supplementary information of potential antimicrobial agents … · 2017-03-06 · supplementary...
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Supplementary Information
Design, synthesis and biological evaluation of novel Schiff base-bridged tetrahydroprotoberberine triazoles as new type
of potential antimicrobial agentsJun-Rong Duan,a Han-Bo Liu,a Ponmani Jeyakkumar,§a Lavanya Gopala,a Shuo Li,*b Rong-Xia Genga and Cheng-He Zhou*a
aInstitute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing Municipality, School of
Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.bSchool of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, PR China.
§ Ph.D candidate from India.
Postdoctoral fellow from Sri Venkateswara University, Tirupati 517502, India.
* Corresponding author: [email protected]; [email protected]; Tel.: +86-23-68254967; Fax: +86-23-68254967.
1 Experimental Protocols1.1 General Methods
TLC analysis was done using pre-coated silica gel plates. FT-IR spectra were carried out on Bruker RFS100/S spectrophotometer (Bio-Rad, Cambridge, MA, USA) using KBr pellets in the 400–4000 cm-1 range. 1H NMR, 13C NMR, 1H-1H COSY and 13C-1H COSY spectra were recorded on a Bruker AV 300 spectrometer using TMS as an internal standard. The high-resolution mass spectra (HRMS) were recorded on an IonSpec FT–CR mass spectrometer with ESI resource. UV spectra were recorded at room temperature on a TU-2450 spectrophotometer (Puxi Analytic Instrument Ltd. of Beijing, China) equipped with 1.0 cm quartz cells.
The stock solution of compound 7a was prepared in DMF. Calf thymus DNA (Sigma Chemical Co., St. Louis, MO) was used without further purification, and its stock solution was prepared by dissolving an appropriate amount of DNA in doubly distilled water. The solution was allowed to stand overnight and store at 4 oC in the dark for about a week. The concentration of DNA in stock solution was determined by UV absorption at 260 nm using a molar absorption coefficient ξ260 = 6600 L mol-1 cm-1 (expressed as molarity of phosphate groups) by Bouguer-Lambert-Beer law. The purity of DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of > 1.8 at A260/A280, which indicated that DNA was sufficiently free from protein. NR stock solution was prepared by dissolving its solid (Sigma Chemical Co.) in doubly distilled water and was kept in a cool and dark place. All the solutions were adjusted with Tris-HCl buffer solution (pH = 7.4), which was prepared by mixing and diluting Tris solution with HCl solution. All chemicals were of analytical reagent grade, and doubly distilled water was used throughout. HSA was dissolved in Tris-HCl buffer solution (0.05 M Tris, 0.15 M NaCl, pH = 7.4). Sample masses were weighed on a microbalance with a resolution of 0.1 mg. All other chemicals and solvents were commercially available, and were used without further purification.
The pH of the solution was determined by using a Sartorius PB-10 pH meter (Sartorius Scientific Instrument (Beijing) Co., Ltd., P.R. China). DNA cleavage was analysed by gel electrophoresis on a DYY-12 electrophoresis meter (Beijing Liuyi Biotechnology Co., Ltd., P.R. China) and gel image analysing system (Vilber Lourmat BIO-1D, France). Plasmid pUC19 DNA, 50×TAE, 6×loading buffer, gold view dye, and agarose were purchased from Beijing Changsheng Biotechnology Co., Ltd. Trishydroxymethylaminomethane (Tris), HCl, NaCl, and NaOH were analytical grade products and used as supplied. All other chemicals were purchased from Chongqing chemical Co. and, unless otherwise indicated, were of analytical grade. The water used for experiments was doubly distilled.
1.2 Biological Assay ProceduresMinimal inhibitory concentration (MIC, μg/mL) is defined as the lowest concentration of target compounds that completely inhibited the
growth of bacteria, by means of standard two-fold serial dilution method in 96-well microtest plates according to the National Committee for Clinical Laboratory Standards (NCCLS). The tested microorganism strains were provided by the School of Pharmaceutical Sciences, Southwest University and the College of Pharmacy, Third Military Medical University. Berberine, Chloromycin, Norfloxacin and Fluconazole, were used as control drugs. DMSO with inoculation bacterial not medicine was used as positive control to ensure that the solvent had no effect on bacteria growth. All the bacteria and fungi growth was monitored visually and spectrophotometrically, and the experiments were performed in triplicate.
1.2.1 Antibacterial Assays
Electronic Supplementary Material (ESI) for MedChemComm.This journal is © The Royal Society of Chemistry 2017
The prepared compounds were evaluated for their antibacterial activities against Gram-positive bacteria (Staphylococcus aureus ATCC25923, Methicillin-resistant Staphylococcus aureus N315 (MRSA), Bacillus subtilis ATCC6633) and Micrococcus luteus ATCC4698), Gram-negative bacteria (Escherichia coli JM109, Escherichia coli DH52, Shigella dysenteriae, Pseudomonas aeruginosa ATCC27853, Bacillus proteus ATCC13315 and Bacillus typhi). The bacterial suspension was adjusted with sterile saline to a concentration of 1 × 105 CFU. Initially the compounds were dissolved in DMSO to prepare the stock solutions, then the tested compounds and reference drugs were prepared in Mueller–Hinton broth (Guangdong huaikai microbial sci. & tech co., Ltd, Guangzhou, Guangdong, China) to obtain the required concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg/mL. These dilutions were inoculated and incubated at 37 ºC for 24 h.
1.2.2 Antifungal Assays
The newly synthesized compounds were evaluated for their antifungal activities against Candida albicans ATCC10231, Candida mycoderma ATCC9888, Candida utilis ATCC9950, Aspergillus flavus ATCC204304 and Beer yeast. A spore suspension in sterile distilled water was prepared from one day old culture of the fungi growing on Sabouraud agar (SA) media. The final spore concentration was 1−5 × 103 spore mL-1. From the stock solutions of the tested compounds and reference antifungal drug Fluconazole, dilutions in sterile RPMI 1640 medium (Neuronbc Laboraton Technology CO, Ltd, Beijing, China) were made resulting in eleven wanted concentrations (0.5 to 512 μg/mL) of each tested compound. These dilutions were inoculated and incubated at 35 ºC for 24 hours.Table S1 Antifungal data as MIC (μg/mL) for compounds 4–7a.
FungiCompds C.
albicans C.
mycodermaC.
utilis A.
flavusB.
yeast 4 128 128 512 128 1
5 512 512 512 512 256
6a 4 128 256 512 512
6b 32 8 16 8 32
6c 2 8 8 8 32
6d 512 8 8 64 16
6e 256 64 16 256 64
6f 512 256 512 256 256
7a 4 64 32 16 0.5
7b 0.5 256 >512 512 256
7c 16 256 32 16 16
7d 256 16 8 64 64
7e 256 8 512 128 512
7f 8 8 0.5 4 32
7g 8 64 8 32 256
7h 256 256 256 8 512
Fluconazole 1 4 8 256 16aC. albicans, Candida albicans (ATCC76615); C. mycoderma, Candida mycoderma; C. utilis, Candida utilis; S. cerevisia, Saccharomyces cerevisia; A. flavus, Aspergillus flavus.
1. 3 Development of Aqueous Solubility in Phosphate Buffer (pH 7.4)A standard solution was prepared by precisely weighing amount (generally 1 mg) of sample and dissolving in 10 mL CH3OH. The
standard solution was scanned by UV to determine the wavelength of maximum absorbance, diluting as necessary. A saturated solution was prepared by stirring magnetically a small volume of pH 7.4 phosphate buffer solution in the presence of excess sample for 3 h, then the excess solid compound was filtered. Afterwards, the saturated solution was scanned by UV at the wavelength of the absorption maximum. Total solubility was dedermined by the relationship C' = A'C/A. Where C = concentration of standard solution in mg/mL, A = absorbance of the standard solution (correcting for any dilutions), A' = absorbance of the saturated solution (correcting for any dilutions), and C' = concentration of saturated solution in mg/mL.Table S2 ClogP values of compounds 6a, 6d-e, 7b, 7d, 7e and 7gb.
Compds ClogP Compds ClogP6a 1.59 7d 3.546d 4.76 7e 3.546e 5.82 7g 4.257b 2.97
bClogP values were calculated by ChemDraw Ultra 14.0.
1. 4 Time−Kill Kinetics AssayThe bactericidal activity evaluated by performing time kill kinetics assay. This gives the imformation about the rate at which the
compounds are acting on bacteria. Briefly, MRSA bacterium was grown in suitable growth medium at 37 oC for 6 h and diluted. Compound 7a was added to the bacterial solution (MRSA of approximately 1.8 105 CFU/mL) at concentrations of 4 MIC in a 96-well plate and the mixture was incubated at 37 oC. At different time intervals (0, 1, 2, 3, 4, 5, 6, 7h), aliquots (20 μL) from the solution were taken out and serially diluted 10-fold in 0.9% sodium chloride. Then 20 μL of the dilutions was plated on yeast−dextrose agar plates and incubated at 37 °C for 24 h. The bacterial colonies were counted, and results are represented in logarithmic scale: log10(CFU/mL) vs time (in hours).
1. 5 Development of Resistance to Compound 7aConsidering the high-level resistance of Norfloxacin to MRSA strains, we selected the representative compound 7a to investigate the
developing rate of bacterial resistance according to the paper “J. Hoque; M. M. Konai; S. Gonuguntla; G. B. Manjunath; S. Samaddar; V. Yarlagadda and J. Halar. J. Med. Chem., 2015, 58, 5486”. We exposed a standard strain of MRSA towards increasing concentrations of compound 7a from original MIC for sustained passages and determined the MIC values of compound 7a for each passage of MRSA.
1.6 Interactions with Calf Thymus DNA
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.50
1
2
3
4
5
6
7
8
Ab
sorb
ance
10-5[Q ]-1
Fig. S1 The plot of A0/(A-A0) versus 1/[compound 7a].
350 400 450 500 550 6000.0
0.1
0.2
0.3
i
aAbso
rban
ce
Wavelength (nm)
NR
a
i
Fig. S2 UV absorption spectra of NR in the presence of DNA at pH 7.4 and room temperature. c(NR) = 2 × 10-5 mol/L, and c(DNA) = 03.81× 10-5 mol/L for
curves ai respectively at increment 0.48 × 10-5 mol/L.
1.7 DNA Cleavage Eexperiments
The plasmid pUC19 DNA was treated with different compounds in the buffer(5 mM Tris-HCl/50 mM NaCl, pH = 7.0) to a total volume of
20 μL. After incubation at 37 °C for a certain time, the solution was quenched by the addition of 6×loading buffer (2 μL). Then, the resulting
solutions were loaded on a 0.8% agarose gel containing gold view dye. Gel electrophoresis was carried out at 100 V for 0.5 h in 1 × TAE
buffer, and then the plasmid bands were visualized by a transilluminator and photographed.
Fig. S3 Agarose gel electrophoresis patterns for the cleavage of pUC19 plasmid DNA (6.25×10-3 μg/μL) by 7a–Zn2+ and berberine–Zn2+ complexes in buffer
(50 mM Tris-HCl/50 mM NaCl, pH = 7.0) at 37°C after 16 h of incubation. Lane 1: DNA control; lane 2: DNA + berberine (0.0125 mM); lane 3: DNA + 7a
(0.0125 mM); lane 4: DNA + Zn2+ (0.0125 mM); lane 5: DNA + berberine (0.0125 mM) + Zn2+ complex (0.0125 mM); lane 6: DNA + 7a (0.0125 mM) + Zn2+
(0.0125 mM).
1.8 Interactions of Compound 7a with HSA
0.0 0.2 0.4 0.6 0.8 1.0 1.2
0.0
0.5
1.0
1.5
2.0
T = 288 K T = 298 K T = 310 K
F 0/F-1
105 [Q] (mol/L)
Fig. S4 SternVolmer plots of 7aHSA system at different temperatures.
250 300 350 400 450 5000.0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
250 275 300
0.4
0.6
0.8
Abso
rban
ceWavelength (nm)
D
C
Abso
rban
ce
Wavelength (nm)
A
B
CD
A: Compound 7aB: Compound 7a-HSAC: HSAD: Difference
Fig. S5 UV–vis spectra of HSA in the presence of compound 7a: A, absorption spectrum of compound 7a only; B, absorption spectrum of compound 7a/HSA
1:1 complex; C, absorption spectrum of HSA only; D, difference between absorption spectrum of compound 7a/HSA 1:1 complex and compound 7a, c(HSA)
= c(compound 7a) = 1.0 × 10-5 mol/L. The curves C and D for the wavelength ranging from 250–300 nm were depicted in the inset.
0 1 2 3 4 5 61
2
3
4
5
6
7
T = 288 K T = 298 K T = 310 K
F 0/(F
0-F)
10-5 [Q]-1 (L/mol)
Fig. S6 Modified SternVolmer plots of 7aHSA system at different temperatures.
Table S3 Stern–Volmer quenching constants for the interaction of compound 7a with HSA at various temperatures.
pH T (K) KSV (L/mol) Kq (L/mol s-1) Ra S.D.b
288 1.63 × 105 2.55 × 1013 0.995 0.0717
298 1.31 × 105 2.05× 1013 0.996 0.05327.4
310 1.09 × 105 1.70 × 1013 0.999 0.0256
Ra is the correlation coefficient. S.D.b is standard deviation.
Table S4 Binding constants and sites of 7a–HSA system at pH = 7.4
Modified Stern–Volmer Method Scatchard equationT(K)
10-5Ka (L/mol) R S.D 10-5Kb (L/mol) R S.D n
288 1.32 0.999 0.0345 3.35 0.998 0.022 1.07
298 1.19 0.999 0.0639 2.02 0.998 0.021 1.04
310 1.02 0.999 0.0324 1.26 0.999 0.012 1.01
3.20 3.25 3.30 3.35 3.40 3.45 3.5011.50
11.55
11.60
11.65
11.70
11.75
11.80
InK a
1000/T (K-1)
InKa = 1142.4/T + 7.85R = 0.998 S.D. = 0.0117
Fig. S7. Van’t Hoff plots of the compound 7aHSA system.
Table S5 Thermodynamic parameters of 7a–HSA system at different temperatures.
T (K) ΔH (kJ/mol) ΔG (kJ/mol) ΔS (J/mol·K)
288 –28.23
298 –28.96
310
–9.498
–29.72
65.27
2 Some Representative Spectra2.1 Spectra of Compound 5
1H NMR Spectrum
N
O
O
OH
OCH3N
NN
N 5
13C NMR Spectrum
HRMS Spectrum11:01:10 16-Mar-2016DJR-5
m/z340 360 380 400 420 440 460 480 500 520
%
0
100ZCH-702 18 (0.333) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:28)
1.83e3420.1670
362.3281353.2660 406.3533381.2932
421.1712
442.1472458.1253
HR-MS (TOF) calcd for C22H21N5O4: [M+H]+, 420.1672; found, 420.1670.1H1H COSY Spectrum
N
O
O
OH
OCH3N
NN
N 5
N
O
O
OH
OCH3N
NN
N 5
N
O
O
OH
OCH3N
NN
N 5
HMQC Spectrum
HMBC Spectrum
IR Spectrum
N
O
O
OH
OCH3N
NN
N 5
N
O
O
OH
OCH3N
NN
N 5
N
O
O
OH
OCH3N
NN
N 5
2.2 Spectra of Compound 6a
1H NMR Spectrum
13C NMR Spectrum
HRMS Spectrum01-Jul-2016DJR-32
m/z350 375 400 425 450 475 500 525 550 575 600
%
0
100ZCH-835 13 (0.129) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (8:28)
1.19e3448.1986
444.1689
376.1196330.3380
449.2017
470.1821471.1815
HR-MS (TOF) calcd for C24H25N5O4: [M+H]+, 448.1985; found, 448.1986.
N
O
O
O
OCH3N
NN
N 6a
N
O
O
O
OCH3N
NN
N 6a
N
O
O
O
OCH3N
NN
N 6a
IR Spectrum
2.3 Spectra of Compound 6b1H NMR Spectrum
13C NMR Spectrum
N
O
O
O
OCH3N
NN
N 6a
N
O
O
O
OCH3N
NN
N 6b
N
O
O
O
OCH3N
NN
N 6b
HRMS Spectrum12-May-2016DJR-14
m/z350 375 400 425 450 475 500 525 550 575 600 625
%
0
100ZCH-799 16 (0.276) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:17)
1.61e3472.1987
353.2661 404.1484406.1661
476.2295
498.2123
499.2147
516.2148
632.1810520.2228
HR-MS (TOF) calcd for C26H29N5O4: [M+H]+, 476.2298; found, 476.2295.IR Spectrum
2.4 Spectra of Compound 6c1H NMR Spectrum
N
O
O
O
OCH3N
NN
N 6c
N
O
O
O
OCH3N
NN
N 6b
N
O
O
O
OCH3N
NN
N 6b
13C NMR Spectrum
HRMS Spectrum
13-May-2016DJR-18
m/z200 300 400 500 600 700 800 900 1000
%
0
100ZCH-801 12 (0.208) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:14)
2.07e3504.2608
500.2281274.2761123.0931
505.2648
506.2690
HR-MS (TOF) calcd for C28H33N5O4: [M+H]+, 504.2611; found, 504.2608.IR Spectrum
2.5 Spectra of Compound 6d
N
O
O
O
OCH3N
NN
N 6c
N
O
O
O
OCH3N
NN
N 6c
N
O
O
O
OCH3N
NN
N 6c
1H NMR Spectrum
13C NMR Spectrum
HRMS Spectrum
11:07:13 16-Mar-2016DJR-9
m/z375 400 425 450 475 500 525 550 575 600 625 650 675
%
0
100ZCH-703 10 (0.186) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:17)
1.31e3532.2925
528.2620
462.2278437.1956 494.2540
533.2968
622.3449
554.2759
555.2761
623.3511
HR-MS (TOF) calcd for C30H37N5O4: [M+H]+, 532.2924; found, 532.2925.
N
O
O
O
OCH3N
NNN 6d
N
O
O
O
OCH3N
NNN 6d
N
O
O
O
OCH3N
NNN 6d
IR Spectrum
2.6 Spectra of Compound 6e1H NMR Spectrum
13C NMR Spectrum
N
O
O
O
OCH3N
NNN 6e
N
O
O
O
OCH3N
NNN 6e
N
O
O
O
OCH3N
NNN 6d
HRMS Spectrum13-May-2016DJR-20
m/z400 450 500 550 600 650 700 750
%
0
100ZCH-802 17 (0.293) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:21)
1.49e3560.3236
556.2924
494.2922397.1851
561.3257
582.3065
598.2840 663.2737769.5151
HR-MS (TOF) calcd for C32H41N5O4: [M+H]+, 560.3237; found, 560.3236. IR Spectrum
2.7 Spectra of Compound 6f1H NMR Spectrum
N
O
O
O
OCH3N
NNN 6e
N
O
O
O
OCH3N
NNN 6e
N
O
O
O
OCH3N
NNN 6f
13C NMR Spectrum
HRMS Spectrum13-May-2016DJR-21
m/z350 400 450 500 550 600 650 700 750 800 850
%
0
100ZCH-803 1 (0.017) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:26)
1.36e3588.3552
584.3223
411.1914 532.2902
589.3578
663.2714664.2704
HR-MS (TOF) calcd for C34H45N5O4: [M+H]+, 588.3550; found, 588.3552.IR Spectrum
2.8 Spectra of Compound 7a1H NMR Spectrum
N
O
O
O
OCH3N
NNN 6f
N
O
O
O
OCH3N
NNN 6f
N
O
O
O
OCH3N
NNN 6f
13C NMR Spectrum
HRMS Spectrum13-May-2016DJR-22
m/z350 400 450 500 550 600 650 700
%
0
100ZCH-804 1 (0.017) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:38)
1.43e3510.2138
506.1837
332.1371407.1629 472.1790
511.2184
512.2202
663.2775548.1782
HR-MS (TOF) calcd for C29H27N5O4: [M+H]+, 510.2138; found, 510.2141.IR Spectrum
N
O
O
O
OCH3N
NN
N 7a
N
O
O
O
OCH3N
NN
N 7a
N
O
O
O
OCH3N
NN
N 7a
2.9 Spectra of Compound 7b1H NMR Spectrum
13C NMR Spectrum
N
O
O
O
OCH3N
NN
N 7a
N
O
O
O
OCH3N
NN
N
F
7b
N
O
O
O
OCH3N
NN
N
F
7b
HRMS Spectrum
13-May-2016DJR-24
m/z350 400 450 500 550 600 650 700 750
%
0
100ZCH-806 24 (0.416) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:25)
1.91e3528.2045
362.3285 524.1753381.2993
529.2059
530.2082
566.1598 663.2714
HR-MS (TOF) calcd for C29H26FN5O4: [M+H]+, 528.2047; found, 528.2045.IR Spectrum
2.10 Spectra of Compound 7c1H NMR Spectrum
N
O
O
O
OCH3N
NN
N
F
7c
N
O
O
O
OCH3N
NN
N
F
7b
N
O
O
O
OCH3N
NN
N
F
7b
13C NMR Spectrum
HRMS Spectrum13-May-2016DJR-25
m/z350 400 450 500 550 600 650 700 750
%
0
100ZCH-807 21 (0.364) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:22)
1.68e3528.2049
524.1736
362.3267381.2952
529.2080
550.1848
HR-MS (TOF) calcd for C29H26FN5O4: [M+H]+, 528.2047; found, 528.2049.IR Spectrum
2.11 Spectra of Compound 7d
N
O
O
O
OCH3N
NN
N
F
7c
N
O
O
O
OCH3N
NN
N
F
7c
N
O
O
O
OCH3N
NN
N
F
7c
1H NMR Spectrum
13C NMR Spectrum
HRMS Spectrum13-May-2016DJR-23
m/z350 400 450 500 550 600 650 700 750 800
%
0
100ZCH-805 6 (0.103) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:19)
1.72e3544.1750
540.1431362.3285 478.1447510.1979
546.1754
547.1765
568.1559
HR-MS (TOF) calcd for C29H26ClN5O4: [M+H]+, 544.1752; found, 544.1750.
N
O
O
O
OCH3N
NN
N
Cl
7d
N
O
O
O
OCH3N
NN
N
Cl
7d
N
O
O
O
OCH3N
NN
N
Cl
7d
IR Spectrum
2.12 Spectra of Compound 7e1H NMR Spectrum
13C NMR Spectrum
N
O
O
O
OCH3N
NN
N
Cl
7d
N
O
O
O
OCH3N
NN
N
Cl
7e
N
O
O
O
OCH3N
NN
N
Cl
7e
HRMS Spectrum01-Jul-2016DJR-27
m/z350 400 450 500 550 600 650 700 750 800
%
0
100ZCH-833 18 (0.176) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:30)
3.06e3544.1754
540.1469
546.1755
547.1799
548.1823
HR-MS (TOF) calcd for C29H26ClN5O4: [M+H]+, 544.1752; found, 544.1754.IR Spectrum
2.13 Spectra of Compound 7f1H NMR Spectrum
13C NMR Spectrum
N
O
O
O
OCH3N
NN
N
Cl
7e
N
O
O
O
OCH3N
NN
N
Cl
7e
N
O
O
O
OCH3N
NN
N
Cl
7f
HRMS Spectrum01-Jul-2016DJR-28
m/z350 400 450 500 550 600 650 700 750
%
0
100ZCH-834 10 (0.099) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:25)
1.60e3544.1755
540.1461
478.1443
546.1738
547.1797
548.1786
HR-MS (TOF) calcd for C29H26ClN5O4: [M+H]+, 544.1752; found, 544.1755.IR Spectrum
2.14 Spectra of Compound 7g
1H NMR Spectrum
N
O
O
O
OCH3N
NN
N
Cl
7f
N
O
O
O
OCH3N
NN
N
Cl
7f
N
O
O
O
OCH3N
NN
N
Cl
7f
13C NMR Spectrum
HRMS Spectrum
12-May-2016DJR-17
m/z200 300 400 500 600 700 800 900 1000
%
0
100ZCH-800 3 (0.052) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:11)
2.03e3578.1364
274.2744
243.0205318.3005
574.1085332.1318
580.1354
581.1393
582.1352583.1361
738.1051
N
O
O
O
OCH3N
NN
N
Cl Cl
7g
N
O
O
O
OCH3N
NN
N
Cl Cl
7g
N
O
O
O
OCH3N
NN
N
Cl Cl
7g
HR-MS (TOF) calcd for C29H25Cl2N5O4: [M+H]+, 578.1362; found, 578.1364.IR Spectrum
2.15 Spectra of Compound 7h1H NMR Spectrum
13C NMR Spectrum
N
O
O
O
OCH3N
NN
N
Cl Cl
7g
N
O
O
O
OCH3N
NN
N
NO2
7h
HRMS Spectrum01-Jul-2016DJR-26
m/z350 400 450 500 550 600 650 700 750 800 850
%
0
100ZCH-832 1 (0.010) AM (Cen,4, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:43)
1.88e3555.1994
556.2040
557.2073
IR Spectrum
N
O
O
O
OCH3N
NN
N
NO2
7h
N
O
O
O
OCH3N
NN
N
NO2
7h
N
O
O
O
OCH3N
NN
N
NO2
7h