S1

Supporting Information

O-Benzoyl Pyridine Aldoxime and Amidoxime Derivatives:

Novel Efficient DNA Photo-Cleavage Agents

Paraskevi Karamtzioti,a Asterios Papastergiou,a John G. Stefanakis,b Alexandros E. Koumbis,b

Ioanna Anastasiou,a Maria. Koffa,a Konstantina C. Fylaktakidoua,*

a Laboratory of Organic, Bioorganic and Natural Product Chemistry, Molecular Biology and

Genetics Department, 68100 Alexandroupolis, Greeceb Laboratory of Organic Chemistry, Chemistry Department, Aristotle University of Thessaloniki,

54124 Thessaloniki, Greece

E-mail: kfylakta@mbg.duth.gr; tel: ++30-25510-30663; fax: ++30-25510-30613

Electronic Supplementary Material (ESI) for Medicinal Chemistry Communications.This journal is © The Royal Society of Chemistry 2015

S2

Table of contents

1H and 13C NMR spectra of 2 S3-S41H and 13C NMR spectra of 3 S5-S61H and 13C NMR spectra of 4 S7-S81H and 13C NMR spectra of 6 S9-S101H and 13C NMR spectra of 7 S11-S121H and 13C NMR spectra of 8 S13-S141H and 13C NMR spectra of 9 S15-S161H and 13C NMR spectra of 14 S17-S181H and 13C NMR spectra of 15 S19-S201H and 13C NMR spectra of 16 S21-S221H and 13C NMR spectra of 17 S23-S24

Photochemistry S25

Irradiation of N΄-((4-nitrobenzoyl)oxy)picolinimidamide (15) in MeOH/H2O 9:1 S25

Irradiation of N΄-((4-nitrobenzoyl)oxy)picolinimidamide (15) in benzene S27

Gel electrophoresis of plasmid with or without N΄-((4-nitrobenzoyl)oxy)

isonicotinimidamide (17) at various pHs S30

UV-Vis spectra of compounds 4, 8, 14-17 in DMSO S30

S3

S4

S5

S6

S7

S8

S9

S10

S11

S12

S13

S14

S15

S16

S17

S18

S19

S20

S21

S22

S23

S24

S25

PhotochemistryIrradiation of derivative 15 under various conditions at 312 nm.

1. Irradiation of N΄-((4-nitrobenzoyl)oxy)picolinimidamide (15) in MeOH/H2O 9:1.

N΄-((4-nitrobenzoyl)oxy)picolinimidamide 15 (13 mg) was placed into a quartz tube containing

a mixture of MeOH/H2O 9:1 (2 mL). The resulting heterogeneous mixture was degassed for 1 h

and then irradiated at 312 nm for 5 h. The floating solid was removed by filtration to give 9 mg

of unreacted starting material. Samples of the solid and the filtrate were analyzed with LC-MS.

N

N

NH2

O

O

NO2

hv

MeOH/H2O

N

NH2

NH

N

NH2

O

N

NH2

NOH

O2N

O

OH

+ + +

O2N

O O

NO2

+

N

NH2

N

N

NH2

NO

O2N

O

O

+ +

O2N

O+

N

NH2

NO N

NH2N+

15

Scheme 1. Proposed structures for the products obtained by the irradiation N΄-((4-

nitrobenzoyl)oxy)picolinimidamide 15 in MeOH/H2O 9:1. The assignment is based on the

fragments observed by the analysis of the LC-MS.

Solid

UV detection MS detection

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

mAU (x100)254nm,4nm (1.00)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1(x10,000,000)

2:TIC1:TIC

Time 4.9 min

Positive: 287 [M+H]+, 309 [M+Na]+, 341 [M+Na+MeOH]+.

50 100 150 200 250 300 350 400 450 500 550 m/z0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0Inten. (x100,000)

309

287

341

449120

224 403246 489 593378152 552 5674171928067 178

N

N

NH2

O

O

NO2

Chemical Formula: C13H10N4O4Exact Mass: 286,070

S26

Filtrate

UV detection MS detection

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

mAU (x100)254nm,4nm (1.00)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

(x1,000,000)

2:TIC1:TIC

Time 3.1 min

50 100 150 200 250 300 350 400 450 500 550 m/z0.00

0.25

0.50

0.75

1.00

Inten. (x1,000,000)

122

105137

235198 278 326 462169 34062 358 417 486

Positive: 122 [M+H]+.

Time 4.9 min

50 100 150 200 250 300 350 400 450 500 550 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0Inten. (x100,000)

579279

333102

138

192

123 224 528380 438

65 311181 581441400266 525477

Positive: 123 [M1+H]+, 138 [M2+H]+, 279 [M4+Na]+, 333 [M3+H+MeOH]+.

50 100 150 200 250 300 350 400 450 500 550 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25Inten. (x100,000)

279

579333

528309

250 380

477224 441400 524 587438120 152 170102 19158

Positive: 279 [M1+Na]+, 287 [M2+H]+, 309 [M2+Na]+, 333 [M3+H+MeOH]+.

N

NH2

NH

Chemical Formula: C6H7N3Exact Mass: 121,0640

N

NH2

O

Chemical Formula: C6H6N2OExact Mass: 122,048

M1N

NH2

NOH

Chemical Formula: C6H7N3OExact Mass: 137,0589

M2

N

N

NH2

O

O

NO2

Chemical Formula: C13H10N4O4Exact Mass: 286,0702

M2

O2N

O O

NO2

Chemical Formula: C14H8N2O6Exact Mass: 300,0382

M3N

NH2

NO N

NH2N

Chemical Formula: C12H12N6OExact Mass: 256,1073

M1

N

NH2

NO N

NH2N

Chemical Formula: C12H12N6OExact Mass: 256,1073

M4

O2N

O O

NO2

Chemical Formula: C14H8N2O6Exact Mass: 300,0382

M3

S27

Time 6.8 min

50 100 150 200 250 300 350 400 450 500 550 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Inten. (x100,000)

166

355

538522266198 300 431 455 556122 224 48337193 58540313863

Negative: 166 [M-H]-.

The starting material 15 is highly insoluble in the reaction solvent. From the integration

of the UV detection of the filtrate it seems that the N-O cleavage dominates, as fragment with

m/z 166 appears in high percentage. Amidine is consumed in side reactions.

2. Irradiation of N΄-((4-nitrobenzoyl)oxy)picolinimidamide (15) in benzene.

N΄-((4-nitrobenzoyl)oxy)picolinimidamide 15 (13 mg) was placed into a quartz tube containing

benzene (2 mL). The resulting heterogeneous mixture was degassed for 1 h, 1,4-cyclohexadiene

(10 eq) was added and then irradiated at 312 nm for 5 h. The floating solid was removed by

filtration to give 8 mg of unreacted starting material. Samples of the solid and the filtrate were

analyzed with LC-MS.

N

N

NH2

O

O

NO2

hv

1,4-cyclohexadiene,benzene

N

NH2

NH

N

NH2

O

N

NH2

NOH

O2N

O

OH

+ + + +

N

NH2

N

N

NH2

NO

O2N

O

O

+ +

O2N

O+

N

NH2

NO N

NH2NN

NH2

NNH2N

N +

15

Scheme 2. Proposed structures for the products obtained by the irradiation N΄-((4-

nitrobenzoyl)oxy)picolinimidamide 15 in benzene. The assignment is based on the fragments

observed by the analysis of the LC-MS.

O2N

O

OHChemical Formula: C7H5NO4

Exact Mass: 167,0219

S28

Solid

UV detection MS detection

0.0 1.0 2.0 3.0 4.0 5.0 6.0 min-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

mAU (x100)254nm,4nm (1.00)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0(x1,000,000)

2:TIC1:TIC

Time 4.9 min

Same as before for solid.

Filtrate

UV detection MS detection

0.0 2.5 5.0 7.5 10.0 min

-0.25

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

mAU (x100)254nm,4nm (1.00)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

(x10,000,000)

2:TIC1:TIC

Time 3.1 min

50 100 150 200 250 300 350 400 450 500 550 m/z0.0

1.0

2.0

3.0

4.0

5.0

Inten. (x100,000)

241122

105

154 259 275 346 361168 200 322 489418 537 58967 555399 517443

Positive: 122 [M1+H]+, 241 [M2+H]+.

Time 4.6 min

50 100 150 200 250 300 350 400 450 500 550 m/z0.00

0.25

0.50

0.75

1.00

Inten. (x100,000)

123

264202 296

180 505234

390317 360 53365 461102 576343145599

479438

Positive: 123 [M+H]+.

N

NH2

NH

Chemical Formula: C6H7N3Exact Mass: 121,0640

M1

N

NH2

O

Chemical Formula: C6H6N2OExact Mass: 122,0480

N

NH2

NNH2N

N

Chemical Formula: C12H12N6Exact Mass: 240,1123

M2

S29

Time 4.9 min

50 100 150 200 250 300 350 400 450 500 550 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Inten. (x100,000)

138

279

309

341 380224265 528105 557400 477188 587524424 44910467

Positive: 138 [M1+H]+, 279 [M2+Na]+, 309 [M3+Na]+.

Time 6.8 min

50 100 150 200 250 300 350 400 450 500 550 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0Inten. (x100,000)

166

355538522

198 327300266224 450 48337193 585407135122 57262

Negative: 166 [M-H]-.

The starting material 15 is highly insoluble in the reaction solvent. From the integration

of the UV detection of the filtrate it seems that the N-O cleavage dominates, as fragment with

m/z 166 appears in high percentage. Amidine is consumed in side reactions.

References for irradiation of oxime acyl compounds: a) J J.-P. Vermes and R. Beugelmans, Tetrahedron Lett., 1969, 10, 2091–2092; b) J. Lalevée, X. Allonas, J. P. Fouassier, H. Tachi, A. Izumitani, M. Shirai and M. Tsunooka, J. Photochem. Photobiol. A, Chem., 2002, 151, 27–37; c) R. Alonso, P. J. Campos, M. A. Rodríguez and D. Sampedro, J. Org. Chem., 2008, 73, 2234–2239.

N

N

NH2

O

O

NO2

Chemical Formula: C13H10N4O4Exact Mass: 286,0702

M3

N

NH2

NOH

Chemical Formula: C6H7N3OExact Mass: 137,059

M1

O2N

O

OHChemical Formula: C7H5NO4

Exact Mass: 167,022Molecular Weight: 167,119

N

NH2

NO N

NH2N

Chemical Formula: C12H12N6OExact Mass: 256,1073

M2

S30

Gel electrophoresis of plasmid with or without N΄-((4-nitrobenzoyl)oxy)

isonicotinimidamide (17) at various pHs

1 Plasmid + tris buffer pH 42 Plasmid + 17 (500 μΜ) – tris buffer pH 4 + UV3 Plasmid + tris buffer pH 54 Plasmid + 17 (500 μΜ) – tris buffer pH 5 + UV5 Plasmid + tris buffer pH 66 Plasmid + 17 (500 μΜ) – tris buffer pH 6 + UV7 Plasmid + tris buffer pH 78 Plasmid + 17 (500 μΜ) – tris buffer pH 7 + UV 9 Plasmid + tris buffer pH 810 Plasmid + 17 (500 μΜ) – tris buffer pH 8 + UV11 Plasmid + tris buffer pH 912 Plasmid + 17 (500 μΜ) – tris buffer pH 9 + UV13 Plasmid + tris buffer pH 1014 Plasmid + 17 (500 μΜ) – tris buffer pH 10 + UV

UV-Vis spectra of compounds 4, 8, 14-17 in DMSO