Antibody-drug conjugates (ADCs), designed to selectively deliver cytotoxic agents to antigen-bearing cells, are poised to become an important class of cancer therapeutics. Human epithelial growth factor receptor (HER2) is considered an effective target for cancer treatment, and a HER2-targeting ADC has shown promising results. Most ADCs undergoing clinical evaluation contain linkers that have a lysosomal protease-cleavable dipeptide, of which the most common is valine-citrulline (VC). However, valine-alanine (VA), another dipeptide comprising two human essential amino acids, has been used in next generation ADCs loading new toxins, but the druggable properties of ADCs loaded the most popular monomethyl auristatin E (MMAE) remain to be further explored. In this study, we generated VA-based ADCs that connected MMAE to an anti-HER2 antibody. We studied the differences in the preparation process, in vitro stability, cathepsin B activity and in vitro cytotoxicity of VA-based ADC compared to the ADC of VC. VA had comparable performance to VC, which preliminarily displays its practicability. Additional efficacy and safety studies in a xenograft model indicate this novel ADC exerted potent anti-tumor activity and negligible toxicity. The results of this study show the application potential of VA-based ADC with MMAE as the payload. HER2 plt cancer ,9. Despit cancer .®), an anti-HER2 ADC that was designed with this new strategy, further prolonged the overall survival of trastuzumab- resistant patients by more than six months [Antibody-drug conjugates (ADCs), which combine the specificity, pharmacokinetics, and biodistribution of a monoclonal antibody (MAB) with the cytotoxic potency of a drug payload, is a promising new therapy for cancer . Ado-trax months ,12. The x months . In addiA suitable linker not only needs to provide sufficient stability during systemic circulation, but must also allow the rapid and efficient release of cytotoxic drug inside the tumor cells . First-gIn this study, we generated VA-based ADCs that connected MMAE to an anti-HER2 antibody. Compared to the VC-based ADC which payload been from a clinically approved ADC drug, we studied the preliminarily practicability of the VA-based ADCs in the preparation process, in vitro stability, cathepsin B activity and in vitro cytotoxicity. In addition, we further studied the anti-tumor activity and toxicity of VA-based ADC in a xenograft model. The results demonstrate that the VA-based ADC with MMAE as the payload also has application potential as an antitumor agent.The design of the VA-based linker system was based on acetazolamide-drug conjugates . CompareBoth linkers with VA and VC were substrates for cathepsin B, and VA-based ADC could be easily cleaved by enzymes in an antibody-doxorubicin conjugate . The VA We generated VA-based internalizing ADCs with MMAE as the payload, as well as the corresponding VC-based ADC as a comparison. Theoretically, this type of ADC can also release the parent drug in a similar manner A. We rep12a, mil40-12b, and mil40-12c; mil40 is a humanized monoclonal antibody that binds to HER2) allowed resolution of the conjugates into several major peaks corresponding to 0, 2, 4, 6, and 8 drug molecules per antibody , can vary between 0 and 8 drugs per antibody. Reduction of inter-molecule chain disulfide bonds to produce free sulfhydryl groups allowed conjunction at specific residues using maleimide-containing linkers, and generated conjugate mixtures at a limited number of defined positions. Hydrophobic interaction chromatography (HIC) analysis of the three ADCs  was 0.27% in the high-performance liquid chromatography (HPLC) spectrum , whereas that of VC-based ADC (mil40-12c) was 0.83%. To enlarge this effect, we synthesized the two ADCs with an average DAR of about 7. In these samples, VA-based ADC had no obvious increase in dimeric peak; however, the aggregation of VC-based ADC elevated to 1.80% with an increase in DAR from 4 to 7. In addition, the reaction system of VA-based conjugates had higher limpidity in the conjugate process, leading the recovery rate of the antibody to increase by 8.4%. Thus, VA was chosen over VC to reduce aggregation in ADCs with MMAE as the potent payload.Similar to other biologics, it is important to monitor high molecular weight species in ADC. Size exclusion chromatography (SEC) is a long-established method to measure size variants of proteins, especially high molecular weight species. To confirm the effects of dipeptide type on the degree of aggregation, we synthesized two types of ADCs using VA and VC as linkers. Then, we analyzed aggregation of the two prepared ADCs with an average DAR of about 4. The dimer of the aggregated antibody peak of VA-based ADC (mil40-12b and 12c were reduced with excess N-acetyl-l-cysteine (NAC) to afford NAC-12b and NAC-12c respectively [12b and NAC-12c at each time point as a percentage of the AUC at t = 0. After seven days, the AUCs for NAC-12b and NAC-12c were 97.92% and 97.05%, respectively, of the two samples taken immediately at t = 0 . To compare the linker stability of the two dipeptides, the reactive maleimide double bond of ectively . These cat t = 0 A. In addNext, we analyzed the in vitro stability of these two ADCs with an average DAR of about 4 and 7. Analogously, the two ADCs having 4 and 7 drugs per antibody, respectively, were diluted with PBS  and incubated at 37 °C in a shaking incubator. All of the samples were analyzed by HIC HPLC. As shown in 12b and NAC-12c) to enzymatic cleavage was determined by treatment of the acetyl-L-cysteine adduct with cathepsin B from human liver ; NAC-12c was tested in parallel for comparison. The cleavage of dipeptide from both substrates resulted in 1,6-elimination of MMAE, which was identified by liquid chromatography-mass spectrometry . The amount of released MMAE over time is shown in According to previous reports, drug linkers containing VA or VC dipeptides are substrates for cathepsin B . Here, w12a, mil40-12b, and mil40-12c) exhibited more potent activity in the HER2-positive cell line, both IC50 and inhibition, while very weak or no inhibitory effects in the HER2-negative cell line, just as antibody drugs, indicating its significant antigen selectivity. As a comparison, MMAE had potent anti-tumor activity in both HER2-positive and HER2-negative cell lines , the weakly positive cell line MCF-7  , and thell lines .The above-mentioned three ADCs had almost identical cytotoxicity in vitro, indicating that the type of dipeptide and length of the carbon chain did not have significant effects on cytotoxicity and drug release in cells. Overall, VA-based ADCs with MMAE exhibited similar efficacy and selectivity to the VC-based ADC, and the selectivity between HER2-positive and HER2-negative cells increased by about 2400–26,000 fold. In addition, both antibodies exhibited very weak cell inhibitory activity in the HER2-positive ovarian cancer cell line SK-OV-3; however, the associated ADCs exhibited similar cytotoxicity to the other cell lines, with an increase in anti-tumor activity of tens of thousands .12b) was evaluated in the BT-474  breast tumor model in female non-obese diabetes / severe combined immunodeficiency (NOD/SCID) mice, with naked antibody and unconjugated MMAE as the control group. Compared with vehicle, the control group only caused a partial delay in tumor growth, whereas the ADC treatment group with the same therapeutic dose exhibited very obvious tumor inhibition , changes in hematology between vehicle and treatment groups were compared at the end of administration and the observation period. The main test indicators included levels of white blood cells (WBCs), red blood cells (RBCs), platelets (PLTs), neutrophils (Neuts), and lymphocytes (Lymph). In contrast to the vehicle group, all of the above hematological parameters from the three ADC treatment groups were similar on Days 28 and 58  regarding all parameters from both batches of blood samples  analysis was conducted on the hematology parameters of the treatment groups on Days 28 and 58, and at the end of drug administration. Unpaired two-tailed  samples . Thus, tAt the end of the xenograft model test, animals in specific groups were used for histopathological studies. Hematoxylin and Eosin (H&E) staining  and bone marrow smears showed no significant differences in histopathology evaluation, revealing no significant tissue damage produced in animals that received ADC relative to vehicle . This fuUnless otherwise indicated, all anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All of the other reagents and solvents were purchased as the highest grade available and used without further purification.Thin layer chromatography (TLC) was performed using pre-coated silica gel plates . Column chromatography was performed using silica gel . NMR spectra were recorded on a JNM-ECA-400 400 MHz spectrometer  using CDCl3 and DMSO-d6 as solvent. Chemical shifts are expressed in (ppm), with tetramethylsilane (TMS) functioning as the internal reference. MS was performed on an API 3000 triple-quadrupole mass spectrometer  equipped with a Turbo Ion Spray electrospray ionization source  that was used for mass analysis and detection. Analyst 1.4 software  was used for data acquisition.Synthetic routes and analytical data are provided in the experimental protocols.l-Histidine buffer (20 mM), pH 7.5, were treated with tris(2-carboxyethyl)phosphine hydrochloride  at 25 °C for 90 min. To the reduced mAb was added the maleimide drug derivatives  in ice-cold dimethylacetamide (DMAC) (5% v/v). After 40 min, the reactions were quenched with excess NAC . The mixture was placed on ice for 30 min before buffer exchange by elution through Sephadex G25, and concentrated by centrifugal ultrafiltration. The conjugates was sterile filtered through a 0.2 μm filter under sterile conditions and stored at −80 °C before use for analysis and testing.Humanized anti-HER2 IgG1 antibodies mil40 (10 mg/mL) in HIC HPLC was used to determine levels of the molar ratio of drug substitution: 1260 HPLC ; Butyl-NPR column . Here, HIC buffer A was 50 mM potassium phosphate, pH 7.0, and 1.5 M ammonium sulfate; and HIC buffer B was 50 mM potassium phosphate, pH 7.0, 20% isopropanol. The gradient was 100% to 100% B over 15 min; flow rate was 1 mL/min; and UV detection wavelength was 280 nm. The DAR was determined by peak area integration according to the reported method .SEC HPLC was used to determine levels of aggregation within each ADC: 1260 HPLC ; G3000SWXL analytical column . The SEC buffer contained 40 mM sodium phosphate and 150 mM sodium chloride ; and the UV detection wavelength was 280 nm. We performed a needle wash after each injection and include blank runs between each analyte. The aggregation was determined by peak area integration according to the reported method .l-cysteine (NAC) solution  was added to 12b and 12c . The reaction mixture was incubated at room temperature for 10 min, after which HPLC analysis revealed complete conversion to NAC-12b and NAC-12c as previously reported [12b and NAC-12c at each time point as a percentage of the AUC at t = 0.The acetyl-reported . mixtureAnalogously, ADCs that contained different dipeptide linkers were incubated in PBS  at 37 °C in a shaking incubator. Aliquots (200 μL) were collected at different time points  and then stored at −20 °C. All samples were thawed at room temperature before HIC analysis. The DAR of the samples at different time points were calculated as previously described .12b and NAC-12c (500 μg/mL) were prepared according to the above method and the enzymatic reaction temperature was set at 37 °C to mimic physiological conditions. The buffer mixture contained 25 mM sodium acetate, and 1 mM EDTA, at pH 5.5. To 880 μL of the buffer was added the NAC-conjugates solution respectively (100 μL) followed by a solution of Cathepsin B , and the reaction mixture was incubated at 37 °C [t = 0, 5, 10, 20, 40, 80, 160, and 320 min), and analyzed by LC-MS. Results of the released MMAE was quantified with the standard curve method.NAC-at 37 °C ,35. Aliq4 cells/mL) were added to each well of a 384-well plate after which 10 μL compound was also added to the assay plate. The plate was incubated for 4 days at 37 °C, 5% CO2, 95% humidity. Then the plates were incubated at room temperature for about 10 min, 40 μL CTG reagent was added to each well, and the plates were incubated for 30 min at room temperature. Luminescence was detected using the EnSpire Plate Reader, and Prism5 for Windows  was used for data analysis, including IC50 calculations.The HER2-positive breast cancer cell line BT-474, SK-BR-3, gastric cancer cell line NCI-N87, ovarian cancer cell line SK-OV-3, weakly positive breast cancer cell line MCF-7 and HER2-negative breast cancer cell line MDA-MB-468 were obtained from ATCC  and maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and Glutamax (Invitrogen). Cells (3.3 × 107 BT-474 breast cancer cells in 0.2 mL DMEM-Matrigel mixture (1:1 ratio) for tumor development. The treatment started when the mean tumor size reached approximately 150 mm3. The animals were given 1, 2.5, and 5 mg/kg ADC (mil40-12b), antibody + free MMAE, and vehicle alone on Days 0, 7, 14, and 21 or given 3 mg/kg ADCs (mil40-12b and mil40-12c) and vehicle on Days 0, 7, and 14. The animals were monitored twice weekly for body weight and tumor size. Tumor volume was calculated using the formula: TV = a × b2/2, where “a” and “b” are long and short diameters of a tumor, respectively. All procedures related to animal handling, care, and the treatment in this study were performed according to guidelines approved by the Institutional Animal Care and Use Committee of Pharmaron following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care under the licence number ON-CELL-XEM-06012016 on 1 June 2016.Female NOD/SCID mice 6–8 weeks of age, with an average weight of approximately 22 g, were approved by Anikeeper Inc. Female NOD/SCID mice were inoculated subcutaneously with 1 × 10The animals in the xenograft model were subjected to whole-blood hematological analysis at the end of drug administration (28 days) and after 1 month of recovery (58 days). Then, 100 μL whole blood from each animal was drawn. Some samples were diluted 3 times for a final CBC count because of the volume limitation. ADC treatment-related changes in hematology parameters included RBC, HGB, WBC, PLT, Neut, and Lymph compared to the control, and results are expressed as mean ± SD. The data were analyzed base on the number with corrected dilution factor.t-test or two-way ANOVA for multiple comparisons was used. The level of significance was set at p < 0.05. Statistical analyses were performed using Prism5 for Windows .For comparison of hematological indicators, unpaired two-tailed At the end of the xenograft model test, animals in specific groups were used for histopathological studies. The organs mainly included the heart, liver, and lung. Tissue samples were fixed in 10% buffered formalin for 24 h and transferred into 70% ethanol. Dehydration through 70% ethanol, 85% ethanol, 90% ethanol, 95% ethanol × 2, 100% ethanol × 2 followed by 3 changes of xylene and then paraffin. Finally, the tissues were paraffin block embedded and sectioned. Then, H&E staining, mounting with permount, observation, and photographing under a microscope were performed in addition to bone marrow smears.12a–12c are shown in The synthetic routes for the linker-MMAE conjugates 3-propanoic acid (3a). Maleic anhydride 1  was added to a solution of the amino acids 2a  in AcOH. The mixture was stirred at 120 °C for 6 h. The reaction mixture was poured into water after cooling to room temperature and extracted with ethyl acetate (3 × 20 mL). The organic layers were combined, washed with brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure to give the crude product. Purification was performed by silica column chromatography in 1:6 ethyl acetate/petroleum ether (v/v). A white solid was obtained . 1H-NMR : δ 2.49 , 3.61 , 7.03 , 12.37 . ESI m/z (M − H)− calculated for C7H7NO4 168.0; found 168.3.6-hexanoic acid (3b). Compound 3b was synthesized with the experimental protocols described for 3a. Yield was 74%, as a white solid; 1H-NMR : δ 1.24–1.17 , 1.51–1.44 , 2.17 , 3.39 , 7.01 , 11.98 . ESI m/z (M − H)− calculated for C7H7NO4 210.1; found 210.0.2,5-Dioxopyrrolidin-1-yl 3-propanoate (4a). A solution of 3a , 2,4,6-trimethylpyridine  and N-Hydroxysuccinimide (SuOH)  in THF (100 mL) was cooled to 0 °C; the trifluoroacetic anhydride  was added dropwise over 30 min. The reaction mixture was stirred for 1 h at room temperature for 1 h, and then evaporated under reduced pressure and re-dissolved in ethyl acetate (200 mL). The organic layers was washed with 1N HCl and brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure to give the crude product. Purification was performed by silica column chromatography in 1:2 ethyl acetate/petroleum ether (v/v) to give 4.71 g (70%) of 4a as a colorless oil, low temperature placed and converted to a white solid. 1H-NMR : δ 2.79 , 3.04 , 3.74 , 7.05 . ESI m/z (M + H)+ calculated for C11H11N2O6 267.1; found 267.1. ESI m/z (M + Na)+ calculated for C11H10N2NaO6 289.0; found 289.1.2,5-Dioxopyrrolidin-1-yl 6-hexanoate (4b). Compound 4b was synthesized with the experimental protocols described for 4a. Yield was 69%, as a white solid; 1H-NMR : δ 1.45–1.37 , 1.63 , 1.78 , 2.61 , 2.84 , 3.53 , 6.69 . ESI m/z (M + H)+ calculated for C14H17N2O6 309.1; found 309.4. ESI m/z (M + Na)+ calculated for C14H16N2NaO6 331.1; found 331.2.l-valyl-l-alanine(((9H-Fluoren-9-yl)methoxy)carbonyl)- (7a). A solution of 5 , SuOH  and DCC  in THF (250 mL) was stirred at room temperature for 16 h. The reaction mixture was cooled to 0 °C for 2 h, and then filtered to remove the insoluble dicyclohexylurea (DCU). The Filter cake was washed with THF; the filtrate was evaporated under reduced pressure to give the crude product as a glassy solid, which was used for the next step directly without further purification. The glassy solid was redissolved in dimethoxyethane (DME) (200 mL) and THF (100 mL), then added the aqueous sodium hydrogencarbonate solution of l-alanine . The reaction mixture was stirred at room temperature for 16 h, poured into aqueous citric acid solution , and filtered after the filter cake was dried under reduced pressure to give the crude product. The crude product was scattered in ether and purification was performed by ultrasound and filtration 3 times to give 16.2 g (53%) of 7a as a white solid. 1H-NMR : δ 0.88 , 1.27 , 1.96 , 3.89 , 4.22 , 7.33 , 7.43 , 7.74 (t 2H), 7.89 , 8.25 , 12.48 . ESI m/z (M + H)+ calculated for C23H27N2O5 411.2; found 411.3. ESI m/z (M + Na)+ calculated for C23H26N2NaO5 433.2; found 433.4.l-valyl-l-Citrulline(((9H-Fluoren-9-yl)methoxy)carbonyl)- (7b). Compound 7b was synthesized with the experimental protocols described for 7a. Yield was 70%, as a white solid; 1H-NMR : δ 0.89 , 1.42 , 1.58 , 1.72 , 1.98 , 2.96 , 3.94 , 4.16 , 4.25 , 5.43 , 5.98 , 7.33 , 7.43 , 7.76 , 7.89 , 8.21 , 12.61 . ESI m/z (M + H)+ calculated for C26H33N4O6 497.2; found 497.6. ESI m/z (M + Na)+ calculated for C26H32N4NaO6 519.2; found 519.6.(9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (8a). p-Aminobenzyl alcohol  and EEDQ  were added to a solution of the 7a  in DCM (150 mL) and methanol (75 mL). The mixture was stirred at room temperature for 36 h, and the reaction mixture was evaporated under reduced pressure to give the crude product. The crude product was scattered in ether and the purification was performed by ultrasound and filtration 3 times to give 5.34 g (77%) of 8a as a white solid. 1H-NMR : δ 0.88 , 1.30 , 2.00 , 3.91 , 4.22 , 4.30 , 4.40 , 4.42 , 5.13 , 7.24 , 7.34 , 7.41 , 7.52 , 7.75 , 7.89 , 8.22 , 9.96 . ESI m/z (M + H)+ calculated for C30H34N3O5 516.2; found 516.4. ESI m/z (M + Na)+ calculated for C30H33N3NaO5 538.2; found 538.3.(9H-Fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (8b). Compound 8b was synthesized with the experimental protocols described for 8a. Yield was 92%, as a light yellow solid; 1H-NMR : δ 0.86 , 1.41 , 1.57 , 1.69 , 1.98 , 3.00 , 3.92 , 4.23 , 4.30 , 4.41 , 5.12 , 5.42 , 5.98 , 7.23 , 7.32 , 7.41 , 7.46 , 7.54 , 7.74 , 7.89 , 8.12 , 9.99 . ESI m/z (M + Na)+ calculated for C33H39N5NaO6 624.3; found 624.5.(S)-2-Amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (9a). To a solution of the 8a  in DMF (40 mL) was added piperidine (2 mL). The mixture was stirred at room temperature for 2 h, and evaporated under reduced pressure to give the crude product. Purification was performed by silica column chromatography in 20:1 DCM/methanol (v/v) to give 2.05 g (90%) of 9a as a white solid. 1H-NMR : δ 0.85 , 1.29 , 1.92 , 2.80 , 3.00 , 4.43 , 4.48 , 5.13 , 7.24 , 7.53 , 8.18 , 10.0 . ESI m/z (M + H)+ calculated for C15H24N3O3 294.2; found 294.2. ESI m/z (M + Na)+ calculated for C15H23N3NaO3 316.2; found 316.2.(S)-2-((S)-2-Amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (9b). Compound 9b was synthesized with the experimental protocols described for 9a. Yield was 84%, as a light yellow solid; 1H-NMR : δ 0.86 , 1.38 , 1.64 , 1.94 , 2.95 , 3.05 , 4.43 , 4.47 , 5.13 , 5.44 , 6.01 , 7.23 , 7.54 , 8.17 , 10.07 . ESI m/z (M+H)+ calculated for C18H30N5O4 380.2; found 380.3. ESI m/z (M+Na)+ calculated for C18H29N5NaO4 420.2; found 402.3.(S)-2-propanamido)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (10a). Compound 4a  was added to a solution of the 9a  in DMF (40 mL). The mixture was stirred at room temperature overnight, and evaporated under reduced pressure to give the crude product. The crude product was scattered in Ether and the purification was performed by ultrasound and filtration 3 times to give 1.84 g (81%) of 10a as a light yellow solid. 1H-NMR : δ 0.83 , 1.30 , 1.93 , 2.44 , 3.36 , 4.12 , 4.37 , 4.42 , 5.12 , 7.01 , 7.23 , 7.54 , 8.05 , 8.19 , 9.83 . ESI m/z (M + H)+ calculated for C22H29N4O6 445.2; found 445.7. ESI m/z (M + Na)+ calculated for C22H28N4NaO6 467.2; found 467.4.6--N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (10b). Compound 10b was synthesized with the experimental protocols described for 10a. Yield was 87%, as a light yellow solid; 1H-NMR : δ 0.84 , 1.17 , 1.30 , 1.47 , 1.96 , 2.14 , 3.38 , 4.16 , 4.37 , 4.42 , 5.11 , 7.01 , 7.22 , 7.53 , 7.83 , 8.16 , 9.87 . ESI m/z (M + H)+ calculated for C25H35N4O6 487.3; found 487.5. ESI m/z (M + Na)+ calculated for C25H34N4NaO6 509.2; found 509.4.6--N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide (10c). Compound 10c was synthesized with the experimental protocols described for 10a. Yield was 79%, as a light yellow solid; 1H-NMR : δ 0.83 , 1.20 , 1.47 , 1.58 , 1.68 , 1.95 , 2.18 , 2.98 , 3.32 , 4.19 , 4.36 , 4.42 , 5.12 , 5.43 , 5.99 , 7.01 , 7.22 , 7.54 , 7.83 , 8.09 , 9.93 . ESI m/z (M + H)+ calculated for C28H41N6O7 573.3; found 573.6. ESI m/z (M + Na)+ calculated for C25H34N4NaO6 595.3; found 595.7.4-((S)-2-((S)-2-propanamido)-3-methylbutanamido)propanamido)benzyl (4-nitrophenyl) carbonate (11a). Bis(4-Nitrophenyl) Carbonate  and DIPEA  were added to a solution of the 10a  in DMF (15 mL). The mixture was stirred at rt for 5 h, and evaporated under reduced pressure to give the crude product. The crude product was scattered in ether and the purification was performed by ultrasound and filtration 3 times to give 0.5 g (86%) of 11a as a light yellow solid. 1H-NMR : δ 0.84 , 1.31 , 1.94 , 2.45 , 3.60 , 4.13 , 4.38 , 5.24 , 7.00 , 7.41 , 7.57 , 7.64 , 8.02 , 8.19 , 8.31 , 9.96 . ESI m/z (M + H)+ calculated for C29H32N5O10 610.2; found 610.4. ESI m/z (M + Na)+ calculated for C29H31N5NaO10 632.2; found 632.6.4-((S)-2-((S)-2-hexanamido)-3-methylbutanamido)propanamido)benzyl (4-nitrophenyl) carbonate (11b). Compound 11b was synthesized with the experimental protocols described for 11a. Yield was 93%, as a light yellow solid; 1H-NMR : δ 0.85 , 1.18 , 1.31 , 1.47 , 1.96 , 2.15 , 3.36 , 4.17 , 4.39 , 5.24 , 7.00 , 7.41 , 7.56 , 7.64 , 7.80 , 8.17 , 8.31 , 10.00 . ESI m/z (M + H)+ calculated for C32H38N5O10 652.3; found 652.6. ESI m/z (M + Na)+ calculated for C32H37N5NaO10 674.2; found 674.6.4-((S)-2-((S)-2-hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (11c). Compound 11c was synthesized with the experimental protocols described for 11a. Yield was 92%, as a light yellow solid; 1H-NMR : δ 0.84 , 1.22 , 1.47 , 1.58 , 1.68 , 1.95 , 2.17 , 3.00 , 3.28 , 4.19 , 4.38 , 5.24 , 5.44 , 6.01 , 7.01 , 7.41 , 7.56 , 7.66 , 7.83 , 8.15 , 8.32 , 10.10 . ESI m/z (M + H)+ calculated for C35H44N7O11 738.3; found 738.6. ESI m/z (M + Na)+ calculated for C35H43N7NaO11 760.3; found 760.6.4-((S)-2-((S)-2-propanamido)-3-methylbutanamido)propanamido)benzyl((S)-1-(((S)-1--1-((S)-2--3--1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (12a). To a solution of the 11a , MMAE , HOBt  in DMF (3 mL) was added DIPEA . The mixture was stirred at rt overnight, and then poured into water (20 mL). The mixture was extracted with ethyl acetate (3 × 20 mL), the organic layers were combined, washed with brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure to give the crude product. Purification was performed by silica column chromatography in 50:1-10:1 DCM/methanol (v/v) to give 47.6 mg (72%) of 12a as a white solid. 1H-NMR : δ 0.88–0.75 , 1.05–0.97 , 1.30–1.23 , 1.54–1.48 , 1.83–1.70 , 1.95–1.91 , 2.14–2.08 , 2.28–2.22 , 2.48–2.38 , 2.89–2.83 , 2.97 , 3.24–3.12 , 3.33–3.30 , 3.47 , 3.64–3.55 , 4.05–3.92 , 4.13 , 4.26 , 4.49–4.35 , 4.76–4.62 , 5.09–4.96 , 5.44–5.36 , 7.01 , 7.16 , 7.35–7.26 , 7.57 , 7.66 , 7.91 , 8.05 , 8.12 , 8.22 , 8.34 , 9.92 . ESI m/z (M + H)+ calculated for C62H94N9O14 1188.6920; found 1188.6913. ESI m/z (M + Na)+ calculated for C62H93N9NaO14 1210.6740; found 1210.6734.4-((S)-2-((S)-2-hexanamido)-3-methylbutanamido)propanamido)benzyl((S)-1-(((S)-1--1-((S)-2--3--1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (12b). Compound 12b was synthesized with the experimental protocols described for 12a. Yield was 80%, as a white solid; 1H-NMR : δ 0.88–0.73 , 1.05–0.93 , 1.30–1.21 , 1.55–1.43 , 1.81–1.71 , 2.00–1.91 , 2.19–2.06 , 2.28–2.21 , 2.43–2.39 , 2.88–2.83 , 2.97 , 3.24–3.12 , 3.33–3.30 , 3.49–3.38 , 3.59–3.53 , 4.01 , 4.17 , 4.26 , 4.49–4.36 , 4.75–4.61 , 5.11–4.96 , 5.43–5.36 , 7.01 , 7.16 , 7.34–7.24 , 7.57 , 7.66 , 7.83 , 7.91 , 8.10 , 8.19 , 8.35 , 9.95 . ESI m/z (M + H)+ calculated for C65H100N9O14 1230.7390; found 1230.7380. ESI m/z (M + Na)+ calculated for C65H99N9NaO14 1252.7209; found 1252.7202.4-((S)-2-((S)-2-hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((S)-1-(((S)-1--1-((S)-2--3--1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (12c). Compound 12c was synthesized with the experimental protocols described for 12a. Yield was 64%, as a white solid; 1H-NMR : δ 0.88–0.73 , 1.07–0.97 , 1.59–1.15 , 1.80–1.66 , 1.99–1.93 , 2.20–2.07 , 2.27 , 2.41 , 2.86 , 3.03–2.94 , 3.32–3.12 , 3.36 , 3.56 , 4.01 , 4.18 , 4.26 , 4.49–4.34 , 4.75–4.62 , 5.11–4.95 , 5.60–5.20 , 6.01 , 7.01 , 7.17 , 7.34–7.24 , 7.41 , 7.58 , 7.66 , 7.82 , 7.92 , 8.12 , 8.35 , 10.00 . ESI m/z (M + H)+ calculated for C68H106N11O15 1316.7870; found 1316.7864. ESI m/z (M + Na)+ calculated for C68H105N11NaO15 1338.7689; found 1338.7678.Mass spectrometry and NMR details of the above synthesized compounds are provided in the Currently, ADC is a promising and rapidly growing area of targeted therapeutics for oncology; however, one of the biggest challenges in their development has been the generation of suitable linkers for the conjugation of antibody and cytotoxins. ADCs that load the popular MMAE usually contain VC-based linkers. In this study, a novel ADC that conjugated MMAE to a humanized anti-HER2 antibody via a VA-based cleavable linker was generated to explore its characteristics when used as an anti-tumor drug.First, VA-containing drug payload exhibits similar performance as VC in terms of in vitro stability and enzymatic activity, and VA-based ADC may be preferred to VC-based ADC on the conjugate process and aggregation. Further, we observed that the VA-based ADC displays favorable potency in vitro and in vivo while not producing significant systemic toxicity during the treatment and recovery period in xenograft studies. In summary, VA-based ADC with MMAE as the payload is shown to be efficacious in the model of HER2-overexpressing cancer, and may have advantages in the generation process and material source of the product compared to current linker technology.


Their structures were established based on spectroscopic methods. The absolute configuration of zosteropenilline A (1) as 4R, 5S, 8S, 9R, 10R, and 13S was determined by a combination of the modified Mosher’s method, X-ray analysis, and NOESY data. Absolute configurations of zosteropenillines B–D (2–4) were determined by time-dependent density functional theory (TD-DFT) calculations of ECD spectra. The effect of compounds 1–3, 7, 8, 10, and 11 on the viability of human drug-resistant prostate cancer cells PC3 as well as on autophagy in these cancer cells and inhibitory effects of compounds 1, 2, and 8–10 on NO production in LPS-induced RAW 264.7 murine macrophages were examined.Twelve new polyketides, zosteropenillines A–L ( Penicillium thomii and P. lividum associated with the brown alga Sargassum miyabei + and by 13C NMR analyses. The general features of the 13C NMR of 2 resembled those of 1 with the exception of the C-9–C-11 and C-15 carbon signals. COSY and HSQC spectra of 2 revealed the partially connectivity sequence of the protons in the A ring as CH2(15)-CH(8)-CH2(9)-CH(10). These data and HMBC correlations from H-9b (δH 0.93) to C-7 (δC 29.1), C-8 (δC 40.5), C-10 (δC 41.4), C-11 (δC 134.4), and C-15 (δC 68.1); and from H2-15  to C-7, C-8 and C-9, established the A ring structure, lacking the hydroxy group at C-9, and indicated the presence of a hydroxymethyl group at C-8 in 2. The NOESY cross-peaks H-4 (δH 2.07)/H-10 (δH 1.74), H3-14 (δH 1.26); H-8 (δH 1.63)/H-9α (δH 1.91), H-10; H-5 (δH 1.78)/H-9β (δH 0.93); H-9β/H2-15  indicated the trans-ring fusion of the A and B ring, cis-ring fusion of the B and C ring, and the β-orientation of hydroxymethyl group at C-8. Compound 2 was named zosteropenilline B.The molecular formula of 3 was established to be C15H22O3 from a HRESIMS peak at m/z 273.1463 [M + Na]+ and by 13C NMR analyses. The 1H and 13C NMR spectra (2) with the exception of the C-6–C-10 and C-15 carbon and proton signals. The HMBC correlations H-9a (δH 1.75)/C-8 (δC 69.7), C-10 (δC 36.9); H3-15 (δH 1.25)/C-7 (δC 24.9), C-8, C-9 (δC 44.5) established the structure of 3 including the hydroxy and methyl groups at the C-8 position. The relative configuration of 3 was defined based on the observed NOESY correlations  with the exception of the C-7–C-9 and C-15 carbon and proton signals. The mutual correlations from H3-15 to C-7 and C-9 established the structure of the A ring and location methyl group at C-8. Compound 4 was named zosteropenilline D.The molecular formula of NMR data  observed2–4) were determined on the basis of the ECD spectroscopy data. The geometry of the stable conformations of compounds 1–4 were optimized using general procedure, described in the experimental section of the paper. The internal rotations of the hydroxyl groups as well as the inversions of the six-membered rings A and C were accounted for during fulfilled conformational analysis. We found that for all these compounds ring C can exist in two stable conformations: “chair” and “boat”. The “chair” conformation is more stable for about ΔE# (chair → boat) ≈ 2 kcal·mol−1 and the barrier for interconversion between conformations is ΔE# (chair → boat) ≈ 6 kcal·mol−1. Analogously, ring A can also exist in “chair” and “boat” conformations. The “chair” conformation is more stable for about ΔE# (chair → boat) ≈ 10 kcal·mol−1 and the barrier for interconversion between conformations is ΔE# (chair → boat) ≈ 14 kcal·mol−1  with PBE1PBE exchange-correlation functional and cc-pvTz basis set − and by 13C NMR analyses. The COSY-45 data and HMBC correlations from H-4 (δH 2.11) to C-5 (δC 38.5), C-6 (δC 29.0), C-10 (δC 53.5), from H-5 (δH 1.95) to C-6, C-10, from H-6a (δH 1.33) to C-4 (δC 63.7), C-7 (δC 31.8) and C-10, from H-7a (δH 1.73) to C-5, C-9 (δC 77.2), from H3-15 (δH 1.06) to C-7, C-8 (δC 39.2), C-9, from H-9 (δH 3.29) to C-5, C-11 (δC 67.3), from H-10 (δH 1.61) to C-5, C-9, C-11, from H-11 (δH 4.38) to C-12 (δC 72.7), and from H3-14 (δH 1.36) to C-4, C-12, C-13 (δC 77.5) revealed the presence of a decalin moiety in 1 and established the location of methyl groups at C-8 and C-13 and hydroxy groups at C-9, C-11 and C-12. HMBC correlations from H-1a (δH 4.22) to C-2 (δC 37.9), C-3 (δC 206.0) and C-13, from H-2a (δH 2.67) to C-1 (δC 60.9), C-3, from H-4 (δH 2.11) to C-2 and C-3 established a saturated γ-pyrone derivative in 5  based on COSY and HMBC correlations. The COSY-45 data and HMBC correlations from H2-1 (δH 3.91) to C-2 (δC 48.6) and C-3 (δC 217.6), from H2-2  to C-1 (δC 57.6) and C-3, and from H-4 (δH 2.59) to C-3 indicated the presence of a 3-hydroxy-1-oxopropyl residue (C-1–C-3 residue numbering) at C-4. The relative configuration of 6 was assigned on the basis of a NOESY experiment and 1H-1H coupling constants  which was earlier isolated from the P. thomii associated with the brown alga Sargassum pallidum + and by 13C NMR analyses. The 1H NMR, 13C NMR, DEPT and HSQC spectra of 8  groups, four methylenes  including one oxygen-bearing , seven methines  including two oxygen-bearing, one carbonyl carbon (δC 214.0) and one 3sp quaternary carbon (δC 131.5). The structure of a 1,2,3,4-tetrasubstituted cyclohexane ring and side chain in 8 were found by extensive NMR spectroscopy  as for zosteropenillines E (5) and F (6). HMBC correlations from H-4 (δH 2.92) to C-5 (δC 37.9), C-12 (δC 129.0), C-13 (δC 131.5), from H-10 (δH 1.28) to C-5, C-11 (δC 73.2), C-12, from H-11 (δH 4.32) to C-12, C-13, and from H3-14 (δH 1.60) to C-4 (δC 61.7), C-12, C-13 established a B ring structure, Δ12 double bond and the location of the hydroxy group at C-11. The relative configuration of 8 was assigned based on NOESY cross-peaks H-4/H-10; H-5 (δH 1.69)/H-9 (δH 3.25), H-11, H3-15 (δH 1.02); H-8 (δH 1.47)/H-10; H-9/H-11 and 1H-1H coupling constants  and F (6). Observed long-range COSY correlations H-4 (δH 3.15)/H2-14 , H-12 (δH 6.20)/H-14b. and HMBC correlations from H-4 (δH 3.15) to C-13 (δC 140.8), C-14 (δC 112.8), from H-9 (δH 2.89) and H-10 (δH 1.85) to C-11 (δC 130.6) and from H2-14  to C-4 (δC 59.6), C-12 (δC 129.3), C-13 indicated the presence of a diene system at the C-11 and C-13(14) in 9. The relative configuration of 9 was assigned based on the NOESY correlations H-4/H-10; H-8 (δH 1.43)/H-10 and H-5 (δH 1.68)/H-9, H3-15 (δH 1.05). Compound 9 was named zosteropenilline I.The molecular formula of 10 was established to be C15H24O4 from a HRESIMS peak at m/z 291.1573 [M + Na]+ and by 13C NMR analyses. The 13C NMR data (10 matched the data reported for zosteropenilline G (7) with the exception of the C-7–C-9 and C-15 carbon signals. These data, together with the molecular mass difference of 16 mass units between 7 and 10, indicated the presence of the hydroxymethyl group at C-8 in 10 instead of a methyl group. NOESY cross-peaks H-4 (δH 2.92)/H-6b (δH 0.95), H-10 (δH 1.87); H-5 (δH 1.52)/H-7b (δH 1.02), H-9b (δH 0.82), H3-14 (δH 1.20); H-7b/H-9b, H2-15 ; H-8 (δH 1.61)/H-6b, H-10 indicated the trans-ring fusion of the A and B rings, a β-orientation of the side chain, H3-14 and hydroxymethyl group at C-8. Compound 10 was named zosteropenilline J.The molecular formula of NMR data  observed11 was established to be C15H22O5 from a HRESIMS peak at m/z 305.1370 [M + Na]+ and by 13C NMR analyses. The UV spectrum exhibits a λmax at 242 nm (log ε 3.46), consistent with the enone system in 11. The COSY and HSQC spectra of 11 revealed the partial connectivity sequences of the protons in the A ring as CH(5)-CH2(6)-CH2(7)-CH(8) and CH2(15)-CH(8)-CH2(9). These data and HMBC correlations from H-9a (δH 2.60) to C-7 (δC 27.9), C-8 (δC 41.1) and C-15 (δC 67.4), from H-9b (δH 2.01) to C-10 (δC 166.3), from H2-15  to C-7, C-8 and C-9 (δC 38.3) established the structure of A ring. Long range correlations from H-4 (δH 3.01) to C-5 (δC 39.4), C-10, C-12 (δC 200.3) and C-13 (δC 74.2), from H-11 (δH 5.90) to C-5 and C-13 and from H3-14 (δH 1.19) to C-4 (δC 61.6), C-12, C-13 determined the structure of B ring and indicated the 10-en-12-one position for the enone chromophore in 11. The structure of side chain in 11 was found by NMR spectroscopy as for zosteropenilline F (6). Observed NOESY correlations H-5 (δH 2.89)/H-9b (δH 2.01), H3-14; H2-15/H-9b indicated these protons to be on the same side of the molecule. These data and the magnitude of the coupling constant of the H-4 signal  indicated H-4 and 13-OH to be on the other side of molecule. Compound 11 was named zosteropenilline K.The molecular formula of 12 was established to be C15H24O5 from a HRESIMS peak at m/z 307.1501 [M + Na]+ and by 13C NMR analyses. The structure of the A ring in 12 was established based on COSY and HMBC correlations as for zosteropenilline K (11). HMBC correlations from H-11 (δH 5.60) to C-5 (δC 40.3), C-9 (δC 37.2), C-12 (δC 68.0), C-13 (δC 72.4), from H-12 (δH 4.36) to C-4 (δC 57.7), C-10 (δC 144.5), C-11 (δC 118.8), C-13, and from H3-14 (δH 1.20) to C-4, C-12, C-13 established the structure of B ring, location oxygenated functions at C-12, C-13 and methyl group at C-13 in 12. The relative configuration of 12 was defined based on NOESY correlations H-5/H3-14; H3-14/H-12; H-4/H-6b; H-6b/H-8 and the magnitude of the coupling constant of the H-4 signal . Compound 12 was named zosteropenilline L. Besides the new zosteropenillines (1–12), a known pallidopenilline A (13) was also isolated from this fungus. For the first time, pallidopenilline A was found in fungus P. thomii KMM 4675 +m/z 251.1636 .Zosteropenilline B (2): colorless crystals; c 0.12, MeOH); UV , λmax (∆ε) 201 (−1.55), 291 (−0.23), 332 (+0.03), 365 (−0.03) nm; IR (CHCl3) νmax 3460, 2957, 2919, 1701, 1602, 1455, 1385, 1108, 1055 cm−1; 1H and 13C NMR data, see +m/z 273.1474 , [M − H]−m/z 249.1496 .Zosteropenilline C (3): white solid; c 0.1, MeOH); UV (MeOH) low intensity band; CD , λmax (∆ε) 204 (−9.86), 297 (−1.63) nm; IR (CHCl3) νmax 3604, 2973, 2927, 1705, 1602, 1453, 1375, 1140, 1044 cm−1; 1H and 13C NMR data, see +m/z 273.1463 , [M − H]−m/z 249.1499 .Zosteropenilline D (4): amorphous; c 0.06, MeOH); UV (MeOH) low intensity band; CD , λmax (∆ε) 204 (−4.01), 296 (−0.96) nm; IR (CHCl3) νmax 2956, 2929, 1706, 1602, 1456, 1376, 1121, 1069 cm−1; 1H and 13C NMR data, see +m/z 257.1510 .Zosteropenilline E (5): amorphous; c 0.05, MeOH); UV (MeOH) low intensity band; CD , λmax (∆ε) 206 (−0.27), 293 (−0.21), 299 (−0.22), 305 (−0.19), 339 (−0.03); 1H and 13C NMR data, see −m/z 283.1538 .Zosteropenilline F (6): white solid; c 0.11, MeOH); UV (MeOH) low intensity band; CD , λmax (∆ε) 194 (+0.70), 204 (−1.14), 230 (−0.12), 293 (+0.08), 319 (−0.02) nm; IR (CHCl3) νmax 3621, 2976, 2928, 1702, 1602, 1450, 1390, 1046 cm−1; 1H and 13C NMR data, see +m/z 291.1579 , [2M + Na]+m/z 559.3242 .Zosteropenilline G (7): white solid; c 0.034, MeOH); UV (MeOH) λmax (log ε) low intensity band; CD , λmax (∆ε) 199 (−1.85), 218 (−1.07), 244 (+0.11), 287 (+0.57) nm; IR (CHCl3) νmax 3623, 3459, 2884, 1704, 1670, 1602, 1455, 1391, 1056 cm−1; 1H and 13C NMR data, see +m/z 275.1621 , [M − H]−m/z 251.1646 .Zosteropenilline H (8): amorphous; c 0.11, MeOH); UV (MeOH) low intensity band; CD , λmax (∆ε) 209 (+3.62), 295 (+1.98) nm; IR (CHCl3) νmax 3617, 3489, 2959, 2929, 1703, 1602, 1456, 1383, 1055 cm−1; 1H and 13C NMR data, see +m/z 291.1571 , [2M + Na]+m/z 559.3236 .Zosteropenilline I (9): white solid; c 0.07, MeOH); UV (MeOH) low intensity band; CD , λmax (∆ε) 204 (−6.61), 247 (−1.13) nm; 1H and 13C NMR data, see +m/z 273.1462 , [2M + Na]+m/z 523.3029 .Zosteropenilline J (10): colorless crystals; c 0.71, MeOH); UV (MeOH) low intensity band; CD , λmax (∆ε) 196 (−3.81), 213 (+0.35), 238 (+0.05), 295 (−2.15) nm; IR (CHCl3) νmax 3605, 3497, 2959, 2929, 1702, 1602, 1456, 1383, 1061 cm−1; 1H and 13C NMR data, see +m/z 291.1573 , [2M + Na]+m/z 559.3236 .Zosteropenilline K (11): amorphous; c 0.09, MeOH); UV (MeOH) λmax (log ε) 242 (3.46) nm; CD , λmax (∆ε) 193 (0.75), 219 (−4.62), 244 (+0.65), 258 (−0.13), 279 (0.46), 294 (0.41), 318 (0.35) nm; IR (CHCl3) νmax 3617, 3464, 2958, 2927, 1702, 1671, 1653, 1604, 1456, 1390, 1059 cm−1; 1H and 13C NMR data, see +m/z 305.1370 .Zosteropenilline L (12): amorphous; c 0.05, MeOH); UV (MeOH) low intensity band; CD , λmax (∆ε) 196 (−1.29), 210 (+0.14), 298 (−1.12), 349 (+0.04) nm; 1H and 13C NMR data, see +m/z 307.1501 .Pallidopenilline A (13): colorless crystals; c 0.1, MeOH); UV (MeOH) λmax (log ε) low intensity band; CD , λmax (∆ε) 196 (−9.89), 213 (+1.14), 294 (−1.78) nm; 1H and 13C NMR data, +m/z 291.1575 .1 (1.75 mg) in pyridine were added 4-dimethylaminopyridine  and (R)-MTPA-Cl (1.2 μL) at room temperature, and the mixture was stirred for 1.5 h. After evaporation of the solvent, the residue was purified by HPLC on an YMC Si  column eluted with n-hexane–acetone (85:15) to afford the (S)-MTPA ester of 1 (1a). The (R)-MTPA ester of 1 (1b) was prepared similarly manner using (S)-MTPA-Cl. NMR data of -MTPA esters of 1 (1a) m/z 489.1864 [M + Na]+ , m/z 955.3830 [2M + Na]+ ; HRESIMS (1b) m/z 489.1858 [M + Na]+ , m/z 955.3815 [2M + Na]+ .To a solution of ers of 1 . HRESIMS15H22O3 were collected at T = 296(2) K (α-C15H22O3) on a BRUKER Kappa APEX2 diffractometer with graphite monochromated Mo Kα radiation (λ = 0.71073 Å). Intensity data were corrected for absorption using the multi-scan method. The structures were solved using direct methods and refined by least-squares calculation in anisotropic approximation for non-hydrogen atoms. Hydrogen atoms were placed at idealized positions and refined using a riding model. Data collection, reduction, and refinement of the lattice parameters were performed using the Apex II software package [http://www.ccdc.cam.ac. uk/data_request/cif .Experimental intensity data for C package . All cal package . Selecte1 in a hexane solvent and for 2–4 in a CH3OH solvent were done using density functional theory (DFT) with the exchange-correlation functional PBE1PBE [radii = UFF). The statistical weights (img) of different conformations of 1–4 were obtained according to Equation (1):1–4 and ∆imG = iG − mG; G = Eel + Gtr,T + Grot,T + Gvib,T is a sum of electronic, translational, rotational, and vibrational contributions to the Gibbs free energy, calculated at temperature T = 298.15 K; the subscript “m” denotes conformation, for which G is minimal. The excitation energies and the rotatory strengths were calculated using time-dependent density functional theory (TDDFT). Theoretical ECD spectra ) were simulated as a superposition of Gauss-type functions, individual for each transition from electronic ground state to the i-th calculated excited electronic state (1 ≤ i ≤ 50). The same value ξ = 0.20 eV for the bandwidths at 1/e peak heights was used. The total theoretical ECD spectra were obtained after statistical averaging:The conformational analyses of  PBE1PBE , the pol PBE1PBE  and 6-31 PBE1PBE . The molε(peakλ)| is a modulo of the peak value for the chosen characteristic band in the corresponding ECD spectrum.The scaled theoretical and experimental ECD spectra were obtained according to Equation (3):2 for 2 h. After adhesion, cells were incubated with tested compounds (10.0 µM) for 24 h. For endogenously generated NO detection studies, the cells were coincubated with 10 µM of FA–OMe fluorescent probe for 8 h [ex = 460 nm and λem = 524 nm. In each experiment LPS from E. coli serotype 055:B5  were used as a positive control. Fluorescent intensity was measured using plate reader PHERAstar FS .RAW 264.7 murine macrophages were plated into 96-well microplates and incubated at 37 °C with 5% CO for 8 h . Prior tt-test was used to evaluate the data with the significance level of p < 0.05. The means and standard errors for each treatment were calculated and plotted using SigmaPlot 3.02 software .All assays were performed at least in triplicate. The results are expressed as the mean ± standard error (SD). A Student’s The human prostate cancer cells line PC-3 was purchased from ATCC. Cells were cultured according to the manufacturer’s instructions in 10% FBS/RPMI media  containing penicillin/streptomycin (Invitrogen). Cells were continuously kept in culture for a maximum of three months, and were routinely inspected microscopically for stable phenotype and regularly checked for contamination with mycoplasma. Cell line authentication was recently performed by DSMZ  using highly polymorphic short tandem repeat loci .The in vitro cytotoxicity of individual substances was evaluated using the MTT -2,5-diphenyltetrazolium bromide) assay, which was performed as previously described .Western blotting was performed as described before . For det1–12) belong to the series of lovastatin-related polyketides that are formed by an enzymatic intramolecular Diels–Alder cycloaddition of a linear hexaketide precursor [1–12), have been isolated from the extract of the marine-derived fungus Penicillium thomii. The absolute configuration zosteropenilline A (1) was determined by a combination of the modified Mosher’s method, X-ray analysis, and NOESY data. Absolute configurations of zosteropenillines B–D (2–4) were determined by time-dependent density functional theory (TD-DFT) calculations of ECD spectra. Zosteropenillines B, G, and I at a non-toxic concentration of 10.0 µM induced a moderate downregulation of NO production in macrophages stimulated with LPS.Zosteropenillines A–L (recursor ,21,22. Precursor ,25,26,27


H)-ones are compounds which possess a wide range of physical and pharmaceutical applications. These compounds can be easily prepared by cyclocondensation of o-phenylenediamines and aroylpyruvates. Unsymmetrically substituted o-phenylenediamines can be obtained form regioisomeric mixtures of 3,4-dihydroquinoxalin-2(1H)-ones. It is often quite difficult to get a pure regioisomer and determine its structure without controlling the reaction selectivity and exploitation of complex NMR techniques . This article examines the regioselectivity of the cyclocondensation between six monosubstituted o-phenylenediamines  and the derivatives of p-chlorobenzoylpyruvate (ester or acid) which we studied. Six regioisomeric 3,4-dihydroquinoxalin-2(1H)-one pairs were selectively prepared and characterised. Based on our experiences, a simplified methodology for determining the structure of the regioisomers was proposed. We developed two general and highly selective methodologies starting from the same o-phenylenediamines and activated 4-chlorobenzoylpyruvates (ester or acid) enabling switching of 3,4-dihydroquinoxalin-2(1H)-one regioselectivity in a predictable manner. For comparison, all regioselective cyclocondensations were performed with the same standardized conditions , differing only by the additives p-TsOH or HOBt/DIC . Both selected methods are simple, general and highly regioselective (72–97%). A mechanism for the regioselectivity was also proposed and discussed. This study can be used as a guide when choosing the most optimal reaction conditions for the synthesis of the desired 3,4-dihydroquinoxalin-2(1H)-one regioisomers with the best selectivity. The demonstrated methodologies in this article may also be applied to differently substituted 3,4-dihydroquinoxalin-2(1H)-ones in general, which could expand the synthetic impact of our results.3-Acylmethylidene-3,4-dihydroquinoxalin-2(1 H)-one moiety is frequently found in compounds that exhibit biological activity, particularly antimicrobial, anticancer, anti-HIV, antithrombotic, analgesic and antidiabetic  in 3.0 mL of DMF (abs) under Ar. The reaction mixture was stirred for 5 min. Then o-phenylenediamine  was added and the mixture was stirred at rt under Ar for 72 h. The precipitated product mixture obtained after filtration (or centrifugation) was triturated by 3 mL of Et2O and crystallized from DMSO (if not otherwise stated) to yield the main solid regioisomer 16 or 17.Diisopropylcarbodiimide 82 μL  was added to a solution of 4-chlorobenzoylpyruvic acid   of 16d (SYN). Mp: 363.0–365.0 °C (DMSO), yellow solid compound. 1H NMR  δ 13.48 ), 12.92 , 12.26 ), 8.04  = 1.5 Hz, 1H, H-CA(5)), 7.99  = 8.4 Hz, 2H, 2 × H-CB(2)), 7.69  = 8.3 Hz, J = 1.5 Hz, 1H, H-CA(7)), 7.57  = 8.4 Hz, 2H, 2 × H-CB(3)), 7.18  = 8.3 Hz, 1H, H-CA(8)), 6.79 ; 13C NMR  δ 187.7 (CB(1)C=O), 167.0 (-COOH), 156.4 (CA(2)), 145.8 (CA(3)), 137.8 (CB(1)), 137.2 (CB(4)), 130.8 (CA(8a)), 129.4 and 129.3 (2 × CB(2 and 3)), 126.4 (CA(6)), 125.6 (CA(7)), 124.4 (CA(4a)), 118.3 (CA(5)), 115.8 (CA(8)), 90.0 (-COCH=). FTIR cm−1 (solid): 3184 , 2925 (m), 1732 (w), 1688 , 1615 (s), 1586 (s), 1550 (w), 1486 (w), 1366 (m), 1247 (m), 1218 (m), 1095 (m), 1065 (w), 1011 (w), 787 (w), 750 (m) cm−1; MS (ESI m/z): 341.2 [M − H]−; anal. calcd for C17H11ClN2O4 (342.73): C, 59.57; H, 3.23; Cl, 10.34; N, 8.17; found: C, 59.75; H, 3.38; Cl, 10.23; N, 8.01.The compound 6d (SYN)  was prep17d (ANTI)  of DMAP as an additive. The crude product was crystallized from DMSO and obtained as salt with DMAP. To liberate the free acid 17d (ANTI), the salt was suspended in 1 M HCl, stirred for 24 hours, the solid material was filtered off, washed with water and dried to yield 48.4 mg  of 17d (ANTI).Compound d (ANTI)  was prep17d (ANTI) was also prepared according to general procedure B from diamine 11d, acid 12b and HOBt/DIC. The crude product was crystallized from DMSO to yield 105.9 mg  of 17d (ANTI). Mp: 391.0–392.0 °C [DMSO], yellow solid compound. 1H NMR  δ 13.44 ), 12.95 , 12.14 ), 8.02  = 8.6 Hz, 2H, 2 × H-CB(2)), 7.73  = 1.6 Hz, 1H, H-CA(8)), 7.67  = 8.4 Hz, J = 1.6 Hz, 1H, H-CA(6)), 7.61  = 8.4 Hz, 1H, H-CA(5)), 7.60  = 8.6 Hz, 2H, 2 × H-CB(3)), 6.87 ; 13C NMR  δ 188.3 (CB(1)C=O), 167.0 (-COOH), 156.1 (CA(2)=O), 145.6 (CA(3)), 2 × 137.6 (CB(1 and 4)), 129.5 and 129.3 (2 × CB(2 and 3)), 128.1 (CA(4a)), 2 × 126.9 (CA(7 and 8a)), 125.2 (CA(6)), 2 × 116.9 (CA(5 and 8)), 90.8 (-COCH=); FTIR cm−1 (solid): 3486 (m), 3206 , 2634 (w), 1706 , 1661 (w), 1628 (m), 1586 , 1522 (w), 1398 (m), 1374 (w), 1291 (m), 1248 (m), 1184 (m), 1093 (w), 1056 (m), 1009 (w), 899 (w), 781 (w), 764 (w), 721 (w) cm−1; MS (ESI m/z): 341.0 [M − H]−; anal. calcd for C17H11ClN2O4 (342.73): C, 59.57; H, 3.23; N, 8.17; found: C, 59.82; H, 3.10; N, 8.32.Alternatively File 1Additional experimental and characterisation data.


The in vitro antimicrobial activity of the synthesized compounds was investigated against various pathogenic strains; the results obtained were further explained with the help of DFT and molecular orbital calculations. Compound 1b and 1f displayed good antimicrobial activity and synergistic effects when used with kanamycin and amphotericin B. Furthermore, in vitro cytotoxicity of compounds 1b and 1f were studied against HeLa cells  and Hek-293 cells. The results of molecular docking study were used to better rationalize the action and prediction of the binding modes of these compounds.The control of antimicrobial resistance (AMR) seems to have come to a dead end. The major consequences of the use and abuse of antibacterial drugs are the development of resistant strains due to genetic mutability of both pathogenic and nonpathogenic microorganisms. We, herein, report the synthesis, characterization and biological activities of coumarin-thiazole-pyrazole (CTP) molecular hybrids with an effort to explore and overcome the increasing antimicrobial resistance. The compounds were characterized by analyzing their IR, Mass,  Staphylococcus aureus, Staphylococcus enteric, Escherichia coli) and fungal  pathogens are developing resistance against widely prescribed antibacterial and antifungal drugs like amphotericin B (Amp B), fluconazole (FLC), chloroamphenicol and penicillin +; Calcd.: 461.92;Anal. Calcd. for C23H16ClN5O2S: C, 59.80; H, 3.49; N, 15.16. Found: C, 59.85; H, 3.50; N, 15.46.4-(6-Chlorocoumarin-3-yl)-2-diazenyl)pyrazol-1-yl) thiazole (1b). Rf: 0.6; mp 220–222°C;Yield: 88%; IR (cm-1): 1719 (C = O), 1602 (C = N); 1H NMR : 8.30 , 8.08 , 7.65–7.61 , 7.49 , 7.31 , 2.74 , 2.20 ; 13C NMR : 167.47 (C = O), 159.23 (C2-thiazole), 154.49 (C8'-coumarin), 149.56 (C4-thiazole), 147.15 (C4-coumarin), 143.40 (C-Ar), 137.33 (C-Ar), 133.24 (C-Ar), 128.86 (C-Ar), 125.07 (C-Ar), 124.26 (C-Ar), 124.16 (C-Ar), 122.74 (C-Ar), 119.53 (C-Ar), 118.64 (C-Ar), 116.89 (C-Ar), 115.04 (C-Ar), 112.54 (C-Ar), 31.16 (5-CH3 pyrazole), 25.30 (3-CH3 pyrazole); MS (m/z) Found: 542.1, 540.1; Calcd.: 540.82; Anal. Calcd. for C23H15BrClN5O2S: C, 51.08; H, 2.80; N, 12.95. Found: C, 51.00; H, 2.82; N, 13.14.4-(Coumarin-3-yl)-2-diazenylpyrazol-1-yl)thiazole (1c). Rf: 0.6; mp 240–241°C; Yield: 80%; IR (cm-1): 1713 (C = O), 1601 (C = N); 1H NMR : 8.32 , 8.02 , 7.87 , 7.71 , 7.49 , 7.62 , 7.43–7.40 , 2.74 , 2.20 , 2.05 ; 13C NMR : 162.73 (C = O), 157.90 (C2-thiazole), 147.90 (C8'-coumarin), 140.92 (C4-thiazole), 140.62 (C4-coumarin), 139.09 (C-Ar), 134.47 (C-Ar), 133.02 (C-Ar), 132.82 (C-Ar), 130.27 (C-Ar), 128.85 (C-Ar), 128.44 (C-Ar), 128.39 (C-Ar), 128.35 (C-Ar), 126.95 (C-Ar), 126.92 (C-Ar), 125.36 (C-Ar), 123.28 (C-Ar), 118.07 (C-Ar), 117.99 (C-Ar), 113.57 (C-Ar), 35.10 (5-CH3 pyrazole), 29.61 (3-CH3 pyrazole), 14.60 (Ph-CH3); MS (m/z) found: 441.3; Calcd. 441.50; Anal. Calcd. For C24H19N5O2S: C, 65.29; H, 4.34; N, 15.86; Found C, 65.69; H, 4.26; N, 15.85.4-(6-Chlorocoumarin-3-yl)-2-diazenyl)pyrazol-1-yl) thiazole (1d). Rf: 0.6; mp 255–256°C; Yield: 83%; IR (cm-1): 1720 (C = O), 1601 (C = N); 1H NMR : 8.39, 8.00, 7.86 , 7.66 , 7.63 , 7.53 , 7.35 , 2.69 , 2.19 , 1.98 ;13C NMR : 164.37 (C = O), 152.86 (C2-thiazole), 151.56 (C8'-coumarin), 144.30 (C4-thiazole), 143.71 (C4-coumarin), 139.30 (C-Ar), 138.86 (C-Ar), 131.69 (C-Ar), 131.60 (C-Ar), 129.69 (C-Ar), 128.69 (C-Ar), 128.41 (C-Ar), 126.37 (C-Ar), 126.20 (C-Ar), 120.61 (C-Ar), 120.24 (C-Ar), 119.41 (C-Ar), 116.44 (C-Ar), 113.44 (C-Ar), 20.94 (CH3-Ph), 24.37 (5-CH3 pyrazole), 13.86 (3-CH3 pyrazole); MS (m/z) Found: 477.6, 475.8; Calcd. 475.95; Anal. Calcd. for C24H18ClN5O2S: C, 60.56; H, 3.81; N, 14.71; Found C, 60.55; H, 3.71; N, 14.63.4-(Coumarin-3-yl)-2-diazenyl)pyrazol-1-yl)thiazole (1e). Rf: 0.6; mp 210–212°C; Yield: 88%; IR (cm-1): 1717 (C = O), 1600 (C = N); 1H NMR : 8.30 , 8.08 , 7.82 , 7.71 , 7.65–7.61 , 7.47 , 7.28–7.24 , 2.72 , 2.32 ;13C NMR : 164.81 (C = O), 159.22 (C2-thiazole), 154.38 (C8'-coumarin), 149.26 (C4-thiazole), 147.14 (C4-coumarin), 141.59 (C-Ar), 136.64 (C-Ar), 133.14 (C-Ar), 129.23 (C-Ar), 128.82 (C-Ar), 125.07 (C-Ar), 123.35 (C-Ar), 121.87 (C-Ar), 119.40 (C-Ar), 118.67 (C-Ar), 116.81 (C-Ar), 110.77 (C-Ar), 25.08 (5-CH3 pyrazole), 14.71 (3-CH3 pyrazole); MS (m/z) found: 506.1, 504.1 (3:1); Calcd.: 506.37; Anal. Calcd. for C23H16BrN5O2S: C, 54.55; H, 3.18; N, 13.83. Found: C, 54.48; H, 3.12; N, 13.82.4-(6-Chlorocoumarin-3-yl)-2-diazenyl)pyrazol-1-yl) thiazole (1f). Rf: 0.6; mp 235–236°C;Yield: 85%; IR (cm-1): 1719 (C = O), 1605 (C = N); 1H NMR : 8.28 , 8.07 , 7.79 , 7.65–7.61 , 7.28 , 2.84 , 2.21 ; 13C NMR , 159.23 (C2-thiazole), 150.22 (C8'-coumarin), 147.40 (C4-thiazole), 142.56 (C4-coumarin), 139.21 (C-Ar), 133.23 (C-Ar), 128.88 (C-Ar), 125.10 (C-Ar), 124.85 (C-Ar), 124.80 (C-Ar), 122.43 (C-Ar), 119.34 (C-Ar), 118.64 (C-Ar), 116.81 (C-Ar), 113.49 (C-Ar), 110.39 (C-Ar), 25.16 (5-CH3 pyrazole), 15.54 (3-CH3 pyrazole); MS (m/z) found: 497.2, 495.2 (3:1); Calcd.: 496.37; Anal. Calcd. for C23H15Cl2N5O2S: C, 55.65; H, 3.05; N, 14.11; Found: C, 55.64; H, 3.10; N, 14.17.4-(Coumarin-3-yl)-2-pyrazol-1-yl)thiazole (1g). Rf: 0.6; mp 205–206°C; Yield: 82%; IR (cm-1): 1715 (C = O), 1603 (C = N); 1H NMR : 8.34 , 8.08 , 7.82–7.78 , 7.78 , 7.65–7.63 , 7.62–7.57 , 7.33 , 2.79 , 2.24 ; 13C NMR : 167.79 (C = O), 159.35 (C8'-coumarin), 154.35 (C2-thiazole), 148.40 (C4-coumarin), 146.95 (C4-thiazole), 142.99 (C-Ar), 138.95 (C-Ar), 135.97 (C-Ar), 132.95 (C-Ar), 128.78 (C-Ar), 124.99 (C-Ar), 123.43 (C-Ar), 122.59 (C-Ar), 120.05 (C-Ar), 119.79 (C-Ar), 118.76 (C-Ar), 116.75 (C-Ar), 110.52 (C-Ar), 24.83 (5-CH3 pyrazole), 15.22 (3-CH3 pyrazole); MS (m/z) found: 427.3;Calcd.: 427.28; Anal. Calcd.forC23H17N5O2S: C, 64.62; H, 4.01; N, 16.38; Found C, 64.55; H, 4.05; N, 16.45.Escherichia coli MTCC 2961, Staphylococcus aureus MTCC 3160, Enterococcus faecalis MTCC 439, Vibrio cholera MTCC 3906, Streptococcus pyogenes MTCC 442 were cultured in Mullar Hinton broth . The fungal strains Candida albicans NCPF 400034, Candida glabrata MTCC3019, Candida krusei NCPF 44002, Candida parapsilosis NCPF 450002, Candida keyfer, NPCPF 410004 and Candida tropicalis NPCPF420007 were cultured in yeast extract-peptone-dextrose  and RPMI 1640 media . For agar plates, 2.5% (w/v) bacteriological agar  was added to the medium. The strains were stored with 15% glycerol at -80°C as frozen stocks. The cells were freshly revived on respective agar plates from the stock before each experiment .,54.6) waat 0–5°C .1a-1g) exclusively, the condensation of 3,5-dimethyl-4-hydrazoarylpyrazole-1-thiocarboxamides (5) with 6-substituted-3-bromoacetylcoumarins (7a-b) was carried out in refluxing ethanol under alkaline condition  and mass.In order to achieve the synthesis of 4-(coumarin-3-yl)-2-thiazoles(ondition . The str1H NMR spectra of compounds (1a-1g) revealed two singlets in the range δ 8.39–8.29 and 8.00–8.10 ppm corresponds to coumarin 4-H and thiazole 5-H, respectively, thereby confirming the formation of 1. Two Singlets in the range δ 2.19–2.32 and 2.69–2.86 ppm appeared due to 3-CH3 and 5-CH3 group attached at pyrazole ring. It has been well established that 5-CH3 is more deshielded compared to 3-CH3 due to a lone pair of electrons present on the nitrogen atom of the thiazole nucleus and therefore appeared at about δ 2.80ppm [1H NMR along with other spectral data has been given in experimental section.The  2.80ppm ,55. More1a-1g) was evaluated in vitro according to Clinical and Laboratory Standard Institute (CLSI) guidelines using five clinical bacterial strains  and six fungal strains . The antimicrobial activity of synthesized compounds (1a-1g) ranged from 250 μM to 15.67 μM and results have been summarized in Tables 1b and 1f have been found to be the most potent antimicrobial agents among all tested compounds. When used as a standalone antibacterial agent, the MIC of 1b was either comparable (S. aureus) or at least 50% reduced (S. pyogens) compared to KAN. In case of 1f, the MIC is 50% less compared to that of KAN when tested against S. aureus, E. faecalis and S. pyogens.The antimicrobial activity of compounds a-1g was 1b and 1f as standalone found to be more active in comparison to other candidates when Amp B was used as reference drug. The results of antimicrobial activity study revealed that incorporation of halogen atoms (Cl and Br) on coumarinyl and phenyl moieties linked to pyrazolylthiazoles may be responsible for higher potential of 1b and 1f. Generally, substitution of hydrogen by halogen in coumarin derivatives compounds increases the lipophilicity and thus increases the rate of cell penetration, which is an important feature of antimicrobial drug efficiency [1b and 1f significantly increased activity against the bacterial and fungal strains. The results demonstrated that the antimicrobial activity of the investigated compounds was influenced by the physicochemical properties of the type and position of substituent on the aromatic ring.Though MIC was much higher in case of antifungal screening, the compounds ficiency . SubstitS. aureus and candida sp. are responsible for major opportunistic infections in clinical settings. There are growing concerns about the ability of S. aureus to acquire resistance, even towards the newest antibacterial available in the market [C. keyfer infections mostly occur in patients with hematologic malignancies or carcinomas who are already undergoing highly cytotoxic chemotherapy [S. aureus [Furthermore, it is noteworthy here that e market . Similarotherapy . KAN has. aureus . Likewis. aureus ,63. Emer. aureus ,65. Ther. aureus .1b or 1f can act as an adjunct to potentiate a reduction in MIC of KAN and Amp B against clinical strains S. aureus and C. Keyfer, respectively. A checkerboard dilution assay has been used to determine the fraction inhibitory concentration index (FICI) for the compounds 1b and 1f, when used in combination with KAN and Amp B against S. aureus and C. Keyfer, respectively. As evident from S. aureus with compounds 1b, and also with 1f. Likewise, in case of Amp B, there is a 5-fold reduction in MIC with compound 1b and ~8 fold reduction with compound 1f against fungal pathogen C. keyfer.As discussed above, we further evaluated whether the compounds 1b and 1f have comparable cytotoxicity  at MIC concentrations  and Hek-293 cells  indicated that the compounds trations  and negl1b and 1f are active as an adjuvant at much lower concentrations , we believe that these compounds can act as an ideal adjuvant to increase the efficacy of KAN, Amp B or possibly another drugs too. Our experiments with another antifungal agent fluconazole showed that usage of 1b and 1f as adjunct reduced the MIC ~5-fold with 1b and 8-fold with 1f against C. keyfer substantiating the broad spectrum effects of these derivatives as adjunct. Compound 1f consistently demonstrated the most potent activity as a standalone as well as in combination to other drugs as an adjunct. So we evaluated this compound for further analysis including a time kill kinetic assay.Since compounds S. aureus and C. keyfer were rapidly reduced after treatment with the 1f (MIC conc. 15.67 μM) and 1f (MIC conc. 62.50 μM), respectively. Maximum killing of S. aureus and C. keyfer cells with 1f were observed after 24 h in comparison to untreated control. Samples withdrawn at the indicated time points were evaluated for colony forming units (CFUs) at MHA (Muller Hinton Agar) and YPEDA (Yeast Extract Peptone Dextrose Agar) medium. At synergy concentrations of treatment with 1f (0.5 μM of KAN and 07.81 μM of 1f) against S. aureus and (0.097 μM of Amp B and 31.25 μM of 1f) against C. keyfer showed killing after 12 h (S. aureus) and 8 h (C. keyfer), respectively . A change in both S. aureus and C. Keyfer cell morphology was observed in treated with compound 1f at MIC conc. (at 15.67 μM for S.aureus and 62.50 μM for C. keyfer). Increased roughness and cell wall damage was observed within 12 h of incubation . The MIC value of Amp B against C. keyfer is 12.50 μM. More promisingly, when used as an adjunct to standard antimicrobial drugs, compounds 1b and 1f significantly enhanced the efficacy of these drugs KAN, Amp B and FLC by significantly reducing the MIC values of these drugs.As a result, compounds (1a-1g), using Khon-Sham’s DFT method that undergo the gradient-corrected hybrid density functional B3LYP method and the 6–31+g bases set [The electron donating and receiving ability of a molecule can be determined by considering HOMO and LUMO energy of that molecule. Higher the energy of HOMO higher will be its electron donating ability. A full geometry optimization was performed for all compounds ases set . It has ases set , HOMO-LU1b and 1f showed illustrious LUMO energy of -2.647 eV and -2.648 eV respectively and also exhibited higher antibacterial & antifungal activity than the other compounds. It was observed that compounds with more stabilized LUMO orbitals have magnificent biological activity /2. This descriptor is used as a measures of resistance to change in the electron distribution or charge in a molecule [HOMO+ ELUMO) /2, which is defined as the power of an atom in a molecule to attract electrons towards it [HOMO+ELUMO)/2. Electrophilicity index (ω), is a measures of the propensity or capacity of a species to accept electrons was introduced by Parr and is calculated using the electronic chemical potential and chemical hardness and given as ω = μ2/2η[1a-1f are summarized in 1a and 1f are comparatively soft molecule with high polarizability than other compounds of this series. The high electronegativity (4.41 eV) and electrophilicity (5.51 eV) of 1a and 1f indicates its high power to withdrawn electrons and hence to act as an electrophile.The chemical reactivity indices like chemical hardness (η), electronegativity (χ), electronic chemical potential (μ), and electrophilicity Index (ω), were also calculated for all the synthesized molecules. The stability and reactivity of a chemical system is represented by chemical hardness which is given by η =  with a root-mean-square deviation (RMSD) of <1 results .1a-1g were performed in the binding pocket of DNA gyrase B (PDB ID:5L3J). Four basic parameters-Glide score, Glide energy, H-bonds and non-bonded interactions  were used to define the binding affinity of ligands 1a-1g for DNA gyrase B. Glide score with more negative value represents minimum energy for the formation of complex between ligand and receptor (Glide energy). The docked poses for compounds 1b, 1g and KAN in the active site of DNA gyrase B are shown in Figs -1 to -84.78 kcalmol-1) and the energy ranges are more than that of reference compounds KAN and CPF  with DNA gyrase B are shown in In the present manuscript, an attempt has been made to study the interactions of novel coumarin-thiazole-pyrazole (CTP) hybrids with DNA gyrase B. For this purpose molecular docking studies of all the synthesized compounds  and CPF . It has (1a-1g) may prove good lead as antibacterial drugs by targeting DNA gyrase B. For drug formulation pharmacokinetic properties are likewise a significant prospect and should be brought into thoughtfulness. The pharmacokinetic parameters of all synthesised hybrids have been studied along with standard drug KAN and CPF using QikProp [(1a-1g) were seems to be acceptable and were incorporated in 1a-1g was highly satisfactory with a high percentage (>80%) of Human Oral Absorption as compared to the reference, indicating their possibilities in oral drug formulation for the treatment of S. aureus infection.Pharmacodynamics results indicated that compounds  QikProp . The majThe bowel-blood barrier was predicted using Caco-2 cell permeability (QPPCaco) as model along with Lipinski’s rule of 5  all the 1b and 1f displayed significant antimicrobial activity without expressing any significant mammalian cell cytotoxic effect. Compound 1b showed the similar level of antibacterial activity with MIC 31.25 μM and 1f found best agent with MIC 15.67 μM particularly against S. aureus when compared with standard drug, kanamycin. Interestingly, as an adjuvant to standard antimicrobial drugs, compounds 1b and 1f significantly enhanced the efficacy of the standard drugs KAN, Amp B and FLC by significantly reducing the MIC values of these drugs.In conclusion, some new coumarinyl linked pyrazolylthiazoles have been synthesized and explored for their potential as standalone or adjuvant antimicrobial agents against pathogenic strains. Compounds 1b and 1f may act as lead compounds for the development of potent antibacterial and antifungal agents and as adjuvant they have potential to unlock the microbial resistance to antibiotics.The results demonstrated that the antimicrobial activity of the investigated compounds was influenced by the physicochemical properties of the type and position of substituent on the aromatic ring. The results based on electron microscopic investigations of cell morphology and cellular structural integrity suggest that the antimicrobial activity of these compounds may in part be attributed to initial cell wall damage, leading to action on the intracellular components of cells, ultimately leading to cell death. The result of DFT studiy of compounds were correlated with the observed antimicrobial activities which indicate that coumarin moiety plays an important role in antimicrobial activities. Moreover, the molecular docking studies further helped in supporting the experimental results and the ADME properties predicted further strengthen the path towards oral drug formulation. Therefore, it is concluded that the compounds S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S13 Fig(TIF)Click here for additional data file.S14 Fig(TIF)Click here for additional data file.S15 Fig(TIF)Click here for additional data file.S16 Fig(TIF)Click here for additional data file.S17 Fig(TIF)Click here for additional data file.S18 Fig(TIF)Click here for additional data file.S19 Fig(TIF)Click here for additional data file.


Ligusticopsis wallichiana (family: Apiaceae) are widely used as traditional medicines. Although many volatile constituents are already identified from the leaves of L. wallichiana, there is no detailed report on the nonvolatile constituents. In the present study, we aimed to isolate and identify the major chemical constituents from the leaves. Bhutkesoside A (1), falcarindiol (2), ferulic acid (3), cnidioside A (4), quercetin 3-O-β-D-glucopyranoside (5), rutin (6), 4′-O-methylquercetin 3-O-β-D-glucopyranoside (7), scopoletin (8), umbelliferone (9), eugenol 4-O-β-D-glucopyranoside (10) and pumilaside A (11) were isolated from the 70% MeOH extract. The structures of isolated compounds were elucidated on the basis of 1H- and 13C-NMR spectroscopic data. Compounds 4–11 are reported for the first time from L. wallichiana. Compounds 5 and 6 showed potent free radical-scavenging activity.Different plant parts of  Ligusticopsis wallichiana (DC.) Pimenov & Kljuykov  Raizada & H. O. Saxena, Selinum tenuifolium Wall. ex C. B. Clarke) is a perennial aromatic herb belonging to family Apiaceae. It is widely distributed in the Himalayan region of Nepal, India, Pakistan, Bhutan, and China between 2700 and 4800 m D20 +99.9° . The 1H-NMR spectrum , 5.47 , and 5.25  assignable to terminal vinyl protons. A set of olefenic protons at δH 5.51  and 5.61  were also present and their coupling constant of 10.6 Hz suggested the cis configuration. Two protons attached to oxygen bearing carbon were present at δH 5.20  and δH 4.93 . Two methylene proton at δH 2.10 to 1.27 ppm and a methyl signal at δH 0.88  were also observed. The 13C-NMR spectra (δC 78.2 (C), 70.3 (C), 79.8 (C), and 68.7 (C) were assignable to a disubstituted acetylene moiety. Two oxygen-bearing carbons at δC 63.4 (CH) and 58.5 (CH) and a methyl group at δC 14.0 ppm were also observed. On the basis of these data and comparison with literature values, compound 2 was identified as falcarindiol D27 −33.4° . The 1H-NMR spectrum , 7.37 , 7.32 , and 6.81 . Seven proton signals attached to oxygen bearing carbons assignable to a sugar moiety were present at δH 4.82 , 3.72 , 3.26–3.30 , 3.16 , and 3.75 . The proton signals at δH 4.82  was assignable to the anomeric proton for a sugar moiety. Further two methylene protons signals coupled each other at δH 2.87  and 2.40  were observed. The 13C-NMR spectra (δC 153.5 (C), 153.4 (C), 144.9 (CH), 126.6 (C), 120.8 (C), 120.6 (CH), 106.2 (CH), and 98.3 (CH) revealed a benzofuran moiety. Six carbon signals at δC 101.5 (CH), 73.3 (CH), 77.0 (CH), 69.9 (CH), 76.6 (CH), and 60.8 (CH2) were assignable to a β-glucopyranosyl moiety which was also supported by the coupling constant of anomeric proton (J = 7.3 Hz). Three carbon signals δC 174.8 (C), 35.3 (CH2), and 26.1 (CH2) revealed a propanoic acid derivative. On the basis of these data and comparison with literature values, compound 4 was identified as cnidioside A D23 −31.9° . The 1H-NMR spectrum , 7.58 , 6.85 , 6.41 , and 6.21  assignable to proton signals of quercetin. Seven proton signals attached to oxygen bearing carbon were present at δH 5.47 , 3.10 , 3.25 , 3.10 , 3.59 , and 3.33  were present. Among them proton at δH 5.47  was assignable to anomeric proton of the sugar moiety. The 13C-NMR spectra of (δC 177.5 (C), 164.3 (C), 161.2 (C), 156.3 (C), 156.2 (C), 144.8 (C), 148.5 (C), 133.4 (C), 121.6 121.2 (C), 115.2 (CH), 116.2 (CH), 104.0 (C), and 98.7 (CH), were assignable to a 3-O-substituted quercetin. The remaining six signals at δC 100.9 (CH), 77.5 (CH), 76.5 (CH), 74.1 (CH), 69.9 (CH), and 61.0 (CH2) for a monosaccharide revealed the β-glucopyranosyl moiety which was supported by the coupling constant (J = 7.0 Hz) of anomeric proton. On the basis of these data and comparison with literature values, compound 5 was identified as quercetin 3-O-β-D-glucopyranoside D23 −35.5° . The 1H-NMR spectrum  and a methyl group at δH 1.00  suggested the presence of rhamnopyranosyl moiety. The 13C-NMR spectra , 76.4 (CH), 75.9 (CH), 74.1 (CH), 70.6 (CH), and 68.2 (CH2) were assignable to a β-glucopyranosyl moiety and other six carbons signals at δC 100.7 (CH), 70.4 (CH), 70.0 (CH), 71.8 (CH), 67.0 (CH), and 17.7 (CH3) were assignable to a α-rhamnopyranosyl moiety. The downfield shift of C-6 of glucopyranosyl moiety at 68.2 ppm suggested Rha-1→Glc-6 linkage. On the basis of these data and comparison with literature values, compound 6 was identified as rutin D27 −14.7° . The 1H-NMR spectrum . The 13C-NMR spectra of compound 7 (δC 177.5 (C), 164.3 (C), 161.2 (C), 156.3 (C), 156.2 (C), 144.8 (C), 148.5 (C), 133.4 (C), 121.6 121.2 (C), 115.2 (CH), 116.2 (CH), 104.0 (C), and 98.7 (CH), were assignable to a 3-O-substituted quercetin. The remaining six signals at δC 101.5 (CH), 72.7 (CH), 70.8 (CH), 68.2 (CH), and 63.7 (CH2) confirmed the presence of a β-glucopyranosyl which was also supported by the coupling constant (J = 7.9 Hz) of the anomeric proton. Signal at δC 55.6 was assigned to a methoxy group. In differential NOE experiment, irradiation of the methoxy signal at δH 3.85  increased the intensity of proton signal assignable to C-5′ at δH 7.04  while no effect was seen in the protons at C-2′ at δH 7.56, 1H , which suggested that the methoxy group was attached at C-4′ position in B-ring of quercetin. On the basis of these data and comparison with literature values, compound 7 was identified as 4′-O-methylquercetin 3-O-β-D-glucopyranoside D27 −35.5° . The 1H-NMR spectrum , 7.07 , and 6.71 , which were assignable to a 1,3,4-substituted aromatic ring. Further three proton signals due to terminal alkene at δH 5.94 , 5.05 , and 5.01  coupled with two proton signals at δH 3.31  were observed, which suggested the presence of allyl moiety. A singlet at δH 3.83  suggested the presence of a methoxy group. Further seven protons assignable to a sugar moiety were present at δH 4.83 , 3.48 , 3.37–3.39 , 3.45 , 3.68 , and 3.86 . The 13C-NMR spectra (δC 150.8 (C), 146.4 (C), 139.0 (CH), 136.5 (C), 122.1 (CH), 118.3 (CH), 115.1 (CH2), 114.2 (CH), and 40.7 (CH2), were assignable to a 1,3,4-trisubstituted aromatic ring with an allyl moiety. Other six signals δC 103.1 (CH), 78.2 (CH), 77.9 (CH), 74.9 (CH), 71.4 (CH), and 62.5 (CH2) were assignable to β-glucopyranosyl moiety as in the case of previous compounds. The carbon signal at δC 56.8 was assigned to a methoxy group. In differential NOE experiment, irradiation of the methoxy signal at δH 3.83  enhanced the intensity of proton at δH 6.79  assignable to C-2 of the aromatic ring, which suggested that the methoxy group was present at C-3 position, and glucopyranosyl moiety was attached to C-4. On the basis of these data and comparison with literature values, compound 10 was identified as eugenol 4-O-β-D-glucopyranoside D27 −25.9°. Its molecular formula was determined to be C21H38O8 on the basis of a HR-FAB-MS peak of [M + Na]+ at 441.2482 . 1H-NMR spectrum of compound 11  and 1.03 , two methyl singlets were present at δH 0.83  and 1.23 . Remaining ten proton signal equivalents were present from δH 2.11 to δH 4.48 ppm. The 13C-NMR spectra (δC 98.3 (CH), 77.0 (CH), 76.9 (CH), 74.3 (CH), 70.4 (CH), and 61.4 (CH2) were assignable to a glucopyranosyl moiety. The rest fifteen signals at δC 77.9 (CH), 76.8 (CH), 71.3 (C), 49.9 (CH), 40.3 (CH), 39.9 (CH2), 35.5 (CH2), 27.9 (CH2), 24.6 (CH), 23.6 (CH3), 23.1 (CH3), 22.6 (CH3), 22.4 (CH2), and 13.8 (CH3) can be assignable to a eudesmane-type sesquiterpenoid moiety. On the basis of these data and comparison with literature values, compound 11 was identified as pumilaside A [Compound pound 11  showed s spectra  showed tlaside A .L. wallichina in this study, a diacetylene glucoside, bhutkesoside A (1); a polyacetylene derivative, falcarindiol (2); and a phenylpropanoid derivative, ferulic acid (3) were also isolated from the roots of same plant, which were reported in a previous paper [4–11) were isolated for the first time from this plant which included a benzofuran derivative, cnidioside A (4); three flavonoid derivatives, quercetin 3-O-β-D-glucopyranoside (5), rutin (6), and 4′-O-methylquercetin 3-O-β-D-glucopyranoside (7); two coumarin derivatives, scopoletin (8) and umbelliferone (9); a phenylpropene derivative, eugenol 4-O-β-D-glucopyranoside (10), and a eudesmane sesquiterpene glucoside, pumilaside A (11). It was the first study on the isolation and identification of nonvolatile compounds from the leaves of L. wallichiana. Regarding coumarin derivatives, three furocoumarins such as bergapten, heraclenin, and heraclenol were isolated from the roots of L. wallichiana [L. wallichiana and presence of coumarin derivatives in the leaves.Among these eleven compounds isolated from the leaves of us paper . All othlichiana . This is6) (EC50 52.4 μM) and quercetin-3-O-β-D-glucopyranoside (5) (EC50 54.5 μM) showed potent free radical-scavenging activity as compared to positive control, Trolox (EC50 96.1 μM). Flavonoids with unsubstituted hydroxyl groups in C3′ and C4′ position (5 and 6) showed potent activity; however, a compound with C-4′ methoxy substitution, 4′-O-methylquercetin 3-O-β-D-glucopyranoside (7), did not show any activity in the free radical-scavenging assay. These results were similar to previous studies on the free radical-scavenging activities of flavonoids [All these isolated compounds were evaluated for their DPPH free radical-scavenging activity. Among them, only two flavonoids, rutin (avonoids , 19.L. wallichiana. Some of the isolated compounds also showed potent free radical-scavenging activity. Further studies should focus on the detailed biological activities of extracts and isolated compounds to provide the scientific evidence for their traditional uses.In conclusion, eleven nonvolatile compounds belonging to different chemical classes were isolated and identified for the first time from the leaves of


O-acetylmalyngamide 2 (1), 6-O-acetylmalyngamide 2 (2), and N-demethyl-isomalyngamide I (3), were isolated from the marine cyanobacterium Moorea producens. Their structures were determined by spectroscopic analysis and chemical derivatization and degradation. These compounds stimulated glucose uptake in cultured L6 myotubes. In particular, 6,8-di-O-acetylmalyngamide 2 (1) showed potent activity and activated adenosine monophosphate-activated protein kinase (AMPK).Three new compounds of the malyngamide series, 6,8-di- Lyngbya sp., showed potent cytotoxicity against HeLa S3 cells  to give five fractions (Fractions 1–5). Fraction 1 was subjected to further HPLC  to yield compound 2 . Fraction 2 was subjected to further HPLC  to yield compound 3  and two fractions (Fractions 2-1 and 2-2). Fraction 2-2 was further subjected to HPLC  to yield compound 1 .Samples of the marine cyanobacterium, 6,8-Di-O-Acetylmalyngamide 2 (1): Colorless oil; c 1.20, MeOH); IR (neat) 3302, 2929, 2856, 1731, 1651, 1539, 1445, 1373, 1232, 1132, 1096 cm−1; 1H NMR, 13C NMR and HMBC data, see m/z 572.2989 [M + H]+ .6-O-Acetylmalyngamide 2 (2): Colorless oil; c 2.68, MeOH); IR (neat) 3375, 2929, 2856, 1730, 1650, 1540, 1446, 1375, 1239, 1066 cm−1; 1H NMR, 13C NMR and HMBC data, see m/z 552.2692 [M + Na]+ .N-Demethyl-isomalyngamide I (3): Colorless oil; c 1.12, MeOH); IR (neat) 3313, 2928, 2856, 1714, 1647, 1541, 1456, 1433, 1094 cm−1; 1H NMR, 13C NMR and HMBC data, see m/z 470.2692 [M + H]+ .Moorea producens  to yield S-MTPA ester (0.8 mg). Using the same procedure as described above, R-MTPA (0.5 mg) ester was obtained from 2 (1.0 mg).Compound S-MTPA ester: 1H NMR  δ 4.03 (H-4), 5.29 (H-6), 2.41 (H-7a), 2.66 (H-7b), 5.28 (H-8), 1.12 (H-10), 2.15 (6-OAc); ESIMS m/z [M + Na]+ 768.3.R-MTPA ester: 1H NMR  δ 4.08 (H-4), 5.11 (H-6), 2.40 (H-7a), 2.61 (H-7b), 5.21 (H-8), 1.24 (H-10), 2.13 (6-OAc); ESIMS m/z [M + Na]+ 768.3.2 (5.0 mg) was reacted with Ac2O (50 μL) in pyridine (50 μL), and the mixture was stirred for 2 h at room temperature. The reaction mixture was subjected to reversed-phase HPLC  to yield acetylated compound of 2 (4.7 mg).Compound 2: c 1.20, MeOH); 1H NMR  δ 6.63 , 6.29 , 5.69 , 5.47 , 5.45 , 5.41 , 5.11 , 4.27 , 4.23 , 3.95 , 3.31 , 3.17 , 2.60 , 2.32 , 2.28 , 2.27 , 2.19 , 2.18 , 2.16 , 1.42 , 1.27 , 1.26 , 0.87 ; ESIMS m/z [M + H]+ 572.3.Acetylated compound of 2 (7.3 mg) was dissolved in a 5.0 mL solution of 10% KOH in 80% aqueous EtOH and refluxed for 14 h. The reaction mixture was concentrated, and the residue was partitioned between EtOAc and H2O. The organic layer was subjected to reversed-phase HPLC  to yield lyngbic acid (1.6 mg). Using the same procedure as described above, lyngbic acid (4.7 mg) ester was obtained from 3 (13.5 mg).Compound 2: c 0.16, CHCl3); 1H NMR  δ 5.49 , 3.32 , 3.15 , 2.42 , 2.35 , 2.19 , 1.43 , 1.27 , 0.88 .Lyngbic acid from 3: c 0.47, CHCl3); 1H NMR  δ 5.49 , 3.32 , 3.15 , 2.42 , 2.35 , 2.19 , 1.43 , 1.27 , 0.88 .Lyngbic acid from 3 (7.3 mg) was reacted with R-MTPACl (10 μL) and DMAP (0.5 mg) in pyridine (50 μL), and the mixture was stirred for 3 h at room temperature. The reaction mixture was concentrated, and the residue was partitioned between EtOAc and 0.1M NaHCO3 (1:1). The extract obtained from the organic layer was subjected to reversed-phase HPLC  to yield compound 4 (2.3 mg).Compound 4: c 0.23, MeOH); 1H NMR, 13C NMR and HMBC data, see m/z 474.2381 [M + Na]+ .Compound 3 cells/well in 96-well plates or 2 × 105 cells/well in 60-mm culture dishes) were maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), streptomycin (100 µg/mL), and penicillin G (100 units/mL) at 37 °C with 5% CO2. After reaching 80% confluence, the cells were cultured in low glucose DMEM containing 2% FBS for 1 week to differentiate into myotubes. The medium was renewed every 2 days.L6 myoblasts  containing 0.1% bovine serum albumin (BSA), 10 mM HEPES, and 2 mM sodium pyruvate (KHH buffer) for 2 h. The myotubes were then cultured in KHH buffer containing 5 mM glucose with or without compounds 1, 2, and 3 (10–40 µM) for 16 h and without or with compound C (30 µM), an AMPK inhibitor for 6 h. Nepodin [L6 myotubes were incubated in filter-sterilized Krebs-Henseleit buffer . Equal amounts of protein samples (50 µg/lane) were electrophoresed on 10% Mini-PROTEAN TGX precast gels  and transferred to nitrocellulose membranes . The membranes were washed with Tris buffered saline (TBS) for 15 min and blocked with 3% nonfat dry milk, or 80% BSA in TBS containing 0.05% Tween 20 (TBST), at room temperature for 1 h. The membranes were then washed with TBST and incubated with anti-AMPK or anti-phosphorylated AMPK primary antibodies  at 4 °C overnight. The membranes were then washed with TBST and incubated with HRP-linked anti-rabbit IgG secondary antibodies (1:2000 in blocking buffer) at room temperature for 1 h. After washing with TBST, immunoreactive bands were detected by a chemiluminescent reagent  and quantified by densitometry analysis using a ChemiDoc XRS Plus system  and Image Lab software .O-acetylmalyngamide 2 (1), 6-O-acetylmalyngamide 2 (2), and N-demethyl-isomalyngamide I (3) were isolated from the marine cyanobacterum Moorea producens collected at Okinawa Prefecture, Japan. The gross structures of these compounds were determined on the basis of spectroscopic analysis. The absolute stereostructures of 1 and 2 were established by NOESY experiments, and chemical derivatization and degradation. The relative configuration of the cyclohexanone ring in 3 was determined by a NOESY experiment. These compounds stimulated glucose uptake in cultured L6 myotubes. Compound 1 was found to activate an insulin-independent AMPK pathway, so 1 may have antidiabetic properties.Three new malyngamide analogs, 6,8-di-