 23.96Yield ; mp 284–286 °C; 6.1.1.2.1HNMR : δ 8.88 , 8.85 , δ 8.13–8.08 , δ 7.95–7.92 , δ 7.46–7.43 , δ 6.83–6.76 , δ 2.78 ; 13CNMR : δ 162.4, 150.5, 135.2, 134.5, 131.2, 130.7, 128.0, 127.3, 125.0, 122.8, 120.7, 120.2, 115.6, 115.3, 18.2; FT-IR : 3385 (NH2), 3321 (NH); MS (Mwt.: 250.12): m/z 250.00 , 249.00 (100.00%), 108.00 (60.92%), 65.00 (77.59%); Anal. Calcd for C15H14N4: C, 71.98; H, 5.64; N, 22.38; Found: C, 72.09; H, 5.78; N, 22.52.Yield ; mp 240–242 °C; 6.1.2.General procedure:  and triethylamine  in acetonitrile (20 ml), the respective benzoyl chloride   was added and the mixture was heated under reflux for 6 h, until the disappearance of starting material as judged by TLC (CHCl3/CH3OH 9.5:0.5). The reaction mixture was filtered and then the filtrate was concentrated in vacuo, to afford the crude product which was purified by flash column chromatography (EtOAc/pet ether 9:1) to afford 5a–d as yellow crystals.To a stirred solution of the respective N1-benzene-1,4-diamine 6.1.2.1.1HNMR : δ 9.66 , δ 9.12 , δ 8.62 , δ 8.11–8.08 , δ 7.98–7.96 , δ 7.93–7.88 , δ 7.64–7.62 , δ 7.62–7.60 , δ 7.59–7.57 , δ 7.50–7.48 ; FT-IR : 3550, 3510 (2NH), 1649 (C=O); MS (Mwt.: 340.38): m/z 340.15 + (9.74%), 172.15 , 122.05 (100.00%); Anal. Calcd for C19H19FN4: C, 70.79; H, 5.94; N, 17.38; Found: C, 71.03; H, 5.99; N, 18.48.Yield , mp 149–151 °C; 6.1.5.9.1HNMR : δ 8.16–8.13, δ 7.62–7.60 , δ 7.58–7.56 , δ 6.91–6.89 , δ 6.74–6.72 , δ 3.75–3.73 , δ 3.12–3.10 , δ 2.08 ; FT-IR  3132 (CH aromatic), 2954 ; MS (Mwt.: 305.38): m/z 305.15 , 185.65 (16.50%), 172.35 , 55.55 (100.00%); Anal. Calcd for C18H19N5: C, 70.80; H, 6.27; N, 22.93; Found: C, 71.04; H, 6.38; N, 23.18.Yield , mp 226–228 °C; 6.1.5.10.1HNMR : δ 8.32–8.30 , δ 8.29–8.27 , δ 7.82–7.81 , δ 6.97–6.96, δ 6.62–6.60 , δ 3.64–3.62 , δ 3.41–3.39 , δ 2.92 ; FT-IR : 1610 (C=O); MS (Mwt.: 322.36): m/z 322.00 , 184.00 (31.70%), 172.00 (100.00%); Anal. Calcd for C18H18N4O2: C, 67.07; H, 5.63; N, 17.38; Found: C, 67.21; H, 5.69; N, 17.52.Yield , mp 90–92 °C; 6.1.6.in vacuo. The resulting orange solid was crystallised from (EtOAc/pet ether 1:1) to afford the title compound (11) as an orange crystals.General procedure:p-Phenylenediamine  and the dichlorophthalazine  were treated with 2-BuOH (7.5 ml) in a tube and heated to 110 °C. The reaction quickly became a solid, yellow mass. After four hours, the reaction was cooled and diluted with water. The resultant slurry was then partitioned between equal volumes of DCM and 1 N NaOH (15 ml). The aqueous layer was extracted into DCM (2 × 15 ml). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated 1HNMR : δ 13.21 , 9.27 , δ 8.30–8.28 , δ 8.07–8.04 , δ 7.46–7.43 , δ 6.83–6.76 , δ 2.48 ; FT-IR : 3385 (NH2), 3321(NH); MS (Mwt.: 270.72): m/z 250.00 , 249.00 (100.00%), 108.00 (60.92%), 65.00 (77.59%); Anal. Calcd for C14H11ClN4: C, 62.11; H, 4.10; N, 20.70; Found: C, 62.37; H, 4.18; N, 20.94.Yield 0.22 g, (88%); mp 170–172 °C; 6.1.7.General procedure:(11)  in DMF (20 ml), the respective phenyl isocyanate   was added and the mixture was heated under reflux for 8–10 h, after which TLC (CH2Cl2/CH3OH 9:1) showed no starting material. The reaction mixture was poured over ice-water; the formed precipitate was allowed to settle, then filtered off and dried to afford the crude product (12a-c) which was further crystallised from EtOAc.To a stirred solution of the N1-benzene-1,4-diamine 6.1.7.1.1HNMR : δ 12.85 , δ 8.63 , δ 8.30–8.27 , δ 8.08–8.05 , δ 8.03–7.02 , δ 7.99–7.97 , δ 7.95–7.94 , δ 7.46–7.43 , δ 7.29–7.24 , δ 6.98–6.94 ; 13CNMR : δ 159.0, 152.5, 146.1, 139.6, 137.0, 134.4, 132.9, 128.7, 128.3, 126.4, 125,4, 121.7, 120.5, 119.5, 118.1; FT-IR : 3296, 3286, 3155 (3NH), 1668 (C=O); MS (Mwt.: 389.84): m/z 389.28 , 367.27 (6.23%), 322.10 (11.84%), 128.10(100.00%); Anal. Calcd for C21H16ClN5O: C, 64.70; H, 4.14; N, 14.96; Found: C, 64.71; H, 3.89; N, 14.52.Yield 0.12 g; (68%), mp 225–227 °C; 6.1.7.2.1HNMR : δ 12.84 , δ 8.81 , δ 8.29–8.27 , δ 8.07–8.05 , δ 8.02–8.00 , δ 7.98–7.96 , δ 7.95–7.94 , δ 7.57–7.56 , δ 7.48–7.47 , δ 7.31–7.30 ; FT-IR : 3446, 3300, 3157 (3NH), 1670 (C=O); MS (Mwt.: 423.07): m/z 424.88 [M + 1H]+ (3.74%), 423.50 , 328.27 (52.58%), 252.97 (20.15%), 119.04(100.00%); Anal. Calcd for C21H15Cl2N5O: C, 59.45; H, 3.56; N, 16.51; Found: C, 59.49; H, 3.37; N, 16.29.Yield 0.20 g; (53%), mp 236–238 °C; 6.1.7.3.1HNMR : δ 12.84 , δ 9.36 , δ 8.29–8.27 , δ 8.27–8.25 , δ 8.09 , 8.05–8.03 , δ 8.01–7.97 , δ 7.59–7.50 , δ 7.39–7.37 ; FT-IR : 3387, 3344, 3290 (3NH), 1664(C=O); MS (Mwt.: 492.28): m/z 493.06 [M + 1H]+ (2.63%), 395.08 (32.95%), 372.90 (35.24%), 137.07 (100.00%); Anal. Calcd for C22H14Cl2F3N5O: C, 53.68; H, 2.87; N, 14.23; Found: C, 53.72; H, 2.68; N, 14.44.Yield 0.22 g; (49%), mp 187–189 °C; 6.1.8.General procedure:10) , and cesium carbonate  in acetonitrile (20 ml), the appropriate 1-aryl-3–(4-hydroxyphenyl)urea (1a-c)  was added and the mixture was heated under reflux for 6 h, after which TLC (CHCl3/CH3OH 9:1) showed no starting material. The solvent was evaporated under vacuum and the resultant solid was stirred with cold 1 M NaOH solution (20 ml), filtered off, dried and recrytallised from acetonitrile to afford compounds (13a-c).To a stirred solution of 1,4 dichlorophthalazine derivative : δ 10.48 , δ 10.31 , δ 8.46–8.44 , δ 8.29–8.27 , δ 7.90–7.88 , δ 7.69–7.66 , δ 7.40–7.37 , δ 7.27–7.25 , δ 6.94–6.92 ; 13CNMR : δ 164.7, 158.8, 153. 8, 152.4, 141.3, 136.6, 135.3, 132.3, 128.4, 128.0, 127.3, 125. 7, 123.8, 121.6, 120.6, 120.2, 118.1, 115.1; FT-IR : 3325, 3295 (2NH), 1662 (C=O); MS (Mwt.: 390.82): m/z 390.97.00 , 346.01 (5.06%), 268.11 (18.11%), 179.11 (100.00%); Anal. Calcd for C21H15ClN4O2: C, 64.54; H, 3.87; N, 14.43; Found: C, 64.97; H, 4.21; N, 14.12.Yield 0.12 g; (46%), mp 191–193 °C; 6.1.8.2.1HNMR : δ 9.06 , δ 8.54 , δ 8.34–8.32 , δ 8.18–8.16 , δ 7.44–7.42 , δ 7.35–7.33 , δ 7.27–7.25 , δ 6.78–6.76 ; FT-IR : 3278, 3155 (2NH), 1666 (C=O); MS (Mwt.: 425.27): m/z 425.28 , 305.13 (7.74%), 201.08 (11.73%), 63.03 (100.00%); Anal. Calcd for C21H14Cl2N4O2: C, 59.31; H, 3.32; N, 13.17; Found: C, 59.62; H, 3.60; N, 13.65.Yield 0.19 g; (50%), mp 172–174 °C; 6.1.8.3.1HNMR : δ 10.20 , δ 10.13 , δ 8.28–8.26 , δ 8.25–8.23 , δ 8.09 , δ 7.93–7.92 , δ 7.76–7.74 , δ 7.54–7.51 , δ 6.69–6.66 ; FT-IR : 3294, 3155(2NH), 1662 (C=O); MS (Mwt.: 493.27): 493.46 , 281.00 (2.26%), 194.97(100.00%); Anal. Calcd for C22H13Cl2F3N4O2: C, 53.57; H, 2.66; N, 11.36; Found: C, 53.81; H, 2.90; N, 11.3.Yield 0.22 g; (49%), mp 210–212 °C; 6.1.9.General procedure:(3b)  in butanol (50 ml), triethylamine  the respective amine   was added and the mixture was heated under reflux for 2 h, after which TLC (CHCl3/CH3OH 9:1) showed no starting material. The mixture was then concentrated in vacuo, the crude product was recrystallised from methanol.To a stirred mixture of the 1-chloro-4-methyl-phthalazine 1HNMR : δ 10.47 , δ 9.10–9.07 , δ 8.45 –8.44 , δ 8.31–8.29 , δ 8.23–8.20 , δ 7.85–7.82  , δ 7.51–7.49 , δ 7.10–7.07 , δ 6.70–6.68  , δ 2.97 ; FT-IR  3360 (NH), 3050 (CH aromatic); MS (Mwt.: 269.73): m/z 269.28 , 239.18 (15.43%), 125.22 (24.70%), 90.97(100.00%); Anal. Calcd for C15H12ClN3: C, 66.79; H, 4.48; N, 15.58; Found: C, 67.03; H, 4.57; N, 15.75.Yield 1.56 g; (88%), mp 213–215 °C; 6.1.10.General procedure:14b) , N-bromo-succinimide  and dibenzoyl peroxide  in carbon tetrachloride (150 ml) was refluxed for 24 h. The reaction mixture was cooled to 40 °C, and then filtered. The filtrate was concentrated under vacuum to afford the crude product, which was triturated with ether to give a yellowish solid product.A mixture of the N-aryl-4-methylphthalazin-1-amine : δ 11.06 , δ 8.07–8.05 , δ 7.99 –7.97 , δ 7.62–7.60 , δ 7.45–7.44 , δ 4.02 ; FT-IR  3450 (NH), 3053 (CH aromatic); MS (Mwt.: 348.62): m/z 350.00 , 348.00 , 268.04 (65.94), 102.03 (100.00%); Anal. Calcd for C15H11BrClN3: C, 51.68; H, 3.18; N, 12.05; Found: C, 51.90; H, 3.16; N, 12.18.Yield 0.10 g; (42%), mp 172.-174 °C; 6.1.11.General procedure:15) , potassium carbonate , potassium iodide  in acetone (20 ml), the respective amine   was added and the mixture was heated under reflux for 4–6 h, after which TLC (CH2Cl2/CH3OH 99:1) showed no starting material. The mixture was then filtered and the filtrate concentrated in vacuo, the residue was crystallised from acetone.To a stirred solution of 4-(bromomethyl)-N-(4-chlorophenyl)phthalazin-1-amine : δ 11.03 , δ 9.65 , δ 8.75–8.72 , δ 8.28 –8.25 , δ 7.90–7.88 , δ 7.85–7.87 , δ 7.46–7.43 , δ 7.05–7.03 , δ 6.83–6.81 , δ 3.89 ; FT-IR  3446, 3383 (2NH), 3068 (CH aromatic), 2976 ; MS (Mwt.: 360.84): m/z 360.02 , 345.88 (13.64%), 268.02 (49.93%), 75.01 (100%); Anal. Calcd for C21H17 ClN4: C, 69.90; H, 4.75; N, 15.53; Found: C, 70.12; H, 4.82; N, 15.69.Yield 0.10 g; (42%), mp 120–122 °C; 6.1.11.2.1HNMR : δ 9.90 , δ 8.86–8.84 , δ 8.34 –8.32 , δ 8.20–8.18 , δ 8.12–8.09 , δ 7.88–7.86 , δ 7.53–7.51 , δ 7.44–7.42 , δ 3.79 ; FT-IR  3379 (2NH), 3080 (CH aromatic), 2954 ; MS (Mwt.: 395.28) m/z 395.99 , 271.01 (16.80%), 268.01 (100%); Anal. Calcd for C21H16Cl2N4: C, 63.81; H, 4.08; N, 14.17; Found: C, 63.94; H, 4.12; N, 14.28.Yield 0.10 g; (42%), mp 164–166 °C; 6.1.11.3.1HNMR : δ 9.70 , δ 8.86–8.84 , δ 8.34 –8.32 , δ 8.20–8.18 , δ 8.12–8.09 , δ 7.88–7.86 , δ 7.53–7.51 , δ 7.44–7.42 , δ 3.79 , δ 2.69 ;): 13CNMR : δ 162.2, 152.9, 151.1, 138.9, 135.0, 133.8, 131.7, 127.3, 127.1, 126.8, 126.4, 126.3, 124.7, 124.3, 124.1, 120.5, 120.2, 117.4, 113.2, 110.5, 45.5, 16.7, FT-IR  3446, 32992 (2NH), 3050 (CH aromatic), 2922 ; MS (Mwt.: 374.13): m/z 374.75 , 268.06 (21.45%), 189.95 (18.36%), 77.06 (100%); Anal. Calcd for C22H19ClN4: C,70.49; H, 5.11; N, 14.95; Found: C, 70.73; H, 5.19; N, 15.08.Yield 0.10 g; (42%), mp 130–132 °C; 6.1.11.4.1HNMR : δ 11.34 , δ 9.78 , δ 8.73–8.70 , δ 8.48 –8.46 , δ 8.06–8.04 , δ 7.87–84 , δ 7.68–7.66 , δ 7.46–7.44 , δ 7.14–7.12 , δ 4.29 , δ 3.63 ; 13CNMR : δ 164.2, 163.9, 148.7, 148.8, 139.2, 132.3, 131.3, 129.6, 128.5, 127.9, 123.19, 122.1, 120.4, 120.4, 115.1, 110.3, 109.2, 106.0, 105.7, 105.0, 51.2, 45.7;FT-IR  3412, 3385 (2NH), 3041(CH aromatic), 2976 ; MS (Mwt.: 390.87): m/z 391.95 , 350.00 (29.42%), 348.01 (100.00%), 313.04 (10.74%); Anal. Calcd for C22H19 ClN4O: C, 67.60; H, 4.90; N, 14.33; Found: C, 67.91; H, 4.95; N, 14.50.Yield 0.10 g; (42%), mp 175–177 °C; 6.1.12.General procedure:15) , sodium hydride  in acetone (20 ml), the respective phenol   was added and the mixture was heated under reflux for 6–8 h, after which TLC (CH2Cl2/CH3OH 99:1) showed no starting material. The mixture was then filtered and the filtrate concentrated in vacuo, the residue was crystallised from acetone.To a stirred solution of 4-(bromomethyl)-N-(4-chlorophenyl)phthalazin-1-amine : δ 9.33 , δ 8.74–8.75 , δ 8.30 –8.28 , δ 8.11–8.09 , δ 7.89–7.87 , δ 7.49–7.47 , δ 7.17–7.15 , δ 6.76–6.74 , δ 3.89 ; FT-IR  3379 (NH), 3066 (CH aromatic), 2978 ; MS (Mwt.: 361.82): m/z 361.00 , 349.01 (17.15%), 310.24 (12.19%), 268.11 (64.63%), 133.44 (100.00%); Anal. Calcd for C21H16ClN3O: C, 69.71; H, 4.46; N, 11.61; Found: C, 69.88; H, 4.39; N, 11.69.Yield 0.10 g; (42%), mp 242–244 °C; 6.1.12.2.1HNMR : δ 9.33 , δ 8.74–8.72 , δ 8.31–8.28 , δ 8.14–8.11 , δ 8.09–8.06 , δ 7.49–7.47 , δ 7.47–7.44 , δ 6.55–6.53 , δ 3.89 , δ 2.19 ; 13CNMR : δ 173.4, 162.4, 151.0, 139.4, 138.4, 133.1, 132.1, 128.9, 128.6, 128.4, 126.9, 126.9, 125.9, 123.3, 123.15, 121.9, 119.4, 115.7, 112.2, 75.4, 20.9, FT-IR  3414 (NH), 3034 (CH aromatic), 2976 ; MS (Mwt.: 375.85): m/z 375.94 , 349.94 (4.65%), 267.15 (100.00%); Anal. Calcd for C22H18ClN3O: C, 70.30; H, 4.83; N, 11.18; Found: C, 70.43; H, 4.88; N, 11.32.Yield 0.10 g; (42%), mp 193–195 °C;


Cancer is one of the leading causes of death around the world and although the different clinical approaches have helped to increase survival rates, incidence is still high and so its mortality. Chemotherapy is the only approach which is systemic, reaching cancer cells in all body tissues and the search for new potent and selective drugs is still an attractive field within cancer research. Naphthoquinones, natural and synthetic, have garnered much attention in the scientific community due to their pharmacological properties, among them anticancer action, and potential therapeutic significance. Many mechanisms of action have been reported which also depend on structural differences among them. Here, we describe some of the most relevant mechanisms of action reported so far for naphthoquinones and highlight novel targets which are being described in the literature. Furthermore, we gather some of the most impressive efforts done by researchers to harness the anticancer properties of these compounds through specifically designed structural modifications. Non-communicable diseases (NCDs) are non-transmittable illnesses affecting all age groups and regions in the world. Cancer is considered one of the four types of NCDs—alongside cardiovascular diseases, chronic respiratory diseases and diabetes- and on its own, is one of the leading causes of death globally, with approximately 9.6 million cases estimated to occur in 2018 alone. Approximately 70% of all cases occur in low- and middle-income countries where cancer diagnostic and treatment services are insufficient  and medulloblastoma cells (ONS76). Telomerase is a reverse transcriptase specialized in synthesizing telomeric DNA, thus, contributing to maintaining functional telomeres [8), such as cell cycle arrest at the G2/M phase and report of repression of telomerase activity and shortening of telomeres after incubation with the compound for 15 days, was carried out by this group.On a different note, Khaw et al. , focusedelomeres . It has 12), in the search for new scaffolds, they interestingly found that it is a rather potent inhibitor of MALT1 (IC50 = 1.9 µM) and, as mentioned before, is an important inhibitor of cell growth. They also suggested the formation of a covalent bond between the enzyme and the compound. When assessing derivatives of β-lapachone (12) synthesized by the group, the addition of an electron-withdrawing substituent at the C8 position led to an increase in activity. The initial hit for this discovery was obtained through high throughput screening and subsequently modified through structure-based drug design strategies. Compound 4d (24)  Fig.  exhibiteAs noted in this review, naphthoquinones as anticancer agents exhibit many mechanisms of action Fig.  and it a25a and 25b were synthesized by Cardoso et al. in 2014 [H-triazoles with the objective of investigating their antileukemic activity in lymphoid (MOLT-4 and CEM) and myeloid (K562 and KG1) leukemia cell lines The former has good potential for further studies since it turned out to be a potent and selective hit towards cancer cell lines also showing preference against leukemia lymphoid cell lines. Compound 25b is also a promising hit with high potency as a cytotoxic agent (IC50 for leukemia cell lines range between 0.05 and 11.07 µM).Compounds  in 2014  as part 10) and plumbagin (8), Fiorito and collaborators synthesized a series of derivatives from which compounds 26a and 26b resulted as the best candidates. 26a is an ether derivative of juglone (10) which showed more cytotoxicity than its parent compound in 3 out of 6 cell lines tested . On the other hand, 26b is also an ether derivative but of plumbagin (8), showing better potency than its precursor in all cell lines tested. To be noted, all the plumbagin (8) derivatives were more active than the ones related to juglone (10) suggesting that the presence of the methyl group on the position 2 of the naphthoquinones moiety is vital for cytotoxic activity [To develop new compounds based on the structures of juglone (activity .27 was identified as the most relevant due to its yield in the synthesis (60%) and its high potency against a panel of different cancer cell lines (exhibiting 100% growth inhibition in MDAMB-435 breast carcinoma cells). It also showed good selectivity towards cancer cells when tested in normal peripheral blood mononuclear cells (PBMC), greater than some of the natural naphthoquinones used as precursors, such as juglone [10).A series of novel 2-N-aminonaphthoquinones were synthesized by de Moraes and collaborators, from which compound  juglone  (10).7) and juglone (10) derivatives, being compound 28 the most potent cytotoxic agent of the series showing the highest effect on the human colorectal adenocarcinoma cell line HT-29 with an IC50 of 2.0 µM in 48 h. They also reported that 28 arrested the cell cycle of these cells at the S phase and that it induced apoptosis [Wang and collaborators synthesized a series of lipophilic lawsone (poptosis .29 was the best hit from cytotoxicity studies with human cancer cell lines of breast (MCF-7), prostate (DU145) and urinary bladder (T24), making it a promising candidate for further investigation [10) and 29 can be used in combination with ascorbate to potentiate their oxidative stress action in vivo in Ehrlich ascites tumor-bearing mice, showed inhibition of tumor progression [Another novel series of aminonaphthoquinones was synthesized by Benites and collaborators in 2010, from which compound tigation . Years lgression .30 showed the best results against acute myeloid leukemia (AML) cell lines  with IC50 values ranging from 0.18 to 1.05 μM. This compound also had moderate selectivity towards normal cells from hematopoietic bone marrow, with selectivity index (SI) ranging from 1.5 to 2.In the search for new anti-HIV agents, Carter-Cooperet al. synthesized a novel series of amino dimeric naphthoquinones with reportedly good anti-integrase and cytotoxic activity against a panel of leukemia cancer cell lines . Trying para-quinone 31 caught the attention of Gopinath and collaborators when testing naphthoquinones for their action as inhibitors of USP2 through ROS production. Until this finding, ortho-quinones were regarded as good inhibitors of this cancer-related enzyme as opposed to para-quinones, precisely for their inability to behave as redox cyclers. This opens a door in this particular field of study for the development of new para-quinones which can target USP2 [The high potency of get USP2 .32 was one of the most potent in various cancer cell lines with IC50 values ranging from 0.41 to 1.59 μM. It also showed good selectivity with an SI of 0.8–3.0 in comparison to that presented by a well-known and widely-used anticancer drug, doxorubicin (2), which was 0.8 [While looking for new trypanocidal agents derived from quinone moieties, Bahia and collaborators synthesized a series of 1,2,3-triazoles and evaluated their cytotoxic activities in a panel of cancer cell lines from different human tissues as well on normal cells . Compound  was 0.8 .33) was originally synthesized while studying the antibiotic nybomycin [12) in killing different cancer cell lines in a NQO1-dependent manner [12) [Finally, deoxynyboquinone (ybomycin  and it ht manner . Kolossoner [12) .34). In 2018, Prasad and co-authors [35a) and (35b), which exhibited potent activity against all cell lines.Another simple naphthoquinone that exhibits anticancer activity and is also widely used as starting material for the preparation of derivatives with cytotoxic potential is menadione (-authors  synthesi36), which was developed by conjugating a TPP (triphenylphosphonium) cation to the C3 position of menadione’s naphthoquinone ring, increasing its selective accumulation in mitochondria as well as leading to alterations of its redox properties and consequent biological outcome  in A549 cells.Oliveira and collaborators , also usome Fig. . MitoK3 Not all the attention which naphthoquinones have drawn to themselves has been on the bright side. Since the publishing of a highly cited article written by Baell and Holloway 7 years ago , which cIn the referred article, the authors coined the term pan-assay interference compounds (PAINs) to refer to compounds which would form aggregates, react with proteins or interfere in screening assays, leading to false positives . In an aAlthough what was proposed by Baell and Holloway is to some extent, true, in the sense that when working with compounds, which have some of these characteristics, scientists should be careful enough not to report false/biased results in order to avoid irreproducibility in research, ruling them out of any kind of structural optimization campaign or from the pharmaceutical pipeline could lead to the loss of valuable chemical material. Still, the question remains of which molecules should be considered as PAINs .In an effort to ameliorate the blind application of these filters, some authors have published data that question their usefulness. One finding which is crucial is the fact that many PAINs substructures can also be found in drugs available in the market , 124, meAlso, Capuzzi et al., have recently published a study in which they hypothesized that PAINs filters have limited extrapolative power. They based on the limitations of the original study, in which a relatively small library of compounds of a proprietary nature was used, meaning that complete chemical structures were not shown, and they were tested for just one type of activity (protein–protein interaction inhibition) in only six HTS campaigns, using a single detection technology, AlphaScreen . They coFinally, something that cannot be overlooked is the potential application of this kind of compounds which elicit various pharmacological actions, in the advancing field of polypharmacology. Accordingly to this concept, compounds which have the ability to interact with multiple targets are actually desired, and this kind of versatility should be clearly distinguished from promiscuity that derives from non-selective activity .Naphthoquinones act as anticancer agents through a variety of mechanisms involved in all types of cancer. Some of the most important explored so far and which have been extendedly reported in the literature are: damage of DNA through generation of ROS, inhibition of topoisomerase II, regulation of the tumor suppressor factor p53 and induction of apoptosis via ERS. In this review, we summarized the most well-known mechanisms as well as those which have been recently described in the literature, providing new interesting targets to be researched in this pharmacological family. There is still a lot to be elucidated about the ways in which these compounds exert their action as anticancer agents and how to enhance their activity in order to exploit their potential to the maximum. Being privileged structures in pharmacology and chemistry, naphthoquinones will keep on fascinating scientists around the globe and will be further studied and researched in the many fields they have the potential to thrive.


These results indicate that RAN and KPNB1 together are associated with drug resistance and GBM poor prognosis. Furthermore, the functional blockade of RAN and KPNB1 by importazole remarkably suppressed cell viability and activated apoptosis in GBM cells expressing high levels of RAN, while having a limited effect on astrocytes and GBM cells with undetectable RAN. Together, our results demonstrate that RAN activity is important for GBM survival and the functional blockade of RAN/KPNB1 is an appealing therapeutic approach.Glioblastoma, the most common malignant tumor in the brain, lacks effective treatments and is currently incurable. To identify novel drug targets for this deadly cancer, the publicly available results of RNA interference screens from the Project Achilles database were analyzed. Ten candidate genes were identified as survival genes in 15 glioblastoma cell lines. RAN, member RAS oncogene family (RAN) was expressed in glioblastoma at the highest level among all candidates based upon cDNA microarray data. However, Kaplan-Meier survival analysis did not show any correlation between RAN mRNA levels and patient survival. Because RAN is a small GTPase that regulates nuclear transport controlled by karyopherin subunit beta 1 (KPNB1), RAN was further analyzed together with KPNB1. Indeed, GBM patients with high levels of RAN also had more KPNB1 and levels of KPNB1 alone did not relate to patient prognosis. Through a Cox multivariate analysis, GBM patients with high levels of RAN and KPNB1 showed significantly shorter life expectancy when temozolomide and promoter methylation of O Glioblastoma (GBM) is an aggressive tumor generally found in the cerebral hemispheres of the brain. Spanning 16% of the cases of all primary tumors in the brain and ~50% of all malignant brain tumors, GBM is the most common malignant type in the central nervous system . The aveTo identify novel therapeutic targets for GBM, we and other research groups used RNA interference (RNAi) screening, a technique that allows a simultaneous analysis of genes in a genome for their functions in a particular setting. For example, we performed a genome-wide RNAi screen using a diphtheria toxin negative selection approach  and uncoRecently, the Broad Institute initiated a program termed Project Achilles –23. ThisIn this report, we analyzed RNAi screen results in 15 GBM cell lines and identified 10 candidate genes that are important for the survival of GBM cells. Further comprehensive analyses revealed one gene, RAN , as the top candidate because this gene was highly expressed in GBM and its activity was robustly associated with drug resistance and poor prognosis in GBM. RAN is a small GTPase protein that provides energy for nucleocytoplasmic transport and mitotic spindle assembly by hydrolyzing guanosine triphosphate into guanosine diphosphate –28. ThroGBM cell lines, primary GBM cells, and normal human astrocytes were cultured as described previously , 14, 47.https://portals.broadinstitute.org/achilles. The screen contains more than 50,000 short hairpin RNAs (shRNAs) that target the human genome and the results were presented as fold changes of shRNA loss (log2). The lower the fold change of a particular shRNA, the stronger the depletion of the shRNA in GBM cells. This shRNA depletion is, as hypothesized, due to the loss of cells over time. Results of these shRNAs in 15 GBM cell lines  were first sorted by two or more shRNAs targeting one single gene. More than 4,000 genes were targeted by two or more shRNAs. Next, the fold changes of shRNA loss were averaged. Candidate shRNAs with an average of fold change <-4.0 and a fold change <-3.0 in all 15 GBM cell lines were selected.RNAi screen results (Achilles_v2.4.6.rnai.gct) were retrieved from the Project Achilles database at the following website: http://biogps.org/#goto=welcome), Oncomine (https://www.oncomine.org/resource/login.html), Glioblastoma Bio Discovery Portal (https://gbm-biodp.nci.nih.gov), and The Cancer Genome Atlas (TCGA) database . Data from BioGPS were reanalyzed. The arbitrary units of mRNAs of candidate genes in GBM cell lines were divided by those in astrocytes, yielding fold changes (GBM/Astrocytes). Regarding data from the Oncomine database, fold changes of candidate gene mRNAs in GBM tissues normalized with those in normal brain tissues from three different studies  were recorded and summarized in Table P-values that determine the statistical significance of fold changes were included as well. mRNA levels of candidate genes in different subtypes of GBM were retrieved from Glioblastoma Bio Discovery Portal. Patient numbers of classical, mesenchymal, and proneural GBM subtypes were 199, 166, and 163, respectively. Levels of candidate gene mRNAs in GBM subtypes were then averaged. RAN levels and MGMT promoter methylation status in GBM patients were retrieved from the TCGA database and were re-analyzed using JMP software.cDNA microarray data were retrieved from BioGPS (http://gliovis.bioinfo.cnio.es), Glioblastoma Bio Discovery Portal, and The Human Protein Atlas (http://www.proteinatlas.org). The survival results were retrieved from these databases and presented together with the Log-rank P-values.Kaplan-Meier survival analyses of GBM patients from the TCGA database have been reported in GlioVis . The correlation between mRNA levels and GBM patient survival was determined by Cox univariate or multivariate analysis using the JMP software as previously described  Candidate genes should be targeted by two different shRNAs; (2) The fold change of shRNA loss (log2) should be <−3.0 in every GBM cell line tested . We further looked into the correlation between RAN and the survival of GBM subtypes and found no statistically significant trend between RAN mRNA levels and the prognosis of classical, mesenchymal, or proneural GBM subtypes .The role of RAN in GBM has not yet been extensively explored. To address this, we tested the hypothesis that RAN, as a survival gene, correlates with GBM patient survival. By querying the TCGA GBM data using The Human Protein Atlas and the Glioblastoma Bio Discovery Portal, we found that RAN levels did not correlate with patient survival  and levels of RAN and KPNB1 . Congruently, KPNA2 and KPNB1 were also enriched in GBM tissues compared to normal brain tissues , consistent with the results for RAN  with a P < 0.001. Hence, the statistical analysis shows that the significance of importazole-induced growth inhibition is much stronger in RAN-positive cells (P < 0.0001) than in RAN-negative cells (P < 0.001). More importantly, importazole only decreased the viability of astrocytes by 15% with no statistical significance (P > 0.05). These results suggest that targeting RAN activity is an appealing approach with potentially low side effects. To corroborate these results, we treated RAN-positive or RAN-negative primary GBM cells with importazole at different doses. While both RAN-positive VTC-103 and RAN-negative VTC-037 cells showed a dose-dependent response, VTC-103 cells were more robustly sensitive to importazole than VTC-037 cells , particularly when cells were treated with importazole at 12.5 or 25 μM. These results were consistent with those obtained from cell lines.The results shown above suggest that targeting RAN is a potentially appealing approach to impeding GBM disease progression. Our results also indicate that RAN activity in nuclear transport is important for GBM patient survival. We therefore chose importazole to test whether a blockade of RAN activity would inhibit GBM cell viability. We first treated astrocytes and GBM cell lines with 12.5 μM of importazole and monitored cell viability using the MTS cell viability assay. As shown in Figure P < 0.05). Hence, the cytotoxic effect of importazole is also time-dependent. Finally, we tested whether the inhibition of cell viability by importazole is due primarily to cell death such as apoptosis. By using the caspase 3/7 activity assay, we found that importazole activated apoptosis, as manifested by the remarkable increase of caspase 3/7 activity in RAN-positive A172 cells, while failing to activate apoptosis in RAN-negative SF-268 cells and astrocytes  of 4.43 and 4.78 μM, respectively . As a coWhile our study unveils the crucial role of RAN in GBM cell survival, important questions remain to be addressed to establish that targeting RAN is an effective treatment option for GBM, particularly those with TMZ resistance. Future studies will reveal whether RAN is a biomarker that predicts MGMT-dependent TMZ resistance in GBM, elucidate how RAN contributes to TMZ resistance, and determine whether importazole or functional blockade of RAN circumvents TMZ resistance and inhibits GBM progression.RTV, SL, and ZS conceived the project and wrote the manuscript. KLS and KJP performed all experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Improving the activity and selectivity profile of anticancer agents will require designing drug carrier systems that employ soluble macromolecules. Olsalazine-PAMAM-dendrimer-salicylic acid-conjugates with dendritic arms of different lengths have shown good stability regarding the chemical link between drug and spacer. In this study, the drug release was followed in vitro by ultraviolet (UV) studies. Evaluation of the cytotoxicity of the olsalazine-PAMAM-dendrimer-salicylic acid-conjugates employing a sulforhodamine B (SRB) assay in PC-3 (human prostatic adenocarcinoma) and MCF-7 (human mammary adenocarcinoma) cell lines demonstrated that conjugate 9 was more active as an antiproliferative agent than cisplatin, and no cytotoxicity towards the African green monkey kidney fibroblast (COS-7) cell line was observed in any of the conjugates synthesized in the present work. Olsalazine  is an anti-inflammatory drug used in the treatment of inflammatory bowel disease (IBD), ulcerative colitis (UC) and other gastrointestinal problems ,2. OlsalThis article reports the rational design of olsalazine as a nucleus of PAMAM-dendrimer-salicylic acid-conjugates to reach human cancer cells and the evaluation of drug release from these macromolecular products. In this study, the olsalazine-PAMAM-dendrimers of second generation (G2) with 8 amino terminal groups and salicylic acid in the periphery were synthesized . The relN,N-Dimethylformamide (DMF) and dimethylsulfoxide (DMSO) (99.8%) were used without any purification.All reagents were purchased from Sigma-Aldrich . Salicylic acid (SA), 5-amino-2-hydroxybenzoic acid (98%), hydrochloric acid (36.5–38%), sodium nitrite (98%), hexamethylenediamine (98%), ethylendiamine titanium(IV) isopropoxide (97%), methyl bromoacetate, methyl acrylate, Triton X-100 solution and phosphate buffered saline pH 7.4 were used without additional purification. Ethanol and methanol were used without additional drying. Tetrahydrofuran (THF) was dried with Na. 1H and 13C NMR spectra were recorded on a Varian Unity-300 MHz with tetramethylsilane (TMS) as an internal reference. Infrared (IR) spectra were measured on a Nicolet FT-SSX spectrophotometer . Elemental analysis was determined by Galbraith Laboratories, INC Knoxville . FAB+ mass spectra were taken on a JEOL JMS AX505 HA instrument . Electrospray mass spectra were taken on a Bruker Daltonic, Esquire 6000 . Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra were taken on a Bruker Omni FLEX using 9-nitroanthracene (9NA) as a matrix. The ultraviolet–visible (UV-Vis) absorption spectra were obtained at room temperature with a Shimadzu 2401 PC spectrophotometer .50) for each tested drug were calculated from at least three experiments. Human glioblastoma, U-251, PC-3 (human prostatic adenocarcinoma), K-562 (human chronic myelogenous leukemia cells), HCT-15 , MCF-7 (human mammary adenocarcinoma) and SKLU-1 (human lung adenocarcinoma) were supplied by the National Cancer Institute (USA). The African green monkey kidney fibroblast (COS-7) cell line was acquired from Thermo Fisher Scientific . Cytotoxicity assays were determined using the protein-binding dye sulforhodamine B (SRB) in microculture to measure cell growth, as described previously ,20. OlsaAt 35 °C in a Synergy HT Microplate Reader , the cellular uptake of olsalazine-PAMAM-dendrimer-salicylic acid-conjugates was quantitatively determined in a pH 7.4 buffer solution. The human mammary adenocarcinoma cancer cells, MFC-7 were seeded in a 96-well black plate at 10,000 cells/well and cultured. After 24 h, MFC-7 cancer cells were incubated with either olsalazine-PAMAM-dendrimer-salicylic acid-conjugates or free olsalazine 1 at a concentration of either 10 or 25 μM for 16 h at 35 °C. After incubation, the suspension was removed and the cells were washed four times with 50 μL cold phosphate buffered saline solution . All the resulting solutions were kept at 37 °C for 24 h prior to measurements. The fluorescence intensity of each sample well was measured by a microplate reader calibrated with standard solutions of olsalazine (0.1–50 μM/mL) under similar conditions. The excitation and emission wavelengths were 300 and 400 nm; and 340 and 500 nm, respectively. All release measurements were carried out in triplicate and the average values were plotted.The hydrolytic release of the drugs was measured at 37 °C using 25 mL PBS, at 37 °C and pH 7.4. The olsalazine-PAMAM-dendrimer-salicylic acid-conjugates were dried in vacuum at room temperature. Olsalazine was obtained from salicylic acid and 5-amino-2-hydroxybenzoic acid (5-ASA) with sodium nitrite in HCl at 0 °C. The olsalazine derivative 2 was obtained using hexamethylenediamine in presence of molecular sieves of 4 Å and titanium (IV) isopropoxide in THF at 70 °C .2-terminated groups were obtained with ethylenediamine in a mixture of methanol, toluene 5:1 and reflux  spectroscopy .The cytotoxic activity of the synthesized olsalazine-PAMAM-dendrimers 4 and 6 and the olsalazine-PAMAM-dendrimer-salicylic acid-conjugates 8 and 9 were chosen for evaluation of their biological activity against cancer cell lines. We screened in vitro against six human cancer cell lines: U251 (human glioblastoma), PC-3 (human prostatic adenocarcinoma), K-562 (human chronic myelogenous leukemia cells), HCT-15 , MCF-7 (human mammary adenocarcinoma) and SKLU-1 (human lung adenocarcinoma). As a control, we also tested against the COS-7 African green monkey kidney fibroblast cell line. Free olsalazine and cisplatin were used to compare the antiproliferative activity of the dendrimers. The dendrimers 4 and 6, and the conjugates 8 and 9 were not active against the cell lines U251 and SKLU-1. The compounds 4–9 showed almost the same activity against cell lines PC-3, K562, HCT-15 and MFC-7 at 25 and 10 μM. The concentration of the conjugates 8 and 9 was diluted four and eight times, respectively, to have the anticancer activity of one molecule of salicylic acid to compare with the free salicylic acid, olsalazine or cisplatin. 50 of 17.5 ± 2.1 and 11.6 ± 3.6, respectively, and the activity was very close to that of cisplatin. Moreover, these compounds were tested against the COS-7 African green monkey kidney cell line , K-562 (chronic myelogenous leukemia) and MCF-7 (human mammary adenocarcinoma) cancer cell lines appeared to be more sensitive to growth inhibition by the olsalazine-PAMAM-dendrimer-salicylic acid-conjugates than the U-251, HCT-15 and SKLU-1 cancer cell lines. The compound 9 was the best inhibitor of this series against the PC-3, K-562 and MCF-7 cell lines with an ICell line  in order9 revealed an IC50 value that was lower than that of cisplatin for the MCF-7 cell line. In addition, the use of olsalazine-PAMAM-dendrimer-salicylic acid-conjugates could be an effective way to diminish the damage towards normal cells.In summary, we report the development of novel water-soluble olsalazine-PAMAM-dendrimer-salicylic acid-conjugates that inhibit the proliferation of the PC-3, K-562, HCT-15 and MCF-7 cell lines in vitro. The drug release was followed by UV spectroscopy at pH 7.4. These conjugates were designed and synthesized for maximal MCF-7 inhibition and exhibited high comparable selectivity against human mammary adenocarcinoma cancer cells. The olsalazine-PAMAM-dendrimer-salicylic acid-conjugate


The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry lines initiated from zygotic embryos . Moreover, we observed similar differences in yield between 2ry and 3ry lines of SD4 (2400 vs 3921 g− 1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2ry vs 1ry and 2ry vs 3ry lines at histo-cytological (using LC-MS/MS) and proteomic levels.To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry and (even more) 3ry lines. Among 2300–2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on “Proteolysis” and “Catabolic process” Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first  and second  embryogenesis cycles. Protein networks established after each induction affected the same “Plant development” and “Defense response” biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3ry lines.Repetitive somatic embryogenesis dramatically improved the proliferating lines’ cellular organization (genotype SD4’s most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2This first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines’ embryogenic status.The online version of this article (10.1186/s12870-018-1337-y) contains supplementary material, which is available to authorized users. Pseudotsuga menziesii (Mirb) Franco), a productive conifer species of the Pinaceae family with globally appreciated wood quality, have been under development for more than 30 years [Plant somatic embryogenesis is the generation of embryos from vegetative cells, usually in vitro. Whenever possible, it is the preferred option for true-to-type vegetative propagation of selected genotypes as both apical and root embryonic meristems are delineated early during establishment of the embryo body plan. Thus, in contrast to other vegetative propagation technologies, there is no need for adventitious organogenesis. In recent years, significant advances have been made in the development of techniques to improve somatic embryogenesis of increasing numbers of tree species, from initiation of embryogenic cultures to maturation of high-quality somatic embryos (SEs). Such progress towards large-scale production of vigorous somatic seedlings has been reported for both hardwood , 2 and s30 years . There i30 years , 6, and Somatic embryogenesis is considered a promising biotechnology for large-scale clonal propagation of forest trees, due to the high multiplication rates it can provide , 8. Morery) embryogenesis, either directly from primary (1ry) embryos in culture or indirectly from various cell aggregates such as proembryogenic masses or nodular calli developing from 1ry SEs [Once somatic embryogenesis has been initiated, embryogenic cultures are proliferated to sustain new embryo formation. In angiosperms, embryogenic potential is maintained during a continuous process of repetitive, secondary . It is still unclear if cleavage polyembryony is the only process involved in early SEs’ proliferation.Embryogenic cultures of gymnosperms proliferate as embryonal masses (EMs), i.e. clusters of multiple attached SEs that become interspersed with singulated SEs at an early stage of late embryogeny , 15. EMsinter alia for Douglas-fir cultures [SE clusters of some conifer species, such as Douglas-fir, develop into polyembryogenic center of various sizes, putatively through continuous but incomplete cleavage polyembryony (somatic polyembryogenesis) and /or de novo somatic embryogenesis from proliferating early SEs . In addicultures , are supQuercus spp. trees [Kalopanax septemlobus trees [in vitro and/or rejuvenation techniques , the embryogenic capacity of initiated lines can usually be maintained for years by repetitive somatic embryogenesis. Moreover, with material of many angiosperm species, 2ry embryogenesis is much more efficient than 1ry embryogenesis [Picea abies is much more responsive to somatic embryogenesis induction treatment than material derived from zygotic embryos . Accordd trees, ), Picea  plants, , 24, 25)ary SEs, ), Larix ted SEs, , 28), Abary SEs, , 30), Piing SEs, ) and moring SEs, ). In mosry somatic embryogenesis has numerous potential applications as it offers a potential means to obtain “immortal” embryogenic lines [ry somatic embryogenesis could be useful for improving the embryogenic potential of some species’ embryogenic lines. For example, 2ry EMs are reportedly more productive than 1ry cultures of some embryogenic lines of hybrid larch [ry lines obtained from recalcitrant genotypes of Douglas-fir with low embryogenic potential (< 500 SEs g− 1 EMs f.w.) have been found to be significantly more productive than 1ry lines [In conifers, 2ic lines . Stable ic lines , 32. Secid larch  and mariid larch . Similarry lines .ry and 1ry lines of conifers are largely unknown, and have only been previously examined in one study focused on Pinus pinaster [ry somatic embryogenesis selective processes may occur that promote development of embryogenic cells and EMs with high capacities to proliferate and regenerate cotyledonary SEs.The reasons for such differences in embryogenic potential between 2pinaster . ClearlyPinus pinaster, resulting in reduced frequencies of immature SEs capable of completing the last stages of late embryogenesis [In contrast to macromorphological observations, cytological features of EMs during proliferation are reliable indicators of embryogenic lines’ ability to produce cotyledonary SEs. In Norway spruce, only EMs showing immature SEs with a dense embryonal head clearly separated from a well-defined suspensor region can reportedly develop further into cotyledonary SEs , at leasogenesis . In bothogenesis , 35.Pinaceae family, such as Picea glauca [Picea abies [Larix spp. [Pinus massoniana [Pinus pinaster [Recently, the development of novel, high-resolution proteomic methods has offered opportunities for both untargeted qualitative proteome coverage and quantitative measurement of proteins involved in plant development. These proteomic analyses have already improved understanding of the metabolic and signaling pathways involved in plant somatic embryogenesis , 36, 37.a glauca , Picea aea abies , Larix srix spp. –42, Pinussoniana  and Pinupinaster , 44.ry lines, and 2ry EMs were compared to 1ry EMs in terms of embryogenic potential, cytologicaly and protein patterns during proliferation. To deepen understanding of repetitive somatic embryogenesis, tertiary (3ry) lines were produced from cotyledonary SEs of 2ry lines via a third cycle of somatic embryogenesis, and 3ry EMs were compared to 2ry EMs. This is the first report of two successive cycles of repetitive somatic embryogenesis in conifers. We first compared 2ry EMs to 1ry EMs, then 2ry to 3ry EMs. However, cells of 3ry EMs do not directly stems from 1ry EMs, and many subcultural steps involving physiological aging of the tissues could occur between them. Thus, we did not compare 1ry and 3ry EMs to avoid possible complexities irrelevant to aims of this study.The main objective in the work presented here was to study cellular and proteomic changes induced by repetitive somatic embryogenesis in Douglas-fir. For this purpose, somatic embryogenesis was induced in cotyledonary SEs to obtain 2ry and 1ry EMs, and between 2ry and 3ry EMs (hereafter “significant” proteins), suggesting that each cycle of repetitive somatic embryogenesis promotes substantial genome-wide rearrangement of gene expression patterns. In addition, Sub-Network Enrichment Analysis (SNEA) was performed to elucidate the functions and interactions of identified proteins, and the results show that this novel approach for studying conifer somatic embryogenesis can yield valuable information.We obtained the first evidence of cytological and proteomic changes in proliferating EMs of Douglas-fir with increasing embryogenic potential following repetitive somatic embryogenesis. Interestingly, the proteomic analysis further revealed different sets of proteins that are significantly differentially expressed between 2Pseudotsuga menziesii trees involved in this work are descendants of trees with provenances in North Bend (genotypes 4455 and 4456) or Enumclaw (4466 and 4477), Washington (USA). They were used as parental trees to perform the following control crosses at INRA : 4455 × 4466 and 4456 × 4477. Somatic embryogenesis was induced from seed explants  following published methodology [− 1 gellan gum. When necessary, EMs were cultured as a thin layer dispersed on a filter paper disc (300 mg f.w. per filter) to promote proliferation, as previously described [EMs were sub-cultured in clumps every 2 weeks on Glitz proliferation medium, consisting of modified Litvay medium , 46 suppry embryogenic lines SD4 and TD17 were isolated and transferred to Glitz initiation medium supplemented with 4.5 μM 2,4-D, 4.4 μM BA and 0.087 M sucrose, solidified with 4 g.L− 1 gellan gum. Each 2ry EM, initiated from a single SE, was then subcultured as described above for proliferation. We obtained 2ry embryogenic lines designated SD4–2, SD4–6 and SD4–8 from the 1ry line SD4, and a 2ry line designated TD17–1 from the 1ry TD17 line. For the second induction cycle, cotyledonary SEs obtained from 2ry line SD4–8 were similarly used as explants to initiate 3ry lines, designated SD4–8-1, SD4–8-2 and SD4–8-3.Two cycles of repetitive somatic embryogenesis were performed, following published protocols . Brieflyw/v) Trypan Blue (Sigma-Aldrich), as previously described [Samples were collected after 10 days of multiplication for morphological and histo-cytological characterizations. Morphology of EMs was documented using a SMZ 1500 stereomicroscope , and their structure was examined using a Jenaval transmission light microscope  after staining fresh material with 0.4%  was estimated. In a first set of experiments, the embryogenic potential of 1ry lines  and 2ry lines  were compared using 96 Petri dishes . In a second set of experiments we compared the embryogenic potential of 2ry (SD4–8) and 3ry  lines, using 6 Petri dishes for each permutation of lines and conditions, and experiments were repeated three times, so 72 Petri dishes were used in total.Proliferating EMs from filter papers were weighed, dispersed in liquid Glitz medium without plant growth regulators and distributed on a filter paper disc placed on the surface of Glitz maturation medium (in a Petri dish) supplemented with 0.2 M sucrose, 60 μM ABA (cis-trans ± abscisic acid), and 10 g Lry, 2ry and 3ry), with EMs cultured as a thin layer dispersed onto filter paper. Soluble proteins extracts were prepared from four biological replicates for each samples (150 mg f.w. of frozen EMs) with 1 ml of urea extraction buffer . Total protein content was determined using the Bradford assay with bovine serum albumin as a standard. Results were expressed as soluble proteins content (μg mg− 1 of dry weight).Analyses were performed for all types of lines  with an AGC target of 3E6 ions collected within 100 ms. Dynamic exclusion was set to 30 s and the top 15 ions were selected from fragmentation in HCD mode. MS/MS scans with a target value of 1E5 ions were collected with a maximum fill time of 100 ms and resolution of R = 17,500. Additionally, only + 2 and + 3 charged ions were selected for fragmentation. Acquired peptide were searched by SEQUEST implemented in Proteome Discoverer 1.4 (Thermo Fisher Scientific Inc.) against a Pseudotsuga menziesii v1 transcriptome – proteome database from PineRefSeq . Spectra from peptides larger than 5000 Da or smaller than 350 Da were rejected and mass accuracy of the monoisotopic peptide precursor and peptide fragments was set to 10 ppm and 0.02 Da, respectively. Only b- and y-ions were considered for mass calculations. Oxidation of methionine (+ 16 Da) was considered as a variable modification and carbamidomethylation of cysteines (+ 57 Da) as a fixed modification. Two missed trypsin cleavages were allowed. Peptide were using the Percolator algorithm [Proteomic and nLC-MS/MS analyses were performed following previously published protocols . Brieflylgorithm  and onlyArabidopsis thaliana database (https://www.arabidopsis.org/) for functional annotation. The proteins were then classified based on their biological functions using Web Gene Ontology Annotation Plot software at level 2 for biological processes  [Arabidopsis thaliana was associated with the corresponding Douglas protein for topGO analysis.Changes in expression, relative to appropriate controls, were calculated based on the cumulative intensity of each peptide (classifying proteins with ≥1.5-fold change ratios as up- or down-regulated). All sequences were mapped against Gene Ontology (GO) terms in the TAIR db.org/) . A binomdb.org/) , based oSub-Network Enrichment Analysis (SNEA) was performed using Pathway Studio® version 11.4 (Elsevier B.V.).ry, 2ry and 3ry lines were evaluated using one-way analysis of variance (ANOVA) and multiple comparisons of means with Tukey contrasts (P < 0.05). For proteomic analysis, differential expression of proteins in 1ry vs 2ry lines, and 2ry vs 3ry lines, was analyzed using two-way ANOVA with interaction and FDR, based on normalized abundance (adjusted P < 0.05).R software  was used for all statistical analyzes. Embryogenic potential and the soluble protein contents of 1Embryogenic potential of Douglas-fir embryogenic lines after repetitive somatic embryogenesis.ry (TD17 and SD4) and 2ry  lines, the primary lines showed significant variations in mean production of cotyledonary SEs (P = 3.01e− 7). SD4 was moderately embryogenic (478 SEs g− 1 f.w.) whereas TD17 showed very weak embryogenic potential . TD17–1 was 8 times more productive  to 6 times (SD4–2) more cotyledonary SEs  and 3ry  lines, SE yields of the three 3ry lines were very high  yields  than SD4–8 (2401 SEs g− 1 f.w.).In the subsequent comparison of the embryogenic potential of 2Macromorphological (EM color and morphotype) and histo-cytological traits  of primary SD4, TD17), secondary  and tertiary  embryogenic lines of Douglas-fir are summarized in Additional file 7, secondry, 2ry, 3ry) had various colors; usually shades of yellow and brown, but some lines  were rather pink . The structure of immature early SEs proliferating in EMs was hardly distinguishable in the 1ry lines. However, the EMs’ surfaces had notable granularity, particularly those of line SD4  in the dead material.Both 17–1 Fig. . NECs wePrimary line SD4 is characterized by the production of large structures resembling polyembryogenic centers Figs. , 4a, b. ry line, 2ry lines produced well-arranged polyembryogenic centers of various sizes formed by compact meristematic parts joined together with elongated suspensor cells. The most typical polyembryogenic centers with distinct embryonal heads were observed in SD4–2  and in both 1ry and 2ry lines  is a multivariate technique that analyzes a data set with inter-correlated quantitative dependent variables to represent it as a set of new orthogonal variables called principal components displaying the pattern of similarities. The first principal component (PC1 axis) obtained from PCA of the 2293 proteins identified in 1ry and 2ry lines , and explained 16% of the total variance. PC1 obtained from PCA of all 2554 proteins identified in 2ry and 3ry lines of genotype SD4 explained 65% of the total observed variance and type of line was the main determinant  we identified 2293 proteins in the comparison of 2nes Fig.  was mainant Fig. .Fig. 6Prry vs 1ry lines and 3ry vs 2ry lines, respectively . Surprisingly, only 33 proteins were members of both sets, moreover 9 of these 33 were up-regulated following both somatic embryogenesis cycles, 3 were down-regulated following both cycles and there were opposite changes in expression of the other 21 proteins. Thus, specific sets of differentially expressed proteins, and changes in expression profiles, were associated with each cycle of somatic embryogenesis. In Additional file ry vs 1ry) and 228 (3ry vs 2ry) significant proteins. The most strongly represented categories are “Metabolic Processes”, “Cellular Processes” and “Response to Stimulus” .In the subsequent two-way ANOVA of changes in protein expression following the first and second cycles of somatic embryogenesis, 162 and 288 significant proteins were identified in the comparisons of 2Arabidopsis thaliana bibliographic database, which implies the network representation with Arabidopsis protein names. The SNEA provided further indications of their functions, interactions and putative targets, as well as regulators involved in metabolic pathways that may be affected during repetitive somatic embryogenesis  or 3ry somatic embryogenesis (51 proteins) were integrated in the constructed networks. They included many proteins associated with metabolism of plant growth signaling compounds, including plant growth regulators such as jasmonic acid, ABA and salicylic acid, together with proteins related to lignin  and flavonoid secondary metabolites . Indole-3-acetic acid (IAA) was revealed as an important regulator associated with both networks. The subsets of significant proteins and targets/regulators in each network were mostly specific for each cycle of somatic embryogenesis, but affected the same major biological processes (“plant development” and “defense response”). In the latter case, plant growth signaling compounds, such as jasmonic acid, salicylic acid and ABA and many other molecules identified in these networks (including secondary metabolites) have known involvement not only in adaptation to stress , but also in non-defensive functions during plant development.Results of GO category enrichment analyses (Term Enrichment) using Panther and Biocoductor R of overall trends of the functional categories enriched in the lines following the cycles are shown in Table sis Fig. . OverallArabidopsis annotation) were down-regulated following the first cycle of repetitive somatic embryogenesis and up-regulated following the second cycle. Interestingly, two others were up-regulated following both cycles and identified as endochitinase EP3 and E3 ubiquitin-protein ligase ARI1, both of which are involved in embryo development and pathways such as controlled proteolysis in conifers.Eleven significant proteins involved in the networks , a ribonucleoprotein. RISC is known to incorporate the microRNAs (miRNAs) MIR165A and MIR166A to cleave the corresponding, targeted mRNA. This gene silencing process, participates to the biological process of mRNA splicing and is apparently modulated only during the third cycle of repetitive somatic embryogenesis.Repetitive somatic embryogenesis enhanced the embryogenic potential of embryogenic lines derived from two Douglas-fir genotypes.Pinaceae species such as pines [The experiments with two unrelated genotypes  with differing embryogenic potential (under the test conditions) provided abundant information about this common “genotype effect”, which has been observed in various conifers . These ias pines  and Dougas pines , 53, andry lines, 2ry lines of both SD4 and TD17 genotypes displayed significantly higher mean embryogenic potential . Increases in embryogenic potential after a second cycle of somatic embryogenesis (secondary somatic embryogenesis) have been previously recorded in experiments with hybrid larch , maritiincrease ) and Douincrease ). This iry coniferous embryogenic lines. Interestingly, two of the three tested 3ry lines (SD4–8-2 and SD4–8-3) exhibited significantly higher embryogenic potential , and the third 3ry line (SD4–8-1) non-significantly higher embryogenic potential (3344 SEs g− 1 f.w.) than the original 2ry line SD4–8 . We conclude that repetitive somatic embryogenesis enhanced the SD4 genotype’s embryogenic potential. However, the increase in maturation performance observed after the third cycle of somatic embryogenesis was weaker than that observed after the second cycle (1.6- vs 4.5-fold increase on average). Long-term stable embryogenic potential has been demonstrated in some Pinaceae species, such as Larix spp. [Pinus spp., [ry lines to younger 2ry and 3ry lines) observed in this study supports the hypothesis that increases in embryogenic potential following repetitive somatic embryogenesis are mainly due to initiation effects rather than ageing. In conifers, cellular and molecular changes in proliferating lines after one or two successive cycles of somatic embryogenesis have been poorly investigated. To our knowledge, there is only one previous report on physiological and molecular aspects of 1ry and 2ry lines with contrasting embryogenic potential, in Pinus pinaster [ry vs 2ry lines and 2ry vs 3ry lines, respectively).In this study, one genotype (SD4) was subjected to a third cycle of somatic embryogenesis (tertiary somatic embryogenesis) to further investigate cumulative effects of repetitive somatic embryogenesis on its embryogenic potential. To our knowledge, this is the first report of the initiation of 3rix spp. , whereaspinaster . In thisry and 2ry lines of genotype TD17 produced all types of EMs that we have recently described [ry and 2ry lines of genotype SD4 markedly differed. The large polyembryogenic centers that predominated in 1ry line SD4 slightly resembled meristemoids that develop during organogenesis or somatic embryogenesis of angiosperms [ry and 3ry lines. Moreover, the meristem-like part consisted of a very thin layer of densely cytoplasmic cells, while other cells were vacuolated and accumulated secondary metabolites. Parts that could be regarded as cleavable embryonal heads were rare. These structures were observed in neither 2ry nor 3ry lines, although some structures similar to those observed in SD4 material occurred in addition to well-organized bipolar SEs in lines SD4–2 and SD4–6. In conclusion, repetition of somatic embryogenesis clearly improved EMs’ organization. The increased yields of cotyledonary SEs after maturation may have been due to increases in frequencies of small SEs together with reductions in sizes of polyembryogenic centers (as we observed in 3ry line SD4–8-2). Alternatively, it could be related to the organization of polyembryogenic centers into clusters of distinct embryonal heads of very similar size, as observed in SD4–8-1 and even more strongly in SD4–8-3.The repetitive somatic embryogenesis markedly improved SEs’ structures, particularly in genotype SD4. Both 1escribed , i.e. poiosperms , 55, or iosperms . Neverthiosperms . Howeverry lines, having a granular appearance . Such NEC clusters, consisting of cells accumulating not only starch grains but also phenolic compounds, have been considered one of the main impediments of in vitro propagation of woody plants [ry SD4 line, cells with phenolic contents were also present in the large meristem-like parts of polyembryogenic centers. Processes resulting in oxidation of phenols in the tissues could have detrimental effects on the embryogenic potential of 1ry lines, especially TD17. Thus, the reduction in numbers of NECs containing phenolic compounds was another desirable effect of the repetitive somatic embryogenesis. We found far fewer NECs in the 2ry lines TD17–1 and SD4–2, and close to zero in 3ry lines induced from SD4–8.When EMs are cultivated in clumps, highly mitotically active and growing polyembryogenic centers or singulated early SEs occur on the surface of the clumps, while inner parts usually consist of dying suspensor cells or whole SEs. Such organization resulted in most of the investigated lines of Douglas-fir EMs, especially 3spensors   than in previous analyses based on two-dimensional gel electrophoresis [Picea glauca database. PCA revealed that the type of line  accounted for most of the total observed variance in expression patterns of this large set of identified proteins, in both genotypes.We compared protein profiles of both 2phoresis , 44 confPicea abies at the beginning of embryogenesis [Pinus pinaster in transitions between stages of embryo development induced by regulatory signal. The latter study highlighted several epigenetic regulation mechanisms involved in stage-to-stage transitions.No significant differences in total protein content among the compared embryogenic lines were detected. However, the expression of substantial numbers of proteins was affected by the somatic embryogenesis cycles . Functional analysis showed that most of these significant proteins are mainly involved in metabolic and cellular processes. It is well known that embryogenic competence is accompanied in plants, including conifers (reviewed in ) by actiogenesis , 62, andArabidopsis thaliana, so resulting networks inevitably miss important elements of woody, perennial species’ networks. Third, embryo patterning is still a poorly understood and complex process involving a regulated network of at least 300–450 genes [Biological processes associated with significant proteins were identified by both GO analyses Table  and scre50 genes .The discussion of significant proteins according to assigned biological processes and functional categories could be complicated by the possible involvement of proteins in several pathways. Therefore, in four sections we discuss the proteomic changes associated with the somatic embryogenesis cycles affecting “plant development”, “proteolysis”, “signaling by growth regulators and polyphenols” and “stress and redox responses”.ry lines, is directly associated with the biological process “Plant development”, and plays well-known roles in oxidative stress regulation in plants [ry lines, the putative methyltransferase DDB could be a key mediator of increases in embryogenic potential through its involvement in epigenetic mechanisms that are known to promote embryonic development [ry lines. Ubiquitin protein ligases have known association with activation of chromatin modifiers called ubiquitin−/small ubiquitin-related modifier (SUMO)-conjugating genes, resulting in global modifications of gene expression . These conclusions are supported by the GO term enrichments Fig.  and prespinaster . The laspinaster . These tpinaster . Finallypinaster , a plantry somatic embryogenesis, by highlighting their interactions with various significant hormone-responsive proteins. Jasmonic acid was the one with most connections. All of the associated proteins except one  were more strongly expressed in 1ry lines than in 2ry lines. Several studies have detected positive effects of jasmonic acid on maturation of SEs [ry lines, which may explain their higher embryogenic potential. These flavonoids play important roles in the cells since they have several links with oxidative stress responses [ry lines, indicating activation of the phenylpropanoid synthesis pathway and phenylalanine metabolism, which could explain their relatively high contents of polyphenols -binding Rossmann-fold protein, involved in redox reactions, was also up-regulated in 2ry and 3ry lines, relative to 1ry lines. In contrast, other proteins possibly involved in ROS responses were down-regulated in 2ry lines, for example the GST ERD9 and PAL-like protein. Relationships have been established between the latter enzyme and both flavonoids and polyphenols, as previously discussed. GSTs are a multigene family that may be involved in auxin responses, production of secondary metabolites (anthocyanins), and confer antioxidant activity to flavonoids [Oxidative stress, one of various stresses affecting cell cultures, appears to be a key factor for somatic embryogenesis , 81, whiesponses , 81, as avonoids .In conclusion, characteristic biological processes of embryogenesis, such as “plant development”, “defense” and “stress”  “metabolism” and “proteolysis” were affected during proliferation by repeated somatic embryogenesis cycles. Moreover, the major result is the identification of numerous proteins that interact with flavonoids and associated secondary metabolites were down-regulated following the second somatic embryogenesis cycle.ry lines had greater embryogenic potential than the 1ry and 2ry lines, and comparison of the 2ry and 3ry lines’ proteomes revealed activation of the same biological processes following the second and third cycles of somatic embryogenesis. Thus, the biological processes “Plant development”, “Proteolysis”, “Stress/water stress”, “Defense response” and “Growth regulators” also appear in the networks derived from the significant proteins associated with the third cycle  among 390 identified significant proteins in total. Furthermore, only 12 were either up-regulated or down-regulated following both cycles. These findings show that embryogenic induction is a complex process that changes the cells’ proteomic composition. Induction is triggered by transfer of the cotyledonary SEs to an appropriate culture medium containing auxin. The repeated intake of auxin during the three somatic embryogenesis cycles presumably affected the EMs, since auxin interacts  with flavonoids, ABA, jasmonic acid and salicylic acid [ry lines and then expression of genes that are sensitive to, or involved in metabolism of, key signaling compounds . The innovative use of protein networks in the proteomic analysis had been very conclusive. It provided valuable information, revealing the general down-regulation and up-regulation of significant proteins following the first and second somatic embryogenesis cycles, respectively. In both cases, interactions with various plant growth signaling agents  were major elements of the shifts, showing the ability of cells to use different protein regulatory pathways to increase embryogenic potential, and resulting in more suitable SEs for maturation. Overall, this first report of cellular and molecular changes in EMs after two successive cycles of somatic embryogenesis in conifers generally, and Douglas-fir specifically, should enhance understanding of the increases in embryogenic potential of 2These findings could also help with practical application. A number of laboratories now have the problem of deterioration in the quality of elite somatic embryogenic lines due to extensive subculture. The methods described in this paper could be of great benefit to reinvigorate these valuable proven production clones.Additional file 1:Table S1. A summary of macromorphological (EM colour and morphotype) and histo-cytological traits (occurrence of polyembryogenic centers (PECs), singulated SEs and NECs) of 1ry , 2ry  and 3ry  embryogenic lines of Douglas-fir. (DOCX 16 kb)Additional file 2:Figure S1. Macroscopic aspect of embryonal masses (EMs) from two Douglas-fir genotypes . These embryonal masses were obtained after 1ry somatic embryogenesis from zygotic embryo and two cycles (2ry and 3ry) of repetitive somatic embryogenesis from somatic embryos. Note the granular aspect of most embryonal masses indicating the occurrence of large polyembryogenic centers (arrowheads) and/or singulated early somatic embryos that are sometimes protruding from the embryonal masses surface. (DOCX 687 kb)Additional file 3:Figure S2. Structure of embryonal masses from primary and secondary lines of genotype TD17. A / TD17; B / TD17–1. Trypan blue staining of squashes of fresh EMs; m – meristem of polyembryogenic centers, s – suspensor. Scale bar = 200 μm. (DOCX 178 kb)Additional file 4:Figure S3. Histology of non-embryogenic cells (NECs) clusters from 1ry and/or 2ry lines of genotypes TD17 and SD4. A / Histology of TD17 groups of loosely arranged NECs (arrow) in the vicinity of small somatic embryos (arrowhead); B / histology of TD17 compact NEC cluster accumulating phenolics  and starch (arrow); C / Lugol staining of TD17 NEC cluster showing starch (arrows) and phenolic compounds ; D / TD17–1 NEC cluster with dividing cells (arrowheads); E /SD4–2 NEC cluster (arrow) within polyembryogenic center besides well-arranged embryonal heads (EH), note phenolic content (in dark blue-grey) of cells separating NEC from suspensor cells (in light blue); F / SD4–6 meristemoid-like NEC cluster; note phenolic content of cells between two meristemoid-like structures (in blue-grey). Scale bar: A, E = 200 μm; B, F = 100 μm; C, D = 50 μm. (DOCX 417 kb)Additional file 5:Table S2. Total protein content  in proliferating 1ry and 2ry embryogenic lines of Douglas-fir. (DOCX 45 kb)Additional file 6:Table S3. Total protein content  in proliferating 2ry and 3ry embryogenic lines of Douglas-fir. (DOCX 45 kb)Additional file 7:ry vs 1ry and 3ry vs 2ry embryogenic lines of two Douglas-fir genotypes. (XLSX 2224 kb)Differentially expressed proteins in 2Additional file 8:Table S4. Functional classification according to gene ontology (GO) of significant proteins identified after two cycles of repetitive somatic embryogenesis in Douglas-fir. (DOCX 72 kb)