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: zhouch@swu.edu.cn; lishuo@cqut.edu.cn; 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