O-fatty acyl derivatives of 3′-fluoro-2′,3′-dideoxythymidine  were synthesized. These conjugates were evaluated for their potential as topical microbicides with anti-HIV activity against cell-free (X4 and R5), cell-associated, and multidrug-resistant viruses. Compared to FLT and 3′-azido-2′,3′-dideoxythymidine (AZT), 5′-O-(12-azidododecanoyl) (5), 5′-O-myristoyl (6), and 5′-O-(12-thioethyldodecanoyl) (8) derivatives of FLT were found to be more active against both cell-free viruses (lymphocytotropic and monocytotropic strains) with EC50 values of 0.4 μM, 1.1 μM, and <0.2 μM, respectively, as well as cell-associated virus with EC50 values of 12.6, 6.4, and 2.3 μM, respectively. Conjugates 5, 6, and 8 exhibited >4 and >30 times better antiviral index than FLT and AZT, respectively. Conjugates 5 and 8 were significantly more potent than FLT against many multidrug-resistant strains. A comparison of the anti-HIV activity with the corresponding non-hydrolyzable ether conjugates suggested that ester hydrolysis to FLT and fatty acids is critical to enable anti-HIV activity. Cellular uptake studies were conducted using fluorescent derivatives of FLT attached with 5(6)-carboxyfluorescein through either β-alanine (23) or 12-aminododecanoic acid (24) spacers. The lipophilic fluorescent analog with a long chain (24) showed more than 12 times higher cellular uptake profile than the fluorescent analog with a short chain (23). These studies further confirmed that the attachment of fatty acids improved the cellular uptake of nucleoside conjugates. In addition, 5, 6, and 8 were the least cytotoxic and did not alter vaginal cell and sperm viability compared to the positive control, a commercial topical spermicide (N-9), which significantly decreased sperm and vaginal cell viability inducing the generation of proinflammatory cytokines.A number of 5′- In June 1981, the very first case of acquired immunodeficiency syndrome (AIDS) in the United States was identified to be caused by the human immunodeficiency virus (HIV) +.O-(tetradecanyl)thymidine (4).3′-Azido-2′,3′-dideoxy-5′- White powder; yield : 7.35 , 6.02 , 4.35 , 3.88–3.96 , 3.85 , 3.76 , 2.40–2.54 , 2.28–2.40 , 1.86 , 1.53 , 1.12–1.30 , 0.82 ; 13C NMR : 163.28 (C-4 C=O), 150.72 (C-2 C=O), 134.58 (C-6), 110.29 (C-5), 87.23 (C-1′), 84.47 (C-4′), 61.89 (C-5′), 59.90 (C-3′), 41.46 (2CHO), 37.35 (C-2′), 31.85, 29.58, 29.53, 29.48, 29.28, 29.24, 27.51, 26.93, 22.62, (methylene carbons), 14.05 (CH3), 13.22 (5-CH3). HR-MS (ESI-TOF) (m/z): C24H41N5O4: calcd. 463.3159; found 464.1528 [M + H]+.O-(Fatty acyl) ester derivatives of FLT and AZT5′-. First, several 5′-O-(fatty acyl) ester derivatives of FLT and AZT were synthesized at a scale of 100 mg according to the previously reported procedure  were synthesized at a larger scale of 5 g and 25 g as we previously reported +.(±O-(2-methoxyteradecanoyl)thymidine (14).)-3′-Azido-2′,3′-dideoxy-5′- Oil; yield ; 1H NMR : 9.00 , 7.28 , 6.17 , 4.50 , 4.35 , 4.20 , 4.10 , 3.38 , 3.39 , 2.50 , 2.37 , 1.95 , 1.70–1.80 ), 1.32–1.42 ), 1.22–1.31 , 0.89 ; HR-MS (ESI-TOF) (m/z): C25H41N5O6, calcd, 507.3057; found, 530.6788 [M + Na]+.O-(3-aminopropanoyl)thymidine (21) and 3′-fluoro-2′,3′-dideoxy-5′-O-(12-aminododecanoyl)thymidine (22). FLT , the appropriate Fmoc-amino acid (1.2 mmol), and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate  were dissolved in dry DMF (10 mL) and dry N-methylmorpholine (2 mL). The solution was stirred overnight at room temperature. The reaction mixture was concentrated and dried under reduced pressure to afford the crude 5′-O-Fmoc-amino acid derivatives of FLT, 19, and 20. The crude intermediates were dissolved as such in THF (10 mL). To the reaction mixture was added piperidine , 1-octanethiol . The reaction mixture was allowed to stir for 1 h at room temperature. The reaction solution was concentrated at reduced pressure. The residue was purified with reversed-phase HPLC using a C18 column and using a gradient system with water/acetonitrile as the solvents, as described above. The chemical structures of the compounds were confirmed by 1H NMR and 13C NMR as shown for compound 22 in General procedure for the synthesis of 3′-fluoro-2′,3′-dideoxy-5′-O-(3-aminopropanoyl)thymidine (21).3′-Fluoro-2′,3′-dideoxy-5′- White powder; yield . HR-MS (ESI-TOF) (m/z): C13H18FN3O5: calcd. 315.1230; found 316.3369 [M + H]+, 631.4397 [2M + H]+.O-(12-aminododecanoyl)thymidine (22).3′-Fluoro-2′,3′-dideoxy-5′- White powder; yield . 1H NMR : 11.38 , 7.81–7.98 , 7.45 , 6.18 , 5.30 , 4.15–4.36 , 3.15 , 2.68–2.82 , 2.21–2.52 , 1.76 , 1.05–1.24 ; 13C NMR : 173.08 (COO), 164.07 (C-4 C=O), 150.87 (C-2 C=O), 136.08 (C-6), 110.03 (C-5), 94.38 , 84.66 (C-1′), 82.03 , 63.49 , 55.32, 51.54 (CH2NH), 36.33 , 33.73 (CH2COO), 29.30, 29.25, 29.12, 28.96, 28.91, 28.84, 27.41, 26.23, 24.79, 23.51 (methylene carbons), 13.86 (5-CH3). HR-MS (ESI-TOF) (m/z): C22H36FN3O5: calcd. 441.5367; found 442.1974 [M + H]+, 883.5248 [2M + H]+.O-(5(6)-carboxyfluorescein) derivatives of FLT (23 and 24).General procedure for the synthesis of 5′- FLT was attached to 5(6)-carboxyfluorescein (FAM) through β-alanine (21) or 12-aminododecanoic acid (22) as linkers. A mixture of 5(6)-carboxyfluorescein , the corresponding FLT-amino acid  and HBTU  was dissolved in dry DMF (10 mL) and diisopropylethylamine  and stirred overnight at room temperature. The reaction mixture was concentrated and dried under vacuum. The final compounds were purified with reversed-phase HPLC using a C18 column and water/acetonitrile as the solvents, as described above. The chemical structures of the compounds were confirmed by 1H NMR and 13C NMR ( 13C NMR .O-(3-(N(5(6)-carboxyfluorescein)aminopropanoyl)thymidine (23).3′-Fluoro-2′,3′-dideoxy-5′- Yield . 1H NMR : 8.48 , 8.19 , 8.04, 8.13 , 7.58 , 7.20–7.42 , 6.96–7.05 , 6.84 , 6.11 and 6.17 , 5.16 and 5.23 , 4.20–4.40 , 3.53 and 3.64 , 2.10–2.74 , 1.72 and 1.78 ; 13C NMR : 172.63 (COO), 172.47 (NHCO), 168.06, 165.63, 156.25 (FAM-Ar-C), 160.49 (C-4 C=O), 151.20, 151.14 (C-2 C=O), 139.64, 133.27, 131.43, 129.24, 129.60, 127.37, 127.82, 126.07 (FAM-Ar-C), 136.46, 136.52 (C-6), 116.84 (FAM-Ar-C), 113.47, 113.58 (C-5), 102.83 (FAM-Ar-C), 94.31, 94.19 , 85.53, 85.57 (C-1′), 82.57, 82.48 , 64.13, 64.00 , 55.11, 49.09 (CH2NH), 36.07, 35.97 (C-2′), 33.76, 33.89 (CH2COO), 11.95, 12.01 (5-CH3). HR-MS (ESI-TOF) (m/z): C34H28FN3O11: calcd. 673.1708; found 674.5083 [M + H]+, 1348.6951 [2M + H]+.O-(12-(N(5(6)-carboxyfluorescein)aminododecanoyl)thymidine (24).3′-Fluoro-2′,3′-dideoxy-5′- Yield ; 1H NMR : 8.45 , 8.17 , 8.10 , 7.64 , 7.43 , 7.30 , 6.76 , 6.70 , 6.61 , 6.16–6.26 , 5.20 , 4.30–4.45 , 4.19 , 3.38 , 2.44–2.63 , 2.15–2.38 , 1.83 , 1.47–1.66 , 1.05–1.24 ; 13C NMR : 174.85 (COO), 170.11 (NHCO), 168.34, 166.35, 155.35 (FAM-Ar-C), 163.45 (C-4 C=O), 152.29 (C-2 C=O), 142.15, 138.28, 137.40, 131.01, 129.46, 126.86, 126.08, (FAM-Ar-C), 135.12 (C-6), 115.14 (FAM-Ar-C), 112.02 (C-5), 103.72 (FAM-Ar-C), 95.32 , 86.92 (C-1′), 84.15 , 64.66 , 54.98, 41.40 (CH2NH), 38.83 , 35.02 (CH2COO), 30.77, 30.74, 30.68, 30.59, 30.54, 30.45, 30.41, 30.34, 30.26, 30.21, 28.18, 26.10 (methylene carbons), 12.81 (5-CH3). HR-MS (ESI-TOF) (m/z): C43H46FN3O11: calcd. 799.3116; found 800.4325 [M + H]+.5 as a model compound.The physicochemical properties including pKa (9.67 ± 0.02), Log D , and solubility were determined for 5 was determined using dip-probe absorption spectroscopy (D-PAS) [5 was found to have an aqueous pKa value of 9.67 ± 0.02 as determined by the spectroscopic method under methanol–water co-solvent conditions.The pKa for  (D-PAS) . The sam5 was initially investigated by the pH-metric (potentiometric) method. The sample was titrated in three triple titrations from pH 2.5 to pH 11.9 at concentrations of 0.7–1.1 mM in various ratios of octanol/water. The results indicated high sample lipophilicity, although the Log P could not be determined due to the apparent pKa in octanol shifting out of the measurable range. Therefore, the Log D of 5 at pH 7.4 was measured as 5.04 using liquid–liquid distribution chromatography. The Profiler LDA is an isocratic chromatography system that uses an octanol-coated column with octanol saturated mobile phases adjusted to pH 7.4. A set of standard compounds with well-known Log D octanol values were run through the column before the samples, and the generated retention times were used as a calibration curve to relate the retention times generated for the sample compounds to Log D. Detection was carried out by using a UV diode array. A multi-wavelength peak location system was used to hone in on the largest peak present in the chromatogram. A Log D value of 5.04 was obtained, which should correspond to the neutral Log P, in consideration of the sample pKa.The Log P of 5 was investigated by the shake-flask methodology with UV-spectroscopic sample detection. To produce a saturated solution of 5, approximately 1 mg of solid material was added to 10 mL of an aqueous solution, which was adjusted to pH 2.3 with 0.5 M HCl. The sample was then sonicated in an ultrasonic bath for several hours (at room temperature) before being left to equilibrate for a period of approximately 3 days. The supernatant was then filtered under vacuum through a 0.2 µm PVDF filter plate, and the UV absorption spectrum of the sample was measured (after adjusting the pH of the solution to 11.8 with 0.5 M KOH). Molar absorption coefficients of 5 were obtained at pH 11.8 for 25 µM, 50 µM, and 100 µM solutions of 5, in order to quantify the concentration of the sample in the saturated supernatant. The UV-absorption signal (0.0039) of the supernatant at the absorption maximum of 5  was close to the detection limit of the apparatus, and the solubility value was determined as an upper limit of <510 nM. Conjugate 5 was completely soluble in ethanol (>30 mg/mL) and the mixture of water/methanol (60:40). However, 5 was less soluble in DMSO (~4.1 mg/mL).Solubility for 50 values.Anti-HIV activities of the compounds were evaluated according to the previously reported procedure . In summ50  was calculated using statistical software and regression analysis. A cellular uptake study was conducted for fluorescence conjugates 23 and 24 using a previously published procedure with minor modifications [For the assessment of compounds against the wild type  and drug-resistant (clones = 4755−5 and A17) HIV-1 clinical isolates, PHA-P stimulated cells from at least two normal donors were pooled, diluted in fresh media, and plated in the interior wells of a 96-well round-bottom microplate. Pooling PBMCs from more than one donor was used to minimize the variability observed between the individual donors, which results from the quantitative and qualitative differences in HIV infection and overall response to the PHA and IL-2 of primary lymphocyte populations. Each plate contained virus/cell control wells (cells + virus), experimental wells (drug + cells + virus), and compound control wells . Test drug dilutions were prepared in microtiter tubes, and each concentration was placed in appropriate wells. Following the addition of the drug dilutions to the PBMCs, a predetermined dilution of virus stock was then placed in each test well . Because HIV-1 is not cytopathic to PBMCs, the same assay plate can be used for both the antiviral efficacy and cytotoxicity measurements. Compounds were incubated with virus and cells in a 96-well format for 6 h. The cells were then washed by removing 75% of the medium 150 μL) and replacing it with 150 μL of fresh (no drug) medium. The plates were then centrifuged (∼200 g) for 10 min, after which 150 μL of the medium was removed, and an additional 150 μL of fresh medium was added to each well and further incubated for 6 days or until peak reverse transcriptase (RT) activity was detected. A microtiter plate-based RT reaction was utilized . Incorpoications ,15,16. A0 μL and Human T lymphoblastoid cells  were grown to 70% confluency in a culture medium. CCRF-CEM cells were incubated with 10 µM of the compounds and mixed with trypan blue (0.1%) to color the dead cells. The percentage of viability was calculated by using a Cellometer Auto T.4 (Nexcelom Bioscience). The cell viability against the vaginal cells (VK-2/E6E7) was conducted according to the previously reported procedure . The effO-fatty acyl derivatives of FLT were designed and synthesized as prodrugs of FLT, a nucleoside reverse transcriptase inhibitor, and their biological activities were evaluated as anti-HIV agents and microbicides. Among all the conjugates, 5, 6, and 8 were found to have the most active anti-HIV activity profile. Compared to AZT and FLT, conjugates 5, 6, and 8 were active against both the cell-free virus (lymphocytotropic and monocytotropic strains) as well as the cell-associated virus. Conjugates 5, 6, and 8 exhibited >4 and >30 times better antiviral index than FLT and AZT, respectively. The increased inhibition by 5, 6, and 8 may be due to their increased rate of uptake, resulting in increased intracellular level of active nucleoside achieved by the conjugate. Hydrolysis of the prodrug to the parent analogs was critical for the generation of anti-HIV activity. Presumably, the intracellular hydrolysis yielded two antiviral agents, FLT and fatty acid analog, with different enzyme targets such as RT and NMT. Conjugates 5 and 8 were active against both the R5 and MDR strains. These conjugates have the potential to be used as topical microbicides to prevent HIV infection during sexual activity. These data provide insights into the more rational design of additional potent and safe anti-HIV microbicides using the FLT or other anti-HIV nucleosides as the parent molecule. When taken together, the results will have significant implications for the design of more potent and innovative anti-HIV agents.Several bifunctional 5′-


These intermediates were reduced to the corresponding 3,7-diazabicyclo[3.3.1]nonane targets. The oxime at C-9 was benzoylated to yield 13. Heating these 3,7-diazabicyclo[3.3.1]nonanes in ethanol with β-CD generated the complexes required. We investigated the ability of such complexes as coatings on seedlings to protect and stimulate growth of three varieties of wheat, namely Kazakhstanskaya-10, Severyanka, and Miras. The complex of 3-[3-(1H-imidazol-1-yl)propyl]-7-(3-methoxypropyl)-3,7-diazabicyclo[3.3.1]nonane (2) promoted growth in the root systems of all three wheat varieties by more than 30% in Kazakhstanskaya-10, 30% in Severyanka and 8.5% in Miras. A complex of 3-Boc-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (9) increased both shoot and root length in only the Severyanka variety. The complex of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (11) stimulated both shoot growth  and root growth  in all three varieties of wheat, respectively. The nature of substituents on the bispidine affect the activity. Solid complexes (1:1) were generated as powders which melted above 240 °C (dec) and were characterized via elemental analyses as 1:1 complexes.We report the syntheses and characterization of novel 3,7-bicycl[3.3.1]bispidines possessing an imidazolpropyl group attached to N-3, and at N-7 a Boc group, as well as a benzoylated-oximated group at C-9. These compounds were complexed with β-cyclodextrin [β-CD] and evaluated as seed protectors of selected wheat seedlings. Using strong acid, condensations of N-substituted piperidones with the appropriate imidazolpropyl groups at N-3 and N-7 led to bispidinones  H-imidazol-1-yl)propyl]piperidine (1-H-imidazol-1-yl)propyl]-7-(3-methoxypropyl)-3,7-diazabicyclo[3.3.1]nonane complex (2) nonan-9-ones 1H-NMR signals of the axial protons of 3,7-diazabicyclo[3.3.1]nonan-9-one (7) at C-2,4 and C-6,8 were multiplets at 2.18–2.33 ppm. The resonance signals of the equatorial protons at C-2,4 and C-6,8 were in the lower-field of the spectrum (2.90–3.01 ppm). In bispidinone (6) with the carboxylate group, signals for axial and equatorial ring protons were at 2.05–2.46 ppm and 3.05–3.51 ppm for H-2,4, and at 3.88–3.95 ppm and 4.24–4.41 ppm for H-6,8.The H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (8) (52%) and 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (10) (40%) were obtained by reduction of the corresponding bicyclic ketones 6 and 7. The absence of the carbonyl IR band in 8 and 10 confirmed that reduction had occurred. The composition and structure of compounds 8 and 10 were further supported from IR, 1H NMR, and 13C-NMR spectral analyses. When comparing the 13C-NMR spectra of compounds 8 and 10 with the spectra of the starting ketones 6 and 7, the appearance of a triplet for H-9 in the 1H-NMR spectrum and the 13C-NMR signals at 35.0 ppm and 35.1 ppm, respectively, for C-9 substantiated the conversion of ketones 6 and 7 to the corresponding 8 and 10 derivatives. Signals for 8 and 10 were visible in the region of 1.53 ppm and 1.88 ppm signals for the H-9 methylene protons, respectively. The signals for H-1,5 protons in 8 and 10 were shifted upfield  in comparison to the initial ketones.3-Boc-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonan-9-one (7) and gave, the corresponding oxime 12 (49%). The appearance in the IR spectrum of bands in the region of 1506 cm−1 and 3200 cm−1 for 12 indicated the formation of an oxime and were assigned, respectively, to the C=N and O-H groups. The most intense band in 13 was the benzoyloxy carbonyl group (1723 cm−1). Oxime 12 was acylated with benzoyl chloride in absolute benzene to give the benzoyloxime 13 (45%). The 13C-NMR spectra of oxime 12 and its ester 13 showed resonances for C-9 at 159.5 ppm and 169.1 ppm, respectively. A second 13C-NMR signal at 166.1 ppm identified the carbonyl carbon for the acyl residue. In the 1H-NMR spectra, H-1.5 protons were observed as separate signals at 2.71 ppm and 2.95 ppm in oxime 12 and at 3.23 ppm and 3.30 ppm for the N-benzoyl derivative. A weak signal at 6.82 ppm was assigned to the proton of the NOH group (12). Signals for the protons of the phenyl ring (13) were observed at 7.49–7.94 ppm.It was reasoned that providing a group at C-9 which had H-bonding capabilities might increase moisture attraction capability in the complexes. Therefore, oximation was initiated with of 3-(3-butoxypropyl)-7-nonane ring  strongly enhanced the bioaction on the growth of wheat seedlings while the shoot growth was slightly inhibited in 2. However, the root length increased. Only example 14 in the 3,7-diazabicyclo[3.3.1]nonanes complex systems involving substitution at C-9 gave an inferior effect on plant development in each variety of wheat. Thus, the nature of the substituent has a dramatic affect for maximum results.The synthetic “assembly” of piperidine and imidazole fragments into one molecule led to specific biologically active substances as growth protectors/regulators of specific wheat seedlings. As can be seen from the test results, the composition of the ring structure of the six-membered azaheterocycle  in complexes with β-CD led to a change in bioactivity , growth was stimulated in both the stem and length of the roots in all three varieties of wheat. Surprisingly, shoot growth in the three wheat varieties was only slightly inhibited by 2 by about 3–5%. Interestingly, experiments revealed that the pre-sowing treatment of seeds with samples 1, 2, 9, and 14 resulted in the plant height being reduced from 0.36% to 24.3%, and the root length decreased from 2.6% to 31.2% . Sample 14 inhibited the growth of the shoot and roots in all three wheat varieties. chnology  and were2 in all three wheat seedlings, experiencing growth of 31–33% (f 31–33% .H-imidazol-1-yl)propan-1-amine (3) and paraformaldehyde with N-substituted piperidones 4 and 5 with N-3-aminopropylimidazole under acidic conditions led to bispidinones 6 and 7 , specific chemical tests, and elemental analyses. The use of β-CDs for encapsulating large molecule is known -3,7-diazabicyclo[3.3.1]nonan-9-one (6). A system composed of a three-necked flask equipped with a stirrer, reflux condenser, and dropping funnel, to which was added 40 mL of methanol that had been deoxygenated with nitrogen. After 30 min, a mixture of 3-(1H-imidazol-1-yl)propan-1-amine , paraformaldehyde , concentrated hydrochloric acid (2.65 mL), and glacial acetic acid (3.77 mL) was added with stirring for 15 min under nitrogen. An addition of 1-Boc-piperidin-4-one  in glacial acetic acid (4 mL) in methanol (15 mL) was added dropwise. After heating the reaction mixture for 10 h at 60–65 °C, a second equivalent of paraformaldehyde was added, and the solution was maintained for 12 h at the same temperature. During the entire reaction, the mixture was purged with nitrogen. The solvent was evaporated, and the residue was dissolved in water (80 mL). Extraction of the neutral products with diethyl ether gave two layers, and the aqueous layer was made alkaline with NaOH. Upon cooling the mixture (at pH 12), the product was extracted with chloroform. The extract was dried (MgSO4), and the solvent was evaporated. The resulting solid was purified by column chromatography over Al2O3 with the eluent being benzene-isopropanol (6:1). The ketone 3-Boc-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonan-9-one  was obtained and possessed an Rf 0.56 , mp 82–85 °C.18H28N4O3: C, 62.05; H, 8.10; N, 16.08.Calculated for CFound: C, 62.07; H, 8.07; N, 16.06−1: 1702 (C=O), 1651 (COO), 1164 (C-O-C).IR spectrum cm1H NMR spectrum, ppm: bicyclononan-9-on: 2.05–2.46 , 3.05–3.51 , 3.88–3.95 , 4.24–4.41 . (1H-imidazol-1-yl)propyl: 6.81–6.83, 7.03–7.08, 7.47–7.54 . Side chains: 1.71–1.78 ; 3.05 3.51 . Boc: 1.22–1.32 .13C NMR spectrum ppm: bicyclononan-9-on: 49.8 , 47.4, 53.7 , 213.7 (C-9). (1H-imidazol-1-yl)propyl: 119.9, 128.7 137.8 (CHimidazol). Side chains: 31.5 (CH2CH2CH), 50.5 (CH2CH2CH2). Boc: 28.5 (CH3), 79.5 (O-C), 154.6 (C=O).3-(3-Butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonan-9-one (7). In a three-necked flask equipped with a stirrer, reflux condenser, and addition funnel, was placed 40 mL of methanol which was then deoxygenated under nitrogen. After 30 min, a mixture was generated by adding 3-(1H-imidazol-1-yl)propan-1-amine , paraformaldehyde , concentrated hydrochloric acid (2.7 mL), and glacial acetic acid (4 mL). This mixture was stirred for 15 min under nitrogen. A solution of 1-(3-butoxypropyl)piperid-4-one  in glacial acetic acid (4 mL) and methanol (17 mL) was added dropwise to the original solution. After heating the reaction mixture for 10 h at 60–65 °C, a second equivalent of paraformaldehyde was added, and the resulting mixture was held at that temperature for another 10 h under nitrogen. The solvent was then evaporated, and the residue was dissolved in water (82 mL). Extraction of the mixture with diethyl ether gave two layers. The aqueous layer was made alkaline with NaOH. Upon cooling the mixture (at pH 12), a product was extracted with chloroform, and the extract was dried (MgSO4). Evaporation of the solvent gave a material which was purified by column chromatography over Al2O3 (eluent-benzene:isopropanol. = 6:1). The ketone 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane-9-one (7) was obtained as an oil in a yield of 12.8 g (68%). The compound had an Rf 0.56  and an nD20 1.5043.20H34N4O2: C, 66.26; H, 9.45; N, 15.46.Calculated for CFound: C, 66.28; H, 9.42; N, 15.45.−1: 1734 (C=O), 1110 (C-O-C).IR spectrum cm1H NMR spectrum, ppm: bicyclononan-9-on: 2.90 , 2.18–2.33 , 2.90–3.01 . (1H-imidazol-1-yl)propyl: 1.56 , 3.27, 3.89–4.04 ; 6.80–6.84, 7.10, 7.44–7.45 ,butoxypropyl: 0.84 , 1.41–1.65 , 2.66, 3.30–3.41 , CH2OCH2CH2CH2CH3).13C NMR ppm: bicyclononan-9-on: 58.4 , 66.9–68.6 , 66.9–68.6 , 214.4 (C-9). (11-yl)propyl:H-imidazol- 28.5 (CH2CH2CH2),53.1 (CH2CH2CH2), 46.6 (CH2CH2CH2), 119.9, 131.3, 137.8 (CHimidazol), butoxypropyl: 14.3 (OCH2CH2CH2CH3), 19.4 (OCH2CH2CH2CH3), 27.7 (NCH2CH2CH2O), 31.9 (OCH2CH2CH2CH3), 53.6 (NCH2CH2CH2O), 63.0 .3-Boc-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (8). To a mixture of 3-Boc-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonan-9-one  and hydrazine hydrate  in triethylene glycol (33.5 mL) at 65–70 °C, was added KOH . The reaction mixture was heated to 160–170 °C and stirred at this temperature for 4 h. Water and excess hydrazine were distilled off at a temperature of 190–200 °C. After cooling the reaction mixture to room temperature, distilled water (56.6 mL) was added, and the mixture was extracted with diethyl ether. The extract was dried (MgSO4), and the solvent was evaporated. The resulting solid was purified by column chromatography over Al2O3 (eluent-benzene:isopropanol-7:1). The 3-Boc-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyc-lo[3.3.1]nonane (8) was obtained  in the form of an oil with an Rf 0.22 ; nD20 1.4680.18H30N4O2: C, 64.64; H, 9.04; N, 16.75.Calculated for CFound: C, 64.67; H, 9.01; N, 16.77.−1: 1112 (C-O-C).IR spectrum cm1H NMR spectrum, ppm: bicyclononane: 1.32 , 1.53 , 2.16, 3.98–4.05 , 2.57–2.58, 4.56–4.67 . (1H-imidazol-1-yl)propyl: 1.55–1.60 , 3.05–3.17, 3.85 , 6.96, 7.03, 7.49 . Boc: 1.48 .13C NMR spectrum, ppm: bicyclononane: 31.2 , 35.0 (C-9), 48.8, 55.3 . (1H-imidazol-1-yl)propyl:29.4 (CH2CH2CH2), 50.1 (CH2CH2CH2), 47.7 (CH2CH2CH2), 120.5, 133.9, 136.3 (CHimidazol). Boc: 28.3 (CH3), 80.2 (O-C); 160.8 (C=O).Complex 3-Boc-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (9). A solution was made of 3-Boc-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane  in ethyl alcohol (25 mL) and β-CD  in distilled water (40 mL). The solution was heated 8 h, and ethanol and water were evaporated at 50–55 °C. The residue 3-Boc-7-[3-(1H-imidazol-1-yl)-propyl]-3,7-diazabicyclo[3.3.1] nonane with β-CD (9)  was formed as a white powder.60H100N4O37: C, 49.04; H, 6.81; N, 3.81.Calculated for CFound: C, 49.01; H, 6.79; N, 3.84.3-(3-Butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (10).To a mixture of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]-nonane-9-one  and hydrazine hydrate  in triethylene glycol (40 mL) at 60 °C was added KOH . The reaction mixture was heated to 160–170 °C and stirred at this temperature for 4 h. Water and excess hydrazine were distilled off at a temperature of 190–200 °C. After cooling the reaction mixture to room temperature, 68 mL of distilled water was added. The product was extracted with diethyl ether, and the extract was dried (MgSO4). Evaporation of the solvent gave a product which was purified by column chromatography on Al2O3 (eluent-benzene:isopropanol = 7:1). 3-(3-Butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (10) was obtained as a yellow oil . The compound  was isolated and had anD20 1.5094.20H36N4O: C, 68.92; H, 10.41; N, 16.08.Calculated for CFound: C, 68.90; H, 10.38; N, 16.06.−1: 1115 (C-O-C).IR spectrum, cm1H NMR spectrum, ppm: bicyclononan-9-on: 1.88 , 1.89 ,2.18–2.25 , 2.57–2.72 . (1H-imidazol-1-yl)propyl,1.58 , 3.11–3.16, 3.87 , 6.86, 7.14, 7.61 , butoxypropyl:0.86 , 1.44–1.71 , 2.46, 3.36–3.38 .13C NMR spectrum, ppm: bicyclononane: 27.7 , 35.1 (C-9), 60.7–62.7 . (1H-imidazol-1-yl)propyl:30.6. (CH2CH2CH2), 44.0 (CH2CH2CH2), 51.3 (CH2CH2CH2), 120.1, 129, 137.8. (CHimidazol),butoxypropyl: 14.3 (OCH2CH2CH2CH3), 19.4 (OCH2CH2CH2CH3), 27.8 (NCH2CH2CH2O), 31.8 (OCH2CH2CH2CH3). 52.3 (NCH2CH2CH2O), 66.9 (CH2CH2CH2O), 70.1 (OCH2CH2CH2CH3).Complex of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo [3.3.1]nonane (11). Solutions of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane  in ethyl alcohol (25 mL) and β-CD  in 40 mL of distilled water were mixed. The mixture was placed in an oven where ethanol and water were evaporated at 50–55 °C. The inclusion complex 11 of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane with β-CD was obtained in the form of a white-brown powder .62H106N4O36: C, 50.19; H, 7.15; N, 3.78.Calculated for CFound: C, 50.17; H, 7.17; N, 3.80.Oxime of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]-nonan-9-one (12). To a three-necked flask equipped with a mechanical stirrer, a reflux condenser with a calcium chloride tube, and a dropping funnel, was placed 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonan-9-one  in ethyl alcohol (97.5 mL) and pyridine (1.96 g 25 mmol). Stirring was initiated, and hydroxylamine hydrochloric acid  was added. The reaction mixture was heated at 110–120 °C for 24 h. Evaporation of the solvent left a residue which was dissolved in water (30 mL), and the mixture was made alkaline with NaOH to pH 12. Extraction (HCCl3) of the aqueous solution gave a solution which was dried (MgSO4). Evaporation of the solvent gave a residue which was purified via column chromatography on Al2O3 (eluent-benzene:isopropanol = 20:1). Oxime 12 of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]-nonane-9-one was obtained as an oil . Supporting date are: Rf 0.20 ; nD20 1.4910.20H35N5O2: C, 63.63; H, 9.34; N, 18.55.Calculated for CFound: C, 63.66; H, 9.31; N, 18.57.−1: 1506 (C=N), 3200 (O-H).IR spectrum, cm1H NMR spectrum, ppm: bicyclononan-9-ketoxime: 2.23–2.46 , 2.71–2.95 , 2.71–2.95 , 6.82 . (1H-imidazol-1-yl)propyl: 1.53–1.60 , 3.23–3.30, 3.90–4.01 , 6.82, 7.11, 7.57 , butoxypropyl: 0.78–0.83 , 1.24–1.26, 1.38–1.40 , 2.23–2.46, 3.48–3.52 , OCH2CH2CH2CH3).13C NMR spectrum, ppm: bicyclononan-9-ketoxime:34.8 (C-5), 58.4 , 57.1 , 159.5 (C-9). (1H-imidazol-1-yl)propyl: 30.5 (CH2CH2CH2), 50.9 (CH2CH2CH2), 46.9 (CH2CH2CH2), 119.9, 128.6,138.0 (CHimidazol),butoxypropyl: 14.3 (OCH2CH2CH2CH3), 27.6 (NCH2CH2CH2O), 31.9 (OCH2CH2CH2CH3), 51.5 (NCH2CH2CH2O), 68.8 (NCH2CH2CH2O), 70.2 (OCH2CH2CH2CH3).O-Benzoyloxime of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonan-9-one (13). A mixture of oxime of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane-9-one  in absolute benzene (42.4 mL) and benzoyl chloride  was stirred for 7 h at 80 °C. The solvent was distilled off, and the residue was treated with aqueous potash. Extraction (HCCl3) of the product gave a solution which was dried (MgSO4). Evaporation of the solvent left a residue which was purified by column chromatography on Al2O3 [eluent-benzene:isopropanol = 7:1] and gave the O-benzoyloxime of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7- diazabicyclo[3.3.1]nonane-9-one (13)  as an oil. Supporting data included an Rf 0.34  and nD20 0.724.27H39N5O3: C, 67.33; H, 8.16; N, 14.54.Calculated for CFound: C, 67.34; H, 8.13; N, 14.52.−1: 1723 (C=O).IR spectrum cm1H NMR spectrum, ppm: ester of bicyclononan-9-ketoxime: 2.26–2.46 , 3.23–3.29 , 3.23–3.29 , 7.49–7.94 . (1H-imidazol-1-yl)propyl: 1.55–161 , 3.67–3.81, 4.25–4.38 , 7.35–7.36, 7.47–7.51 ,butoxypropyl: 0.78–0.81 , 1.20–1.26, 1.38 , 1.55–1.61, 3.67–3.81 .13C NMR spectrum, ppm, ester of bicyclononan-9-ketoxime: 34.5, 35.8 , 58.3–58.6, 61.3 , 169.1 (C-9). (1H-imidazol-1-yl)propyl: 31.8 (CH2CH2CH2), 46.4 (CH2CH2CH2), 51.3 (CH2CH2CH2), 119.9,134.0, 137.8 (CHimidazol),butoxypropyl: 14.3 (OCH2CH2CH2CH3), 19.4 (OCH2CH2CH2CH3), 27.4 (CH2CH2CH2), 31.8 (OCH2CH2CH2CH3), 54.9 (NCH2CH2CH2); 68.7 (CH2CH2CH2O), 70.1 (OCH2CH2CH2CH3), phenyl: 129.3–130.0 (CH), 166.1 (C=O).-one (14)Complex of O-Benzoyloxime of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonan-9O-benzoyloxime  in ethyl alcohol (25 mL) and β-CD  in distilled water (30 mL) were mixed. The mixture was placed in an oven where ethanol and water were evaporated at 50–55 °C. The inclusion complex of 13 with β-CD was obtained as a white powder 14 .To obtain the inclusion complex, solutions of the 69H109N5O38: C, 51.27; H, 6.75; N, 4.33.Calculated for CFound: C, 51.24; H, 6.72; N, 4.36.Triticum aestivum with the varieties Kazakhstanskaya-10, Severyanka and Miras. Severyanka and Miras are drought tolerant varieties, and Kazakhstanskaya-10 was studied as a standard. Presowing treatment of wheat with aqueous solutions of samples at a concentration of 0.01% was carried out in Petri dishes during germination of the seeds for 2 days. The seedlings were then transferred to small floating rafts for germination in hydroponics up to 10 days. Then biometric parameters were taken in comparison with the control.The studies were performed on model samples of spring wheat 1, 2, 9, 11 was studied in different series of experiments; therefore, Water was used as the basis for the germination of the crops. Experimental samples were taken in 15 replicates for each culture and the tested growth regulator. As a control option, tap water was used and the seedlings were allowed to stand for three days. The experiment was conducted over 10 days. For each sample, the degree (%) of stimulation or inhibition was calculated compared to the control series in all experiments  the length of the roots increased in all three wheat seedlings, but markedly so with Kazakhstanskaya-10 and Severyanka, with increases of 31–33% , with varieties and treatments as main effects of shoots and roots length . Sample 14 inhibited the growth of the shoot and roots in all three wheat varieties. In It is clear that the protector/regulator ability of these 3,7-diazabicyclo[3.3.1]nonane systems is very sensitive to the substituents, particularly on C-9 bridge. For example, with 11 , growth was stimulated in both the stem and length of the roots in all three varieties of wheat. Surprisingly, shoot growth in the three wheat varieties was only slightly inhibited by 2 by about 3–5%. Interestingly, experiments revealed that the pre-sowing treatment of seeds with samples


The structures of these derivatives were elucidated by 1H NMR, 13C NMR, infrared and high-resolution mass spectrometry for further validation of the target compound structures. The anticancer activities of the new molecules were evaluated against five human cancer cell lines, including A-549, Hs-683, MCF-7, SK-MEL-28 and B16-F10 cell lines using 5-fluorouracil and etoposide as the reference drugs. Among the tested compounds, 4e and 4f exhibited excellent activities in the same range of the positive controls, 5-fluorouracil and etoposide, against MCF-7 and SK-MEL-28 cancer cell lines, with IC50 values ranging from 1 to 10 μM. The molecular docking studies of 4e and 4f showed a strong binding with some kinases, which are linked to MCF-7 and SK-MEL-28 cancer cell lines.In the present study, new 2-phenyl-5,6,7,8-tetrahydroimidazo [1,2- Cancer is a complex disease characterized by uncontrolled division and proliferation by blocking the process of normal cell division . It is cHeterocycles are an important class of chemical compounds present in a wide variety of drugs, vitamins, natural products and biomolecules, as well as biologically active molecules ,5,6,7. AThe heterocyclic systems bearing sulfonamide groups have had an important place in medicinal chemistry since Gerhard Domagk discovered their antibacterial activity in 1935 . This diProtein kinases are a large family of enzymes (518 in human genome) having a crucial rule of catalyzing protein phosphorylation, which is an important mechanism of cells function, such as proliferation, cell cycle, apoptosis, motility, growth and differentiation. Thus, deregulated kinase functions is often linked to cancer spread and development .The computational docking has been used as a powerful strategy for understanding and predicting the molecular interaction of ligands with various biological receptors, such as protein active sites. This interesting protein–ligand interaction can guide the design of molecules and experiments, providing a large set of candidates in medicinal applications. b]pyridazine derivatives bearing sulfonamides as antiproliferative molecules against five human cancer cell lines, namely A-549 lung cancer, Hs-683 glioma, MCF-7 breast cancer, SKMEL-28 and B16-F10 melanoma cell lines. In order to predict the mechanism of action of the most active compounds, molecular docking studies were also carried out.In continuation of our research interest in the design and discovery of new anticancer agents ,32,33,34b]pyridazine 3 was synthesized in two steps, starting with a condensation reaction between 3-amino-6-chloropyridazine 1 and 2-bromoacetophenone according to the reported method [b]pyridazine 2 in a very good yield (89%) [2 was then involved in a reduction reaction using eight equivalents of sodium borohydride in ethanol at 50 °C to give product 3 in excellent yield (93%).As shown in d method  to obtaild (89%) . Compounb]pyridazine 3 by sulfonyl moieties, we first studied the sulfonylation reaction in the presence of different bases in order to increase its yield using the model reaction between tosyl chloride and 3 (3N) in CH2Cl2. The reaction was stopped after 48 h but the conversion was not complete in leading us to the desired product 4a in acceptable yield (68%)  as a base but we have observed a noticeable drop in the reaction yield to 50% , HS-683 (glioma cancer), MCF-7 (breast carcinoma), and SK-MEL-28 and B16-F1 (melanoma cancer), using MTT  assay. The reference drugs were selected to be 5-Fluorouracil (5-FU) and etoposide. The results listed in 50 value, defined as the concentration, inducing a 50% decrease in cell growth after 3 days of incubation), show that the compounds 4e and 4f display high inhibitory activities, similar to those obtained with the positive controls 5-FU and etoposide, against cancer cell lines MCF-7 and SK-MEL-28 with IC50 values between 9 and 7.8 µΜ, respectively. Moreover, a moderate activity was obtained for compound 4f against cancer cell lines SK- B16-F1 with IC50 value of 10.8 μM. Compounds 4a, 4c, 4d and 4g demonstrated low activity against cancer cell lines A549, Hs-683, MCF-7 and B16-F1 with IC50 values between 10 and 100 μM. Unfortunately, compound 4b has no activity against the cell line panels tested (IC50 > 100 μM).The synthesized compounds 4a–g toward cancer cell lines have shown that the anticancer activity in compound 4e against the MCF-7 cell line, increased when electron-donating groups, OMe and Cl, are present at ortho and meta positions of the phenyl moiety, respectively. In addition, the presence of an electron withdrawing groups CF3 attached to the benzene ring at the para position, increased the anticancer activity of compound 4f against SK-MEL-28 cell line. However, the results have revealed that the presence of OMe (an electron-donating group) at the para position of the phenyl moiety did not show any inhibitory activity on the cancer cell lines at low concentration. Meanwhile, the membrane–water partition coefficient (cLog P) was calculated using ChemBioDraw Ultra v.12. The compounds 4a–g have shown clog P between 2.9 and 4.4. These values correlate with those needed to develop drug-like compounds −2 < LogP ≤ 5 according to Lipinski’s rule ) were carried out by open capillary tubes and are uncorrected using Thermo scientific digital melting point IA9200. The NMR spectra were recorded on a Bruker AC 300 or 400 MHz instruments at room temperature, using TMS as the internal standard and chloroform-d as a solvent. Chemical shifts (δ) are measured in parts per million (ppm). Infrared spectra were recorded on a Thermo Scientific, Nicolet IS50 FT-IR. High-resolution mass spectra (HRMS) was performed on a Maxis Bruker 4G.2).6-Chloro-2-phenylimidazo pyridazine  was prepared as described in the literature pyridazine ( To a solution of compound (2)  in ethanol (20 mL), sodium borohydride  was added and the mixture was stirred at 50 °C for 48 h. The reaction was quenched with drops of water, then the solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2 (20 mL), washed three times with water and the organic phase was separated, dried over magnesium sulfate and evaporated to dryness to afford compound (3) as a white solid (yield 93%). Mp: 118–119 °C. 1H NMR : δ 7.70 , 7.35 , 7.21 , 7.08 , 3.24–3.20 , 2.96–2.88 , 1.94–1.86 . 13C NMR : δ 139.9, 137.3, 134.4, 128.6 (2*CH), 126.6, 124.5 (2*CH), 113.1, 45.0, 22.3, 22.2. IR: ν (cm−1) = 3165 (N–H), 2954(C–H), 1600 (C=C), 1443 (C=N), 1070 (C–N), 755 (C–C). HRMS (ESI): m/z calcd for C12H14N3 [M + H]+ 200.1182; found: 200.1182.4a–g).General procedure for the synthesis of compounds ((3)  was dissolved in CH2Cl2 (6 mL), then pyridine  was added. After 5 min of stirring, sulfonyl chloride (0.55 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. After evaporation, the medium was extracted with CH2Cl2, and the organic phase was washed with water (3 × 10 mL), dried over anhydrous MgSO4 and then concentrated under reduced pressure. The obtained products were purified by silica gel chromatography to give 4a–g as a solid. Compound 4a).5-((4-Methyphenyl)sulfonyl)-2-phenyl-5,6,7,8-tetrahydroimidazo pyridazine ( Compound (4a) was prepared according to the general procedure; the desired product was obtained after chromatography on silica gel (eluent: DCM/EtOAc 7/3) as a pale yellow solid (86%). Mp: 143–144 °C. 1H NMR : δ 7.78 , 7.55 , 7.48–7.36 , 7.33–7.27 , 3.99–3.92 , 2.65 , 2.43 , 1.60 . 13C NMR : δ 146.1, 140.6, 137.3, 132.5, 130.4 (2*CH), 128.7 (2*CH), 128.1 (2*CH), 127.5, 125.2, 124.9 (2*CH), 114.7, 47.5, 21.7, 20.6, 16.8. IR: ν (cm−1) = 2358 (C–H), 1362 , 1166 , 1067 (C–N), 998 (C–C). HRMS (ESI): m/z calcd for C19H20N3O2S [M + H]+ 354.1272; found: 354.1270.4b).5-((4-Methoxyphenyl)sulfonyl)-2-phenyl-5,6,7,8-tetrahydroimidazo pyridazine ( Compound (4b) was prepared according to the general procedure; the desired product was obtained after chromatography on silica gel (eluent: DCM/EtOAc 6/4) as a brown solid (80%). Mp: 142–143 °C. 1H NMR : δ 7.80–7.72 , 7.53 , 7.50–7.44 , 7.39 , 7.37–7.27 , 6.95–6.91 , 3.97–3.91 , 3.85 , 2.61 , 1.68–1.57 . 13C NMR : δ 164.4, 140.4, 138.1, 133.4, 130.4 (2*CH), 128.6 (2*CH), 127.1, 126.9, 124.7 (2*CH), 114.8 (2*CH), 114.7, 55.7, 47.5, 20.9, 17.1. IR: ν (cm−1) = 2952 (C–H), 1592 (C=C), 1362 , 1160 , 1070 (C–O). HRMS (ESI): m/z calcd for C19H20N3O3S [M + H]+ 370.1226; found: 370.1219.4c).5-sulfonyl)-2-phenyl-5,6,7,8-tetrahydroimidazo pyridazine ( Compound (4c) was prepared according to the general procedure; the desired product was obtained after chromatography on silica gel (eluent: DCM/EtOAc 6/4) as a brown solid (70%). Mp: 132–133 °C. 1H NMR : δ 7.78–7.71 , 7.59 , 7.41–7.34 , 7.27 , 7.26–7.23 , 6.92 , 6.75 , 3.99–3.94 , 3.93 , 3.66 , 2.65–2.58 , 1.65–1.55 . 13C NMR : δ 154.1, 149.4, 140.5, 138.0, 133.2, 128.6 (2*CH), 127.2, 126.8, 124.7 (2*CH), 122.1, 114.7, 110.9, 109.7, 56.2, 56.2, 47.6, 21.0, 16.9. IR ν (cm−1) = 2359 (C-H), 1585 (C=C), 1508 (C=C), 1361 , 1263 (C–O), 1159 , 1013 (C–N). HRMS (ESI): m/z calcd for C20H22N3O4S [M + H]+ 400.1324; found: 400.1325.4d).5-((4-Nitrophenyl)sulfonyl)-2-phenyl-5,6,7,8-tetrahydroimidazo  pyridazine ( Compound (4d) was prepared according to the general procedure; the desired product was obtained after chromatography on silica gel (eluent: DCM/EtOAc 6/4) as a brown solid (75%). Mp: 218–219 °C. 1H NMR : δ 8.37–8.29 , 7.80–7.73 , 7.55 , 7.43–7.37 , 7.32–7.27 , 4.06–3.98 , 2.65 , 1.67–1.54 . 13C NMR : δ 151.0, 141.7, 140.0, 138.8, 133.0, 129.5 (2*CH), 128.7 (2*CH), 127.4, 124.8 (2*CH), 124.7 (2*CH), 114.1, 47.9, 21.0, 17.6. IR: ν (cm−1) = 2359 (C–H), 1529 (C=C), 1346 , 1168 , 1066 (N–O), 1002 (C–C). HRMS (ESI): m/z calcd for C18H17N4O4S [M + H]+ 385.0973; found: 385.0965.4e).5-((5-Chloro-2-methoxyphenyl)sulfonyl)-2-phenyl-5,6,7,8-tetrahydroimidazo pyridazine ( Compound (4e) was prepared according to the general procedure; the desired product was obtained after chromatography on silica gel (eluent: DCM/EtOAc 7/3) as a brown solid (65%). Mp: 135–136 °C. 1H NMR : δ 7.94 , 7.79–7.72 , 7.59–7.51 , 7.38 , 7.29–7.21 , 6.88 , 4.00–3.93 , 3.51 , 2.82 , 1.76–1.66 .13C NMR : δ 156.4, 139.9, 137.4, 136.0, 133.7, 131.3, 128.6 (2*CH), 126.9, 126.9, 125.8, 124.7 (2*CH), 115.1, 113.9, 56.7, 47.6, 21.2, 17.7. IR: ν (cm−1) = 2939 (C–H), 1479 (C=C), 1277 , 1159 , 1004 (C–C). HRMS (ESI): m/z calcd for C19H19ClN3O3S [M + H]+ 404.0828; found: 404.0830.4f)5-((4-(Trifluoromethyl)phenyl)sulfonyl)-2-phenyl-5,6,7,8-tetrahydroimidazo pyridazine (. Compound (4f) was prepared according to the general procedure; the desired product was obtained after chromatography on silica gel (eluent: DCM/EtOAc 7/3) as a white solid (78%). Mp: 157–158 °C. 1H NMR : δ 7.77 , 7.70 , 7.55 , 7.40 , 7.29 , 4.07–3.89 , 2.62 , 1.71–1.52 . 13C NMR : δ 140.2, 139.5, 138.5, 136.1 , 133.0, 128.7, 128.6, 127.4 (2*CH), 126.8 , 124.8 (2*CH), 122.8 , 114.2 (2*CH), 47.8, 20.9, 17.5. 19F NMR : δ −63.2 . IR: ν (cm−1): 3167 (C–H), 1612 (C=C), 1372 , 1324 , 1061 (C–N). HRMS (ESI): m/z calcd for C19H17F3N3O2S [M + H]+ 408.0989; found: 408.0988.4g).5--2-phenyl-5,6,7,8-tetrahydroimidazo pyridazine ( Compound (4g) was prepared according to the general procedure; the desired product was obtained after chromatography on silica gel (eluent: DCM/EtOAc 7/3) as a white solid (60%). Mp: 79–80 °C. 1H NMR : δ 8.34 , 8.20 , 7.96 , 7.64–7.52 , 7.37–7.30 , 7.23 , 6.89 , 4.07–3.95 , 2.63 , 1.79–1.66 . 13C NMR : δ 141.1, 138.1, 136.2, 134.2, 133.5, 132.2, 131.7, 129.0, 128.9, 128.7, 128.5 (2*CH), 127.5, 127.0, 124.8 (2*CH), 124.3 (2*CH), 114.1, 47.1, 20.7, 18.1. IR: ν (cm−1): 1609 (C=C), 1505 (C=C), 1368 , 1166 . HRMS (ESI): m/z calcd for C22H20N3O2S [M + H]+ 390.1277; found: 390.1270.4 cells/mL suspension) in 96-well culture plates  and incubated for 24 h. Each compound  was assessed in serial dilution (four concentrations in 0.1% DMSO at the highest concentration) in three replicates (n = 3) and incubated for 72 h. Thereafter, MTT (5 mg/mL solution in PBS) was added to each well (10% v/v) and cells were further incubated for 4 h. Then, after removing the culture medium, the blue crystals were dissolved in 100 µL 100% DMSO and absorbance measured at 540 nm using a 620 nm reference. Absorbance of the serial dilution of each cell line treated under the same conditions, but without the tested compounds, was measured to generate a standard curve, allowing IC50 determination (IC50 is defined as the concentration reducing cell growth by 50%).The growth level of four cancer cell lines was determined using a colorimetric MTT  assay. Cancer cell lines and growth medium were obtained from CLS Cell Line Service GmbH, Eppelheim, Germany). Human skin melanoma SK-MEL-28, mouse melanoma skin B16-F1 and human brain glioma HS683 were grown in DMEM, supplemented with 4.5 g/L glucose, 2 mM L-glutamine and 10% FBS. The human lung carcinoma cell line A-549 was grown in DMEM: Ham’s F12 (1:1) supplemented with 2 mM L-glutamine and 5% FBS and human breast adenoma carcinoma MCF-7 in EMEM supplemented with 2 mM L-glutamine, sodium pyruvate, NEAA, 10 µg/mL insulin human and 10% FBS. MTT assay is based on the reduction of the yellow product thiazolyl blue tetrazolium bromide (MTT) to purple-blue formazan by mitochondrial dehydrogenase of metabolically active cells. The number of living cells after incubation in the presence  of the tested molecule is directly proportional to the blue color, which was measured by spectrophotometry. Briefly, cells were seeded  and converted to PDBQT format using AutoDockTools-1.5.6. Vina executable was run after specifying X, Y and Z coordinates of the macromolecule, as well as the radius value in the configuration file. The resulting conformations were separated using vina split executable and visualized in the Discovery Studio software.The approach starts by retrieving the PDB structure of the candidate kinases from RSCB PDB database. Discovery Studio software (V 21.0.20298) was used to prepare the downloaded molecules by removing water molecules, already existent ligands and adding polar hydrogens. In the next step we used AutoDockTools-1.5.6 to convert PDB structure of the prepared macromolecule to PDBQT format after adding Kollman, Gasteiger charges and AD4 type atoms. The ligand b]pyridazine derivatives bearing sulfonamides were designed and synthesized in a three-step sequence, starting with a condensation reaction between 2-bromoacetophenone and 3-amino-6-chloropyridazine. Then, the obtained products were subjected to a reduction reaction. The final step, the N-sulfonylation reaction, was achieved by using a variety of sulfonyl chlorides. The in vitro cytotoxic potential of these new compounds were screened against A549, HS-683, MCF-7, SK-MEL-28 and B16-F1 cell lines. The compounds 4e and 4f displayed good cytotoxic activities against cancer cell lines, MCF-7 and SK-MEL-28, with IC50 values in low micromolar range. Molecular docking studies of the 4e have showed a strong binding with CSF1R, ErbB2, BRAF and MEK2 kinases. On the other hand, the compound 4f also presents high binding affinities with BRAF, CDK4, KIT, MEK2 and PDGFRA kinases. Importantly, the overexpression of these kinases is linked to MCF-7 and SK-MEL-28 cancer cell line proliferations. The obtained results are very promising and demonstrate that the new compounds 4e and 4f could lead towards anticancer drug development.In conclusion, a new series of 2-phenyl-5,6,7,8-tetrahydroimidazo [1,2-


N-acylhydrazone derivatives have been synthesized as potential topoisomerase I/II inhibitors, and their binding  and biological activities as potential anticancer agents on proliferation of A549 and CCD-18Co have been evaluated. The acridine-DNA complex 3b (-F) displayed the highest bK value (bK = 3.18 × 103 M−1). The HSA-derivatives interactions were studied by fluorescence quenching spectra. This method was used for the calculation of characteristic binding parameters. In the presence of warfarin, the binding constant values were found to decrease , suggesting that derivative 3a could bind to HSA at Sudlow site I. The effect of tested derivatives on metabolic activity of A549 cells evaluated by the 3--2,5-diphenyltetrazolium bromide or MTT assay decreased as follows 3b(-F) > 3a(-H) > 3c(-Cl) > 3d(-Br). The derivatives 3c and 3d in vitro act as potential dual inhibitors of hTopo I and II with a partial effect on the metabolic activity of cancer cells A594. The acridine-benzohydrazides 3a and 3c reduced the clonogenic ability of A549 cells by 72% or 74%, respectively. The general results of the study suggest that the novel compounds show potential for future development as anticancer agents.A series of novel acridine  Shao et al. benzohydrazides 7b–d in 80–90% yields ((Acridine-4-yl)benzohydrazides reaction . The der of lit.  for the 5% yield . To synt% yields .1H, 13C, and 15N NMR spectra for derivatives 3a–e measured in DMSO-d6, only a single set of signals was present -N2 conformation was performed based on the heteronuclear two bond spin–spin coupling constants (2JC1H2 = 10.8 Hz for 3a and 11.4 Hz for 3c) and the NOESY enhancements from 1D NOESY spectra measured in DMSO-d6 for derivative 3a benzohydrazides 7b–d can be attributed to the existence of EN-N/ZN-N conformers. The hydrogen bond N10′···H2 produce a low-field shift of the H2 lines to 15.00 ppm and an up-field shift of the N10′ ···H4, which also stabilizes the ZN-N form of derivative 7 , but a small preference for the ZN-N conformer was observed for derivatives 7b and 7d , 10:5 (7d), The structural characterization of the synthesized compounds was performed using a combination of 1D and 2D NMR techniques. Hydrazones are suitable compounds for examining the stereospecificity of NMR parameters. Their configuration and conformation can be determined by NMR spectroscopy, as evidenced by a number of published scientific articles . It is w present , while iplicated . The geo0–170 Hz . Heteronand 7b–d . The dete 3a see  and 2D Nvative 7 . The sec3a–d and 7b–d) and the assignment of characteristic absorption bands with corresponding wavenumbers (in cm−1) are listed in 3a–d and 7b–d are evident from several stretching vibrations of aromatic C-H (ν(CH)ar) and C=C (ν(C=C)ar) located in the range 3089–3010 cm−1 and 1603–1506 cm−1, respectively. The presence of these components is also confirmed by the scissoring (γ(CCH)ar) and out-of-plane (δ(CCH)ar) vibrations of CCH which occur in the IR spectra of the compounds at about 750 cm−1 and 1020 cm−1. The successful preparation of the hydrazides through a condensation reaction of carbohydrazide and aldehyde derivatives was confirmed by the presence of a stretching vibration of the secondary amine in the range of 3190–3369 cm−1. The wide range of wavelengths observed in these results can be explained by the involvement of the amine group in the hydrogen bond system in solid state. The presence of a hydrazide bond in the case of compounds 3a–d and 7d was also confirmed by the presence of an azomethine vibration (ν(C=N)) at approximately 1620 cm−1  is the quencher concentration. The values of bK and n were determined using the linear regression of a plot of log(F0 − F/F) to log[Q] (Kb and n at the three temperatures are summarized in bK were 105 M−1 (3a–3c) or 104 M−1 (3d), it would therefore be suitable for distribution in the plasma in vivo.In order to evaluate the magnitude of the interaction, other parameters such as the binding constant ( tion (2) :(2)logF0o log[Q] . The val3a, 3b and 3d with HSA are accompanied by a decrease in bK values and n values at increasing temperatures, a trend which indicates the destabilization of the drug-HSA complex under these conditions. In addition, Kb decreased at increasing temperatures, indicating that the interaction process also involves a static quenching mechanism   and tyrosine (Tyr) as chromophore residues of protein . Synchroresidues . The ratuencher] . The resex = λem) is second-order scattering, while peak b (2λex = λem) is Rayleigh scattering. The change at peak 1 (280/339 nm) reflects a change in the polarity of the microenvironment around the Trp and Tyr residues, and peak 2 (230/336 nm) characterizes the polypeptide backbone structure  was used to obtain Stern-Volmer quenching constant values (KSV) and apparent binding constant values (Kb). Previous studies have shown that if a drug has the potential to bind to the same site as the marker, the interaction of this drug with HSA shows an apparent reduction in the Kb value  is the concentration of ctDNA. The binding constant bK was estimated from the intercept-to-slope ratios of A0/(A − A0) vs. 1/[DNA] plots benzohydrazide compounds was that of the fluoro-substituted derivative 3b (bK = 3.18 × 103 M−1), a result which is in accordance with other recent studies. Accordingly, the K values obtained for acridine derivatives 3a–3d were in the range 1.01–3.18 × 103 M−1, since previous research has reported bK values from 104 to 106 M−1 for intercalation complexes, binding constants which are conspicuously smaller than those of groove binders (105–109 M−1) benzohydrazide (3a). Yield 71 mg, 90%; bright yellow solid; mp 235–236 °C; 1H NMR  δ 12.25 , 9.93 , 9.22 , 8.51 , 8.30 , 8.23 , 8.19 , 8.04 , 7.93 , 7.76 , 7.68 , 7.63 , 7.56 . 13C NMR  δ 163.1 (C-4), 147.9 (C-10′a), 146.2 (C-4′a), 144.7 (C-1), 137.1 (C-9′), 133.4 (C-1″), 131.8 (C-4″), 131.2 (C-6′), 131.0 (C-4′), 130.5 (C-1′), 128.8 (C-5′), 128.7 (C-8′), 128.4 (C-3″), 127.8 (C-2″), 126.8 (C-3′), 126.2 (C-9′a), 126.2 (C-8′a), 126.2 (C-7′), 125.7 (C-2′). 15N NMR  δ −206.0 (N-3), −83.3 (N-10′), −57.3 (N-2).N′-[(E)-Acridin-4-ylmethylidene]-4-fluorobenzohydrazide (3b). Yield 68 mg, 82%; bright yellow solid; mp 229–230 °C; 1H NMR  δ 12.26 , 9.91 , 9.23 , 8.51 , 8.31 , 8.23 , 8.19 , 8.11 , 7.93 , 7.76 , 7.68 , 7.40 ; 13C NMR  δ 164.2 , 162.1 (C-4), 147.9 (C-10′a), 146.2 (C-4′a), 144.8 (C-1), 137.1 (C-9′), 131.3 (C-6′), 130.9 (C-4′), 130.6 (C-1′), 130.5 , 129.8 (C-1″), 128.8 (C-5′), 128.7 (C-8′), 126.8 (C-3′), 126.2 (C-9′a), 126.2 (C-8′a), 126.2 (C-7′), 125.7 (C-2′), 128.6 ; 15N NMR  δ −206.3 (N-3), −57.6 (N-2).N′-[(E)-Acridin-4-ylmethylidene]-4-chlorobenzohydrazide (3c). Yield 79 mg, 91%; bright yellow solid; mp 266–267 °C; 1H NMR  δ 12.30 , 9.92 , 9.23 , 8.51 , 8.31 , 8.23 , 8.19 , 8.06 , 7.93 , 7.76 , 7.68 , 7.64 ; 13C NMR  δ 162.1 (C-4), 147.9 (C-10′a), 146.2 (C-4′a), 145.0 (C-1), 137.1 (C-9′), 136.6 (C-4″), 132.1 (C-1″), 131.3 (C-6′), 130.9 (C-4′), 130.6 (C-1′), 129.7 (C-2″), 128.8 (C-5′), 128.7 (C-8′), 128.6 (C-3″), 126.8 (C-3′), 126.2 (C-9′a), 126.2 (C-8′a), 126.2 (C-7′), 125.7 (C-2′). 15N NMR  δ −206.0 (N-3), −57.9 (N-2).N′-[(E)-Acridin-4-ylmethylidene]-4-bromobenzohydrazide (3d). Yield 71 mg, 73%; bright yellow solid; mp 269–270 °C; 1H NMR  δ 12.30 , 9.92 , 9.22 , 8.50 , 8.30 , 8.23 , 8.19 , 7.99 , 7.93 , 7.78 , 7.75 , 7.67 ; 13C NMR  δ 162.2 (C-4), 147.9 (C-10′a), 146.2 (C-4′a), 145.1 (C-1), 137.1 (C-9′), 132.4 (C-1″), 131.5 (C-3″), 131.3 (C-6′), 130.9 (C-4′), 130.6 (C-1′), 129.9 (C-2″), 128.8 (C-5′), 128.7 (C-8′), 126.8 (C-3′), 126.2 (C-9′a), 126.2 (C-8′a), 126.2 (C-7′), 125.7 (C-2′), 125.6 (C-4″); 15N NMR  δ −206.0 (N-3), −58.0 (N-2).N′-[(E)-Acridin-4-ylmetylidene]-4-methoxybenzohydrazide (3e). Yield 70 mg, 82%; bright yellow solid; mp 249–250 °C; 1H NMR  δ 12.12 , 9.90 , 9.22 , 8.49 , 8.29 , 8.23 , 8.20 , 8.03 , 7.93 , 7.75 , 7.68 , 7.09 , 3.86 ; 13C NMR  δ 162.5 (C-4), 162.0 (C-4″), 147.9 (C-10′a), 146.2 (C-4′a), 144.0 (C-1), 137.1 (C-9′), 131.2 (C-6′), 131.1 (C-4′), 130.4 (C-1′), 129.7 (C-2″), 128.8 (C-5′), 128.7 (C-8′), 126.6 (C-3′), 126.2 (C-9′a), 126.2 (C-8′a), 126.2 (C-7′), 125.7 (C-2′), 125.4 (C-1″), 113.7 (C-3″), 55.4 (CH3O); 15N NMR  δ −207.3 (N-3), −83.3 (N-10′), −56.6 (N-2).2S2O3 × 5 H2O . The resulting yellow precipitate was filtered off and washed with cold water (2–10 mL). The crude product 4 was crystallized from methanol.Methanolic solutions (each 8 mL) of iodine  and KOH  were added alternately at 0 °C to a solution of aldehyde 2  in absolute methanol (8 mL) cooled to 0 °C. The reaction mixture was stirred for 2 h at 0 °C, and then for 4 h at room temperature. The reaction mixture was poured into an aqueous solution of Na4, 330 mg, 1.39 mmol) in ethanol (3 mL). The reaction mixture was refluxed for 3 h. Subsequently, a further quantity of hydrazine monohydrate  was added. The reaction mixture was refluxed until the methyl acridine-4-carboxylate  was fully consumed. The reaction mixture was cooled, and the precipitate was filtered off and washed with cold absolute ethanol. The filtrate was concentrated to produce another portion of product 5. The crude product 5 was crystallized from ethanol.Hydrazine monohydrate  was added to a stirred suspension of methyl acridine-4-carboxylate  δ 12.12 , 9.36 , 8.72 , 8.39 , 8.30 , 8.26 , 7.99 , 7.77 , 7.73 , 4.91 . 13C NMR  δ 164.0 (C-1), 147.2 (C-10′a), 145.2 (C-4′a), 138.8 (C-9′), 134.2 (C-3′), 132.7 (C-1′), 132.1 (C-6′), 128.6 (C-8′), 128.5 (C-5′), 127.9 (C-4′), 126.6 (C-7′), 126.5 (C-9′a), 125.8 (C-4′a), 125.3 (C-2′).Yield 515 mg, 75%; brown solid; mp 225–226 °C; 6 (0.422 mmol) was added to a stirred suspension of acridine-4-carbohydrazide  in ethanol (2 mL). The reaction mixture was refluxed until the benzaldehyde 6  was fully consumed. The reaction mixture was cooled, and the precipitate was filtered off and washed with cold absolute ethanol. The crude product was crystallized from methanol to give carbohydrazide 7.Benzaldehyde N′-[(E)-4-Fluorophenylmethylidene]acridine-4-carbohydrazide (7b). Yield 130 mg, 90%; bright yellow solid; mp 255–256 °C. Ratio of isomers: ZN-N: EN-N = 1.0: 0.65 (60.6%: 39.4%). Isomer ZN-N: 1H NMR  δ 14.98 , 9.32 , 9.00 , 8.88 , 8.49 , 8.44 , 8.27 , 8.02 , 7.99 , 7.82 , 7.75 , 7.27 ; 13C NMR  δ 164.68 (C-4″), 162.62 (C-1), 148.54 (C-10′a), 147.73 (C-4), 146.68 (C-4′a), 139.95 (C-9′), 136.81 (C-3′), 134.40 (C-1′), 132.94 (C-6′), 132.74 (C-1″), 130.34 , 129.63 (C-5′), 129.45 (C-8′), 128.92 (C-4′), 127.98 (C-9′a), 127.61 (C-7′), 127.23 (C-8′a), 126.36 (C-2′), 116.54 ; 15N NMR  δ −200.56 (N-2), −95.88 (N-10′), −60.19 (N-3). Isomer EN-N: 1H NMR : δ 14.98 , 9.32 , 9.00 , 8.86 , 8.49 , 8.44 , 8.27 , 8.02 , 7.99 , 7.82 , 7.75 , 7.27 ; 13C NMR  δ 164.68 (C-4″), 162.52 (C-1), 148.50 (C-10′a), 147.71 (C-4), 146.68 (C-4′a), 139.98 (C-9′), 136.81 (C-3′), 134.37 (C-1′), 132.96 (C-6′), 132.74 (C-1″), 130.34 , 129.59 (C-5′), 129.45 (C-8′), 128.92 (C-4′), 127.98 (C-9′a), 127.61 (C-7′), 127.23 (C-8′a), 126.37 (C-2′), 116.54 ; 15N NMR  δ −199.45 (N-2), −95.88 (N-10′), −59.69 (N-3).N′-[(E)-4-Chlorophenylmethylidene]acridine-4-carbohydrazide (7c). Yield 121 mg, 80%; bright yellow solid; mp 243–244 °C. Ratio of isomers ZN-N: EN-N = 1.0: 1.21 (45.2%: 54.8%). Isomer ZN-N: 1H NMR  δ 15.00 , 9.28 , 8.98 , 8.85 , 8.46 , 8.41 , 8.24 , 7.99 , 7.94 , 7.80 , 7.73 , 7.52 ; 13C NMR  δ 162.59 (C-1), 148.49 (C-10′a), 147.56 (C-4), 146.61 (C-4′a), 139.96 (C-9′), 136.83 (C-3′), 136.01 (C-4″), 135.09 (C-1″), 134.45 (C-1′), 132.93 (C-6′), 129.75 , 129.56 (C-5′), 129.43 (C-8′), 128.78 (C-4′), 127.93 (C-9′a), 127.59 (C-7′), 127.20 (C-8′a), 126.32 (C-2′); 15N NMR  δ −199.98 (N-2), −96.30 (N-10′), −57.86 (N-3). Isomer EN-N: 1H NMR  δ 15.00 , 9.28 , 8.98 , 8.83 , 8.46 , 8.41 , 8.24 , 7.99 , 7.94 , 7.80 , 7.73 , 7.52 ; 13C NMR  δ 162.69 (C-1), 148.44 (C-10′a), 147.54 (C-4), 146.61 (C-4′a), 139.92 (C-9′), 136.83 (C-3′), 136.00 (C-4″), 135.09 (C-1″), 134.43 (C-1′), 132.95 (C-6′), 129.75 , 129.60 (C-5′), 129.43 (C-8′), 128.78 (C-4′), 127.93 (C-9′a), 127.59 (C-7′), 127.20 (C-8′a), 126.34 (C-2′); 15N NMR  δ −198.95 (N-2), −96.30 (N-10′), −57.34 (N-3).N′-[(E)-4-Bromophenylmethylidene]acridine-4-carbohydrazide (7d). Yield 140 mg, 82%; bright yellow solid; mp 240–241 °C. Ratio of isomers ZN-N: EN-N = 1.0: 0.48 (67.6%: 32.4%). Isomer ZN-N:1H NMR  δ 15.05 , 9.32 , 9.00 , 8.85 , 8.49 , 8.44 , 8.26 , 8.01 , 7.88 , 7.82 , 7.74 , 7.69 ; 13C NMR  δ 162.54 (C-1), 148.48 (C-10′a), 147.58 (C-4), 146.61 (C-4′a), 139.99 (C-9′), 136.85 (C-3′), 135.46 (C-1″), 134.47 (C-1′), 132.94 (C-6′), 132.73 , 129.95 , 129.60 (C-5′), 129.43 (C-8′), 128.75 (C-4′), 127.93 (C-9′a), 127.60 (C-7′), 127.19 (C-8′a), 126.34 (C-2′), 124.32 (C-4″); 15N NMR  δ −199.57 (N-2), −96.30 (N-10′), −57.54 (N-3). Isomer EN-N: 1H NMR  δ 15.05 , 9.32 , 9.00 , 8.84 , 8.49 , 8.44 , 8.26 , 8.01 , 7.88 , 7.82 , 7.74 , 7.69 ; 13C NMR  δ 162.64 (C-1), 148.44 (C-10′a), 147.56 (C-4), 146.61 (C-4′a), 139.96 (C-9′), 136.85 (C-3′), 135.46 (C-1″), 134.45 (C-1′), 132.96 (C-6′), 132.73 , 129.95 , 129.56 (C-5′), 129.43 (C-8′), 128.75 (C-4′), 127.93 (C-9′a), 127.60 (C-7′), 127.19 (C-8′a), 126.35 (C-2′), 124.31 (C-4″); 15N NMR  δ −198.88 (N-2), −96.30 (N-10′), −57.17 (N-3).3a–3d (0–6.2 μM) at three different temperatures  in 2 mL of 100 mM NaCl and a 10 mM Tris-HCl buffer (pH = 7.4).Steady-state fluorescence (STF) emission spectra, synchronous fluorescence (SF) spectra, and 3D fluorescence spectra (3DF) were measured using a Varian Cary Eclipse spectrofluorimeter with a xenon flash lamp and a 1.0 cm quartz cuvette, with the slits set at 5 nm for the excitation observations and 10 nm for the emission spectra. The STF spectra were recorded at a range of 285–550 nm with a fixed excitation wavelength at 280 nm. The change in fluorescence intensity of HSA at a concentration of 4 μM was observed by titrating varying concentrations of acridine derivatives 3a–3d in the same concentration range as that used in the STF studies. The spectra were recorded at a range of 200–400 nm by setting ∆λ = 60 nm and ∆λ = 15 nm for tryptophan and tyrosine residues, respectively, at room temperature (25 °C).Synchronous fluorescence spectra for HSA (4 μM) were recorded at increasing concentrations of compounds 3a–3d using an excitation wavelength range of 200–350 nm and an emission wavelength range of 200–600 nm at room temperature (25 °C). The 3D spectra were recorded for 4 μM of HSA in a 2 mL buffer solution (100 mM NaCl and 10 mM Tris-HCl buffer pH = 7.4) and for the HSA:3a–3d complexes at a concentration ratio 1:1. The data from 3D measurements were processed into a 3D graphics plot using Origine 8.5 software, (2020) produced by OriginLab Corporation, Informer Technologies, Inc. .The 3DF spectra of HSA were performed in the absence and presence of compounds 3a–3d) complexes at a concentration ratio of 1:1 ([HSA] = 4 μM) were titrated into specific side markers (0–20 μM). The molar ratio of the HSA:marker complexes were 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 and 1:5. The reaction mixture of the HSA-acridine derivatives complexes with the side markers were preincubated for 15 min prior to spectral measurements. Additionally, a reverse titration competitive experiment was performed for derivative 3a. The site probe-HSA complex  was titrated with derivative 3a at a concentration gradient of 0.6–6 μM. In order to determine the binding site of compound 3a, the fluorescence quenching data were analysed using Stern-Volmer and modified Stern-Volmer equations to calculate the value of the Stern-Volmer constants (KSV) and binding constants bK, and to determine the number of binding sites n. All spectra measurements were performed at 280 nm excitation wavelength and the slits were set at 5 nm for excitation and 10 nm for emission spectra at a range of 290–550 nm and at 25 °C.Competitive experiments of HSA were performed using warfarin and ibuprofen as standard site marker ligands. The selected markers for the site I were warfarin, whereas site II was probed using ibuprofen. HSA-acridine derivative  buffer at room temperature. The titration experiment was carried out in the presence of a fixed concentration of compounds 3a–3d (25 μM) and was performed by titrating varying concentrations of ctDNA ranging from 0 to 680 μM. The solution was incubated for 5 min and then tested.UV-Vis absorption spectrum of the drug-ctDNA complexes were measured on a Varian Cary 100 Bio UV-Vis Spectrophotometer. The UV-Vis spectra of free compounds 2. The media were supplemented with 10% fetal bovine serum  and antibiotics . Prior to the selected treatments, cells were seeded on 6- and/or 96-well plates  and left to settle for 24 h. The acridine compounds solutions (at concentrations ranging from 5–75 µM) were then added to cells for 24 or 48 h, and analysis was subsequently performed.Human lung carcinoma cell line A549 and CCD-18Co colon fibroblasts were purchased from the American Type Culture Collection . The A549 cells were cultured in a complete RPMI-1640 medium  and CCD-18Co cells were cultured in a minimum essential medium (MEM)  at 37 °C, 95% humidity and 5% CO3 cells/cm2) and CCD-18Co (15.625 × 103 cells/cm2) were seeded in 96-well microplates. The A549 cells were treated for 24 and 48 h, and the CCD-18Co cells were treated for 48 h with different concentrations  of the derivatives. After the treatment, the MTT -2,5-diphenyltertrazolium bromide) solution in PBS (5 mg mL−1) was added to each well. The reaction was stopped after 4 h incubation, and the formazan was dissolved by the addition of SDS at a final concentration of 3.3%. The absorbance of formazan (λ = 584 nm) was measured using a BMG FLUOstar Optima spectrometer . The results were evaluated as percentages of the absorbance of the untreated control. Results are presented as the average percentage of cells from three independent experiments. IC50 values for the derivatives were extrapolated from a sigmoidal fit (dose-response curve) to the metabolic activity data using OriginPro 8.5.0 SR1 .MTT assays were performed in order to evaluate changes in the metabolic activity of cells that had occurred as a consequence of treatment with the acridine compounds. A549 cells (15 × 103 cell/well into a 6-well plate and treated with different concentration of derivatives (25 and 50 µM) for 48 h. The total cell number was expressed as a percentage of the untreated control of the total cell number. Viability was expressed as a percentage of viable, eosin negative cells. The results are presented as the average percentage of cells from three independent experiments.For the assessment of total cell numbers and viability within individual experimental groups, floating and adherent cells were harvested after treatment with the studied compounds and evaluated using a Bürker chamber with eosin staining. A549 cells were plated at a density of 135 × 10For the colony forming assay, floating and adherent cells were harvested together 48 h after treatment with the studied compounds (25 and 50 µM). The cells were then counted using a Bürker chamber with eosin staining and 500 viable cells per well were seeded in 6-well plates. After seven days of cultivation under standard conditions, the cells in the plates were fixed and stained with 1% methylene blue dye in methanol. Visualized colonies were scanned and counted by Image software, and the results were evaluated as percentages of the untreated control. The results are presented as the average percentage of colonies from three independent experiments.−1 ribonuclease A and 0.02 g L−1 propidium iodide (PI) and incubated in dark conditions at RT for 30 min. The DNA content was analysed using a BD FACSCalibur flow cytometer  with a 488 nm argon-ion excitation laser, and fluorescence was detected using a 585/42 nm band-pass filter (FL-2). ModFit 3.0 software  was used to generate DNA content frequency histograms and to quantify the percentage of cells in the individual cell cycle phases. The results are presented as the average ratio of cells in the individual phase to all cells from three independent experiments.For flow cytometric analysis of the cell cycle distribution, floating and adherent cells were harvested together 48 h after treatment with the compounds (25 and 50 µM), washed in cold PBS, fixed in cold 70% ethanol, and stored overnight at −20 °C. Prior to analysis, the cells were washed twice in PBS, resuspended in a staining solution : p ˂ 0.01, (***): p ˂ 0.001.The obtained data were analysed using a one-way ANOVA with Tukey’s post-test, and are expressed as the mean ± standard deviation (S.D.) of at least three independent experiments. The experimental groups treated with derivatives were compared with the control group: (*): 3a–3d containing acridine moiety were designed, synthesized and characterized in detail using NMR, IR and HR mass spectroscopy techniques. NMR derived parameters  allowed us to determine configuration and conformation in solution for all synthesized compounds. We determined distinct configurational and conformational preferences to form EC4=N3ZC(O)-N2-3 and EC4=N3ZC(O)-N2-7. The duplicate signals in the NMR spectra of 7b–d were attributed to the present EN-N/ZN-N conformers. The major factor that controls the conformation of the studied compound 7 are hydrogen bonds N10′···H2 and C(O)···H4.A series of novel benzohydrazide derivatives 3c and 3d in vitro act as potential dual inhibitors of hTopo I and II with a partial effect on the metabolic activity of A594 cancer cells. The values of binding constant for the interaction of acridine derivatives with ctDNA increased as follows: 3d < 3c < 3a < 3b. The higher values of K from the acridine-benzohydrazone series are present in fluoro-substituted derivative 3b (K = 3.18 × 103 M−1). The effect of tested derivatives on the metabolic activity of A549 cells evaluated by MTT assay decreased as follows: 3b(-F) > 3a(-H) > 3c(-Cl) > 3d(-Br). In the case of 3d, no significant activity against CCD-18Co fibroblasts was observed. The clonogenic survival was analysed after treatment of A549 cells with the compounds 3a and 3c. The acridine-benzohydrazides 3a and 3c reduced the clonogenic ability of A549 cells by 72% or 74%, respectively. The difference in the substituted benzene ring in 3a, 3c plays a slight role in clonogenicity survival  of A549 cell. The results indicated that interaction between acridine derivatives and HSA could lead to the change of protein secondary structure. In the presence of warfarin, the values of binding constants decreased, which suggest that derivative 3a could bind to HSA at the Sudlow site I. The findings presented in this paper suggest that these acridine derivatives exhibit promising potential as topoisomerase I and II inhibitors with anticancer activity against A549 human adherent lung carcinoma cells, and may also serve as DNA and HSA-interacting agents. These features would be of considerable use in the development of drugs with enhanced or more selective effects and greater clinical efficacy.In vitro antiproliferative activities of these compounds against A549 and normal fibroblast cells CCD-18Co were studied. The compounds have been undergoing against topoisomerase I and II, and their binding properties  have been evaluated. The derivatives


However, few studies exist on the structure-activity relationship of 13-amino derivatives of dehydrocostus latone. In this study new 13-amino derivatives of dehydrocostus lactone DHLC (1–4) were synthesized through Michael addition reactions, and were screened against three different breast cancer cell lines, namely hormone receptor positive breast cancer (MCF-7), triple-negative breast cancer (HCC70), and non-tumorigenic mammary epithelial (MCF-12A) cell lines. Dehydrocostus lactone (DHLC) exhibited IC50 values of 1.11 (selectivity index (SI) = 0.06), 24.70 (SI = 0.01) and 0.07 μM against HCC70, MCF-7 and MCF-12A cells, respectively. All the amino derivatives, except DHLC-3 displayed low micromolar IC50 values (ranging from 0.07–4.24 μM) against both breast cancer cell lines, with reduced toxicity towards MCF-12A non-tumorigenic mammary epithelial cells . DHLC-1 and DHLC-2 demonstrated the greatest selectivity for the MCF-7 cells (with SI of 121 and 126.86 respectively) over the MCF-12A cells. This reveals that, overall, the derivatives display greatly improved selectivity for breast cancer over non-tumorigenic mammary epithelial cells, with between 100-fold and 12 000-fold higher SI values. The improved docking scores were recorded for all the 13-amino dehydrocostus lactone derivatives for the enzymes analyzed. Compounds DHLC-4, and DHLC-3 recorded higher docking scores of -7.33 and -5.97 Kca/mol respectively, compared to the parent structure, dehydrocostus lactone (-5.34 Kca/mol) for protein kinase (PKC) theta (1XJD) and -6.22 and -5.88 Kca/mol, respectively for protein kinase iota (1RZR). The compounds further showed promising predicted adsorption, distribution, metabolisms and excretion (ADME) properties. Predicting the ADME properties of these derivatives is of importance in evaluating their drug-likeness, which could in turn be developed into potential drug candidates.The biological activities of dehydrocostus lactone and its analogues are suggested to be mediated by the lactone ring and  Sesquiterpene lactones are some of the most active compounds with a broad structural and functional diversity and are classified based on their skeletal structures into guaianolides, eudesmanolides, hyptocretenolides, pseudo- guaianolides, and heliangolides. However, the guaianolides are the most often reported sesquiterpene lactones with anticancer properties , 2.α,β or α,β,γ-unsaturated carbonyl structure such as α-methyl-γ-lactone, α,β-unsaturated cyclopentanone or some conjugate esters  for 96 hours at 37°C in a 9% CO2 incubator. Thereafter, 0.54 nM of resazurin solution was added and the cells were incubated for 2–4 hours at 37°C in a 9% CO2 incubator. The fluorescence was then measured on a Spectramax spectrophotometer with excitation and emission wavelength set at 560 and 590 nm respectively. The experiment was done in technical triplicate and the data were analyzed using GraphPad Prism Inc, (USA) with half-maximal inhibitory concentration  determined by non-linear regression. Selectivity index values for the compounds were calculated as follows: (IC50 of compound against MCF12A cells)÷(IC50 of compound against breast cancer cells) where a SI > 1 is indicative of selective toxicity towards cancer cells vs non-cancerous cells.Cytotoxicity and the ICn assay, , 13. TheIn silico docking was done on protein kinases (1ZRZ) and the PKC theta (1XJD) transferase enzymes. The enzymes were chosen based on promising scientific evidence showing that these isoenzymes can be activated by sesquiterpenes lactones D was performed on Jasco P-2000 polarimeter. The angle of rotation α of polarized light was recorded at 200 ± 0.50 in chloroform solution and was expressed in degrees (°) of the plane of polarization at the wavelength of 546.3 nm of the D-line of sodium.Specific optical rotation decahydro-6,9-bis(methylene)azulenofuran-2(3H)-one (DHLC-2): 2.78 , 2.40 , 3.82 , 2.51 , 2.71 , 2.39 , 3.87 , 2.15 , 2.77 , 2.48 , 4.79 , 5.12 , 2.41 , 2.34 , 0.92 . 13C NMR : 47.1 (C-1), 32.6 (C-2), 30.2 (C-3), 151.8 (C-4), 52.1 (C-5), 85.4 (C-6), 45.9 (C-7), 33.0 (C-8), 37.7 (C-9), 150.2 (C-10), 45.9 (C-11), 177.9 (C-12), 52.9 (C-13), 109.1 (C-14), 111.6 (C-15), diethylamine  (S1 Table in Guaia-4(15),10(14)-dien-12-oic acid, 13-(ethylamino)-6-hydroxy-γ-lactone (DHLC-3). 1H NMR : 2.91 , 1.39 , 2.54 , 2.85 , 3.98 , 2.25 , 2.16 , 2.51 , 2.73 , 2.91 , 5.07 , 4.90 , 1.37 , 1.16 . 13C NMR : 47.8 (C-1), 32.5 (C-2), 30.2 (C-3), 151.8 (C-4), 51.9 (C-5), 85.8 (C-6), 45.4 (C-7), 32.7 (C-8), 37.6 (C-9), 149.7 (C-10), 47.8 (C-11), 178.0 (C-12), 47.8 (C-13), 109.2 (C-14), 111.9 (C-15), ethylamine  (S1 Table in 13-ethylenediamine dehydrocostus lactone (DHLC-4). 1H NMR : 2.37 , 2.54 , 1.97 , 2.83 , 3.96 , 2.42 , 2.50 , 2.07 , 2.73 , 2.88 , 5.06 , 4.89 , 2.71 , 2.82 . 13C NMR : 47.5 (C-1), 32.5 (C-2), 30.2 (C-3), 151.9 (C-4), 51.9 (C-5), 85.4 (C-6), 45.0 (C-7), 32.7 (C-8), 37.7 (C-9), 150.7 (C-10), 49.4 (C-11), 177.8 (C-12), 47.7 (C-13), 109.2 (C-14), 112.0 (C-15), ethylenediamine  (S1 Table in DHLC was achieved through amination (Scheme 1) of the double bond.The desired decrease in toxicity and increase in activity of Scheme 1. Amination of the double bond.DHLC was achieved using primary, secondary, and tertiary amines. Nitrogen atoms from different amines; dimethylamine, diethylamine, ethylamine, triethylamine, ethylenediamine, diisopropylamine, benzylamine, and 2-methoxybenzylamine were introduced at the C-13 double bond of DHLC to yield respective amino derivatives as in Scheme 2, DHLC even after refluxing the reaction mixture for a further 24 hrs as the starting materials were recovered. Previous studies reported that cyclic and secondary amines possess more predictable conformation and can be used to construct stereoselective amine derivatives decahydro-6,9-bis(methylene)azulenofuran-2(3H)-one (DHLC-2), guaia-4(15),10(14)-dien-12-oic acid, 13-(ethylamino)-6-hydroxy-γ-lactone (DHLC-3), and 13-ethylenediamine dehydrocostus lactone (DHLC-4). The structures of the synthesized compounds are provided in The amination of the C-13 double bond of DHLC-1 was obtained as a colourless oil with Rf value of 0.53 , 45.1mg, 65% yield with its specific optical rotation recorded at[α]D20–15.54 {CHCl3, Conc = 31.00  stretches and 1460 cm-1 due to C = C stretches , 42.2 mg, 62% yield with its specific optical rotation recorded as [α]D20–107.46 {CHCl3, Conc = 4.10 (w/v%)}. The IR spectrum showed N-H absorption bands at 3300 cm-1. The absorptions bands at 2913 and 2858 cm-1 were attributed to C-H  stretches while bands at 1636 and 1460 cm-1 were due to C = C stretches ), 55.1mg with its optical specific rotation recorded as [α]D20–40.60 {CHCl3, Conc = 2.50 (w/v%)}. The IR spectrum showed N-H stretches at 3275 cm-1. The absorptions bands at 2922 and 2847 cm-1 were attributed to C-H  stretches while 1642 cm-1 was due to C = C stretches  and δH 2.72 (H-16) confirmed the introduction of an amine group at C-13 in dehydrocostus lactone. This was further confirmed by the presence of a methyl carbon signal at δC 14.9 (C-17) and δC 44.3 (C-13), attributed ethyl group of the ethylamine. The absence of olefinic carbon signal at δC 120.9 and 139.1 confirmed the successful addition of ethylamine at this position as in S1 Table in Analysis of the DHLC-4 was obtained as a colourless oil with Rf of 0.51 (C6H14: CH2Cl2), 35.7 mg, 54% yield with its optical specific rotation recorded as [α]D20–43.15 {CHCl3, Conc = 8.50 (w/v%)}. The IR spectrum showed N-H stretches at 3240 cm-1. The absorptions bands at 2898 cm-1 were attributed to C-H  stretches while 1 due to C = C stretches (Appendix A44 in + at m/z 291.9881 which indicated a molecular formula of C17H26N2O2 (Appendix A43 in DHLC-4 are provided in Appendices A36-A42 in Compound H 2.71 and δH 2.82 due to the methylene protons of C2H4(NH2)2. The absence of olefinic proton signals at δH 5.52 and δH 6.52 of dehydrocostus lactone and the presence of signals at δH 2.71  and δH 2.82  confirmed the presence of an ethylenediamine group at C-13 in dehydrocostus lactone (S1 Table in C 47.7 (C-13), δC 41.6 (C-17), and δC 52.7 (C-16), which were attributed to the ethylenediamine substituent at C-13. In addition, the absence of olefinic carbon signals at δC 120.9 and 139.1 confirmed the addition of C2H4(NH2)2 at C-13.Analysis of the proton spectrum exhibited signals at δDHLC) and all synthesized amino derivatives were evaluated in vitro for their cytotoxicity against breast cancer cells including the HCC70 and MCF-7 cell lines, and non-tumorigenic mammary epithelial (MCF-12A) cells using camptothecin as a positive control. The cytotoxicity data for all the synthetic derivatives are summarized in DHLC) displayed cytotoxic activities against HCC70, MCF-7, and MCF-12A with IC50 values of 1.11 ± 1.31, 24.70 ± 1.25, and 0.07 ± 0.07 μM respectively. Compounds DHLC-1 and DHLC-2 exhibited cytotoxic activities against HCC70, MCF-7 and MCF-12A with IC50 values of 0.64 ± 1.47, 0.07 ± 0.07 and 8.47 ± 1.24, respectively in the case of DHLC-1 and 1.48 ± 1.49, 0.07 ± 1.31 and 8.88 ± 1.10 μM, respectively, in the case of DHLC-2, as illustrated in DHLC-3, all of the derivatives displayed low micromolar IC50 values (ranging from 0.07–4.24 μM) against HCC70, and MCF-7 breast cancer cell lines, with reduced toxicity towards non-tumorigenic mammary epithelial (MCF-12A) cells. The derivatives DHLC-1, DHLC-2, DHLC-3 and DHLC-4 demonstrated selectivity for the HCC70  and MCF-7 breast cancer cells  over the MCF-12A non-cancerous cells. The parent compound displayed SI values of 0.06 and 0.01 for HCC70 and MCF-7 cells, respectively. This reveals that, overall, the amino derivatives of dehydrocostus lactone display greatly improved selectivity for breast cancers  over non-tumorigenic mammary epithelial cells (MCF-12A) compared to the parent compound, ranging from 100- to over 12 000-fold higher SI values.Dehydrocostus lactone (DHLC-3) exhibited reduced activity against MCF-12A (IC50 = 188.20 ± 1.35μM) and MCF-7 cells (IC50 = 222. 60 ± 1.01) compared to dehydrocostus lactone , whilst 13-ethylenediamine derivative (DHLC-4) displayed improved cytotoxicity against MCF-7 cells  compared to the parent molecule (DHLC), with IC50 value of 24.70 ± 1.25 μM against the cell line. This suggests that addition of ethylene diamine at C-13 of dehydrocostus lactone improves anticancer activities on breast cancer cells compared to non-tumorigenic mammary epithelial cells (MCF-12A). In addition, primary amines derivatives including 13-dimethylamine (DHLC-1), and 13-diethylamine (DHLC-2) showed improved cytotoxicity activities against HCC70 and MCF-7 cells compared to secondary amines derivatives as summarized in The type of amine group used produced 13-amino derivatives with varying cytotoxicity activities. For instance, 13-ethylamine derivative (50 = 0.604 μg/mL) compared to helenalin (ED50 = 0.083 μg/mL) against Hep-2 (Human epidermoid carcinoma of the larynx) cells. Furthermore, dimethylamino adducts of costunolide displayed enhanced antiproliferative activities against leukemia cells (K562) with GI50 value of 4.4 μM compared to the parent molecule, costunolide (G150 = 14.5 μM) [50 values of 9.1 μM compared to costunolide with G150 value of 7.8 μM [50 value of 3.3 μM compared to costunolide (IC50 = 7.8 μM). Dehydrocostus lactone derivative having α-methylene-γ-lactone moiety was synthesized using Heck reactions by Ding et al. [50 of 6.2 μM [50 > 50 μM) or reduced activity with IC50 values of between 31 and 52 μM against the cultured acute myelogenous leukemia cell line HL-60. It was therefore concluded that derivatives having substituents on the α-benzylidene-γ-lactone moiety can selectively inhibit doxorubicin-resistant cell line HL-60/A [In related studies, synthesized 13-dimethylamino derivative of helenalin sesquiterpenes showed improved cytotoxicity activities (ED14.5 μM) . Howeverf 7.8 μM  reportedg et al.  and was f 6.2 μM . The der HL-60/A , 22.β-methylene-γ-lactone, the addition of an amine into this moiety could of help to mask it from other nucleophiles and to increase the water solubility. Hence, the adsorption, distribution, metabolisms, and excretion properties (ADME) of the 13-amino derivatives of dehydrocostus lactone were further analyzed using SwissADME tools [Since the biological activities of dehydrocostus lactone are attributed to α, ME tools , 24. ThiThe physiochemical, pharmacokinetic properties, drug nature, and medicinal chemistry friendliness of the 13-amino derivatives were analyzed and results are summarized in DHLC-3) and 13-ethylenediamine dehydrocostus lactone (DHLC-4) showed the best bioavailability properties while dehydrocostus lactone (DHLC) showed poor flexibility properties [The bioavailability radar in operties .DHLC-4), and guaia-4(15),10(14)-dien-12-oic acid, 13-(ethylamino)-6-hydroxy-γ-lactone (DHLC-3) recorded the better binding energies of -7.33 and -5.97 kcal/mol, which were higher than the parent structure, dehydrocostus lactone  with the pivotal residues for PKC theta (1XJD) and -6.22 and -5.88 kcal/mol with the residues for 1ZRZ respectively.DHLC-1-4) posed in the active site pockets of the protein PKC theta (1XJD). The findings suggest that the activity of these compounds is associated with the ability of their skeletal structures to bind to specific enzymes.The high binding affinities of these derivatives were enhanced by the addition of amino groups on C-13, which were observed to form hydrogen bonds and pi interactions with the enzymes. 1–4) were synthesized through Michael’s addition reactions and were screened against breast cancer cell lines including hormone receptor-positive breast cancer (MCF-7), triple-negative breast cancer (HCC70), and non-tumorigenic mammary epithelial (MCF-12A) cell lines. The amino derivatives of dehydrocostus lactone, while displaying low micromolar toxicity overall to the breast cancer cells, also showed greatly improved selectivity for these breast cancer cells compared to the non-cancerous breast epithelial control cell line and were, in general more toxic to MCF-7 hormone receptor-positive breast cancer cells than the parent dehydrocostus lactone. The compounds further showed improved binding energies to PKC enzymes. Promising predicted ADME properties were also reported for all the compounds. Predicting the ADME properties of these derivatives is of importance in evaluating their drug-likeness, which could in turn be developed into potential drug candidates. The findings from the current study shows the promising potential of 13-amino derivatives of dehydrocostus lactone that can be investigated further to understand their structure-activity relationships about anticancer activities.In summary, new 13-amino derivatives of dehydrocostus lactone (S1 File(DOCX)Click here for additional data file.S2 File(ZIP)Click here for additional data file.S3 File(PNG)Click here for additional data file.


C-glucosidic xanthone originally isolated from the Mangifera indica (mango) plant. Mangiferin exhibits a wide range of pharmaceutical activities. However, mangiferin’s poor solubility limits its applications. To resolve this limitation of mangiferin, enzymatic glycosylation of mangiferin to produce more soluble mangiferin glucosides was evaluated. Herein, the recombinant maltogenic amylase  from a thermophile Parageobacillus galactosidasius DSM 18751T (PgMA) was cloned into Escherichia coli BL21 (DE3) via the expression plasmid pET-Duet-1. The recombinant PgMA was purified via Ni2+ affinity chromatography. To evaluate its transglycosylation activity, 17 molecules, including mangiferin (as sugar acceptors), belonging to triterpenoids, saponins, flavonoids, and polyphenol glycosides, were assayed with β-CD (as the sugar donor). The results showed that puerarin and mangiferin are suitable sugar acceptors in the transglycosylation reaction. The glycosylation products from mangiferin by PgMA were isolated using preparative high-performance liquid chromatography. Their chemical structures were glucosyl-α-(1→6)-mangiferin and maltosyl-α-(1→6)-mangiferin, determined by mass and nucleic magnetic resonance spectral analysis. The newly identified maltosyl-α-(1→6)-mangiferin showed 5500-fold higher aqueous solubility than that of mangiferin, and both mangiferin glucosides exhibited similar 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activities compared to mangiferin. PgMA is the first MA with glycosylation activity toward mangiferin, meaning mangiferin glucosides have potential future applications.Mangiferin is a natural antioxidant  C-glucosidic xanthone originally isolated from the Mangifera indica (mango) plant. Mangiferin has been reported to possess diverse health-promoting activities, such as antioxidant −, 565.3, 331.0, 300.9, 259.3; 1H-NMR : Hδ 3.05 , 3.15 , 3.20 , 3.29 , 3.33 , 3.35 , 3.38 , 3.46 , 3.52 , 3.62 , 3.70 , 4.02 , 4.58 , 4.73 , 6.36 , 6.86 , and 7.37 . 13C-NMR : Cδ 60.6 (C-6″), 66.9 (C-6′), 70.0 (C-4″), 70.2 , 72.1 (C-2″), 72.5 (C-3″), 73.2 (C-1′), 73.3 (C-5″), 78.9 (C-3′), 79.7 (C-5′), 93.3 (C-4), 98.7 (C-1″), 101.3 (C-9a), 102.6 (C-5), 107.5 (C-2), 108.1 (C-8), 111.8 (C-8a), 143.7 (C-7), 150.8 (C-10a), 154.0 (C-6), 156.2 (C-4a), 161.7 (C-1), 163.8 (C-3), and 179.1 (C-9).The composition of compound (2) was as follows: light yellow powder; mp 227-229 °C; ESI/MS m/z: 745.3 [M-H]−, 727.3, 403.3, 385.0, 331.0, 313.2, 301.2; 1H-NMR : Hδ 3.03 , 3.19 , 3.21 , 3.23 , 3.30 , 3.34 , 3.36 , 3.38 , 3.41 , 3.42 , 3.44 , 3.46 , 3.55 , 3.60 , 3.64 , 3.71 , 4.58 , 4.75 , 4.95 , 6.36 , 6.86 , and 7.37 . 13C-NMR : Cδ 60.0 (C-6″), 60.7 (C-6‴), 67.2 (C-6′), 69.8 (C-4‴), 70.2 , 70.8 (C-5″), 71.6 (C-2″), 72.6 (C-2‴), 73.1 (C-3″), 73.2 (C-1′), 73.3 (C-3‴), 73.4 (C-5‴), 78.9 (C-3′), 79.7 (C-5′), 79.8 (C-4″), 93.3 (C-4), 98.6 (C-1″), 100.9 (C-1‴), 101.3 (C-9a), 102.6 (C-5), 107.4 (C-2), 108.1 (C-8), 111.7 (C-8a), 143.7 (C-7), 150.8 (C-10a), 154.0 (C-6), 156.2 (C-4a), 161.8 (C-1), 163.8 (C-3), and 179.1 (C-9).The composition of compound /(OD517 of the control reaction).The assay was performed as previously described  with minBacillus stearothermophilus  is a well-studied MA for the glycosylation of bioactive molecules − ion peak at m/z: 583.4 in the electrospray ionization mass spectrum (ESI-MS) corresponding to the molecular formula C25H28O16 (− ion peak at m/z: 745.3 in the ESI-MS corresponded to the molecular formula C31H38O21 (2) contains two glucosyl moieties attached to the mangiferin structure. To identify the structures in advance, the structures of both compounds were determined using NMR spectroscopy. 1H and 13C NMR, including the DEPT, HSQC, HMBC, COSY, and NOESY spectra, were obtained.The glycosylation of mangiferin by 25H28O16 . The mas31H38O21 , which i1H and 13C NMR sugar signals in compound (1) were assigned to C-glucosyl and O-glucosyl moieties by one-dimensional (1-D) and 2-D NMR experiments. The 1H spectrum of compound (1) in DMSO-6d showed three singlets at 6.36, 6.86, and 7.37 ppm and a complex 10-spin system between 3.0 and 5.0 ppm. Analysis of this second-order system revealed coupling constants typical of two glucose moieties. The compound (1) glucosidic linkage of the C-glucosyl moiety on the xanthone C-2 was revealed by the presence of HMBC correlations between C-2/H-1′ (107.5/4.58 ppm), and the anomeric proton H-1′ at 4.58  indicated a C-β- configuration of mangiferin that was confirmed by the data reported in the literature [O-glucosyl moiety was a doublet signal at H-1″  with the corresponding carbon atom at C-1″ (98.7 ppm) assigned to the anomeric proton and indicating an O-α-configuration by HSQC, which is in the O-α-configuration. The H-1″ (δ = 4.73 ppm) of mangiferin and the HMBC cross signaled H-1″/C-6′ (4.73/66.9 ppm) and H-6′a, 6′b/C-1″ . The significant downfield shift of the 13C signal of C-6′ indicated the connection of the second glucosyl moiety, which confirmed the α-(1→6) linkage of the second glucosyl moiety. The NMR signals were identified as shown in α-(1→6)-mangiferin (The characteristic terature . The manngiferin .1H spectrum of compound (2) in the same compound (1) solvent also showed three singlets at 6.36, 6.86, and 7.37 ppm and a complex 11-spin system between 3.0 and 5.0 ppm. Analysis of this second-order system revealed coupling constants typical of three glycose moieties, which included the chemical shifts listed in J = 9.8 Hz) according to the corresponding HMBC indicated a C-C bond between the sugar and the aglycone of mangiferin (C-glucosyl-xanthone) and were confirmed by the data reported in the literature [O-maltosyl moiety connected to mangiferin was confirmed by HMBC from the anomeric carbon C-6′ (66.9 ppm), and the corresponding anomeric proton H-1″ at 4.75  indicated an O-α-configuration. The maltose doublet signal at δH H-1″  and H-1‴ 4.95  with the corresponding carbon atom at C-1″ (98.6 ppm) and C-1‴ (100.9 ppm) was assigned to the anomeric proton and indicated two O-α-configurations by HSQC. The HMBC cross peaks of C-1″/H-6′  and C-1‴/H-4″ (100.9/3.34 ppm) confirmed the α-(1→4) between the two-glucosyl moiety. Our experimental 1H and 13C chemical shifts listed in 2) as maltosyl-α-(1→6)-mangiferin -mangiferin (1) [α-(1→6)-mangiferin (1) was 2300-fold higher than that of mangiferin. In this study, the amount of purified glucosyl-α-(1→6)-mangiferin (1) was too low to repeat the solubility experiment. The solubility of the newly identified maltosyl-α-(1→6)-mangiferin (2) was determined. The results showed that the solubility of maltosyl-α-(1→6)-mangiferin (2) was 5500-fold higher than that of mangiferin (α-(1→6)-mangiferin (2) possesses higher solubility than glucosyl-α-(1→6)-mangiferin (1). It has been reported that the more sugar moieties in the glycosylated compounds, the higher the aqueous solubility of the glycosylated compounds [The low aqueous solubility of mangiferin restricts its usage as a pharmaceutical agent. However, the glycosylation of mangiferin may mitigate such a restriction. Only a few studies have reported different glycosylation agents for mangiferin. Wu et al. (2013) used β-fructofuranosidase  to glycosylate mangiferin into fructosyl-β-(2→6)-mangiferin and found that its DPPH radical scavenging activity was similar to that of mangiferin . Nguyen erin (1) . They fongiferin . Thus, mompounds ,35,36,37ortho-dihydroxyl groups on the benzene ring of the mangiferin structure have been reported to play a key role in exerting its antioxidant activity [Mangiferin exhibits a wide pharmacological profile, and its antioxidant property is well known from previous studies. Furthermore, it has been associated with the redox aromatic system of the xanthone nucleus ,9,10,12.PgMA from P. galactosidasius DSM 18751T was confirmed to exhibit the bifunctions of hydrolysis and transglycosylation activities. The recombinant PgMA can glycosylate mangiferin and produce glucosyl-α-(1→6)-mangiferin and maltosyl-α-(1→6)-mangiferin with a high maltodextrin concentration. The novel maltosyl-α-(1→6)-mangiferin showed much higher aqueous solubility than that of mangiferin. The two mangiferin glucosides exhibited similar DPPH antioxidative activity compared to mangiferin. To our knowledge, PgMA is the first MA identified with glycosylation activity toward mangiferin. With higher water solubility and compatible antioxidant activity, the two mangiferin glucoside derivatives have better pharmaceutical applicability.The recombinant