Synthesis of Cephalosporin- -Diazeniumdiolates: Biofilm ...Synthesis of Cephalosporin-3′-Diazeniumdiolates: Biofilm Dispersing NO-Donor Prodrugs Activated by -Lactamase Nageshwar

Synthesis of Cephalosporin-3′-Diazeniumdiolates: Biofilm

Dispersing NO-Donor Prodrugs Activated by -Lactamase

Nageshwar Rao Yepuri,a Nicolas Barraud,

b Nasim Shah Mohammadi,

b Bharat G. Kardak,

a

Staffan Kjelleberg,bc

Scott A. Ricebc

and Michael J. Kelso*a

a School of Chemistry, University of Wollongong, NSW, 2522, Australia. Fax: +61 2 4221

4287; Tel: +61 4221 5085; *E-mail: [email protected]

b School of Biotechnology and Biomolecular Sciences and Centre for

Marine Bio-Innovation, University of New South Wales, 2052, Australia.

c The Singapore Centre on Environmental Life Sciences Engineering,

Nanyang Technological University, Singapore.

Electronic Supplementary Information

Table of Contents

1. Synthesis – General Information.

2. Alkylation of Diazeniumdiolate 3 with DPM and PMB-protected-3′-chloro-

cephalosporin esters 2.

3. Experimental Procedures, Characterisation Data and 1H NMR Spectra.

4. Amperometric Measurements of NO Release from 1, 15-19.

5. Microtitre Plate Biofilm Dispersion Assays.

6. References

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

1. Synthesis – General Information

3′-chloro-cephalosporin esters 2 (i.e., (6R,7R)-benzhydryl 3-(chloromethyl)-8-oxo-7-(2-

(thiophen-2-yl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate and (6R,7R)-4-

methoxybenzyl-3-(chloromethyl)-8-oxo-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-

azabicyclo[4.2.0]oct-2-ene-2-carboxylate) and (6R,7R)-4-methoxybenzyl-3-(chloromethyl)-

8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo-[4.2.0]-oct-2-ene-2-carboxylate 5 were

purchased from Shanghai Jinglan Chemical Co. Ltd. (6R,7R)-4-methoxybenzyl-3-

(chloromethyl)-8-oxo-7-amine-5-thia-1-azabicyclo-[4.2.0]-oct-2-ene-2-carboxylate.HCl 26

was purchased from Ark Pharma Inc. All cephalosporin starting materials were used without

further purification. Anhydrous acetone was prepared by refluxing analytical reagent (AR)

grade solvent overnight with CaSO4 (Drierite) before distilling onto 4Ǻ molecular sieves

(pre-dried overnight at 300 °C) under N2. Sodium iodide, trifluoroacetic acid and anisole

were purchased from Sigma Aldrich Chemical Company. Sodium diazeniumdiolates 3,1 9,

2

113 and 13

3 were synthesized according to the literature methods immediately prior to use.

NO(g) used in the preparation of sodium diazeniumdiolate salts was obtained from Sigma

Aldrich. Diethylamine and pyrollidine were refluxed over KOH pellets for 3 h and distilled

onto 4Ǻ molecular sieves under N2 immediately prior to reaction with NO(g). The term

petroleum spirit (Pet. Spirit) refers to petroleum spirit within the boiling range 40-60 oC.

Column chromatography was performed using silica gel 60 (230-400 mesh, Merck). Reaction

monitoring by thin layer chromatography (TLC) was carried out using Merck Silica Gel 60

F254 (0.2 mm) plates. Compounds were visualized by examination under UV light and/or by

staining with cerium ammonium molybdate. 1H and

13C NMR spectra were recorded on a

Varian-Inova-500 MHz spectrometer. Chemical shifts (δ) are relative to tetramethylsilane

(TMS, 0 ppm). IR spectra were recorded neat using a Nicolet Avatar 360 FT-IR spectrometer

with a germanium crystal sample loader. Characteristic absorption bands are quoted in

wavenumbers [cm-1

]. High resolution electrospray mass spectra were recorded using a factory

modified Waters QToF Ultima Mass Spectrometer (Wyntheshawe, UK). Melting points were

determined using a Reichert melting point apparatus and are uncorrected.


2. Alkylation of Diazeniumdiolate 3 with DPM and PMB-protected-3′-chloro-

cephalosporin esters 2.

Scheme S1. Optimisation of reaction conditions for alkylation of sodium diazeniumdiolate

salt 3 with DPM and PMB-protected-3′-chloro-cephalosporin esters 2.


3. Experimental Procedures, Characterisation Data and 1H NMR Spectra

(a) Synthesis 3′-chloro-cephalosporin PMB ester 7.

A pre-mixed solution of triethylamine (0.99 g, 9.78 mmol) and 2,6-lutidine (1.05 g, 9.80

mmol) was added dropwise to a suspension of 3′-chloro-cephalosporin PMB ester.HCl 26

(4.0 g, 9.87 mmol) and 2-(1H-tetrazol-1-yl)acetyl chloride (1.44 g, 9.83 mmol) in dry

CH2Cl2 (20 mL) with stirring at 0 0C. After complete addition the mixture was allowed to

warm to 25 0C and stirred for a further 5 h, after which TLC analysis (6:4 Pet Spirit: EtOAc)

confirmed the reaction was complete. The reaction mixture was diluted with CH2Cl2 (50 mL)

and washed with brine (2 x 30 mL). The organic fraction was dried over MgSO4 and

concentrated in vacuo to give the crude product which was purified by column

chromatography (Pet Spirit:EtOAc, 6:4) to give 7 (3.07 g, 65%) as a white powder.

Characterisation Data for Compound 7:

1H NMR (500 MHz, CD3COCD3): δ 9.17 (s, 1H), 8.60 (d, 1H J = 6 Hz), 7.40 (d, 2H, J = 8.5

Hz), 6.94 (d, 2H, J = 8.5 Hz), 5.91 (q, 1H, J = 5 Hz), 5.54 (s, 2H), 5.29 and 5.20 (ABq, 2H, J

= 12 Hz), 5.23 (d, 1H, J= 5 Hz), 4.61 and 4.59 (ABq, 2H, J = 11.5 Hz), 3.82 and 3.66 (ABq,

2H, J = 18 Hz), 3.80 (s, 3H).

13C NMR (125 MHz, CD3COCD3): δ 166.2, 164.9, 161.9, 160.7, 145.3, 131.1, 127.9, 126.4,

126.3, 114.5, 68.2, 60.2, 58.3, 55.4, 50.0, 44.0, 27.1.

FTIR (cm-1

, Neat): 3283, 3136, 2966, 1771, 1705, 1662, 1555, 1378, 1236, 1170, 1093,

1025, 800.

[α]D (c = 1.0, MeOH) = +117.0

ESI-HRMS (m/z) Calcd. for 501.0718 [M +Na]+

C19H19ClN6NaO5S+, Found 501.0742.


(b) General Procedure A: Alkylation of sodium diazeniumdiolates with ester-protected 3′-

chloro-cephalosporins.

The general procedure is exemplified with sodium diazeniumdiolate 3 and 3′-chloro-

cephalosporin PMB ester 2 to produce 4:

Sodium iodide (0.912 g, 6.08 mmol) was added to a suspension of the 3′-chloro-

cephalosporin PMB ester 2 (3.00 g, 6.08 mmol) in anhydrous acetone (25 mL) under N2 and

the mixture was stirred in the dark at room temperature for 1 h. Sodium diazeniumdiolate 3

(0.944 g, 6.08 mmol) was then added in one shot and the mixture stirred at room temperature

for a further 1.5 hrs (TLC analysis; Pet Spirit:EtOAc 7:3). The solvent was removed under

reduced pressure and the residue diluted with CH2Cl2 (75 mL) and washed with 10% aq.

sodium thiosulphate (2 x 40 mL) and water (1 x 40 mL). The organic fraction was dried over

anhydrous MgSO4 and concentrated in vacuo. The crude product was purified by silica gel


column chromatography (Pet. Spirit:EtOAc, 7:3) and recrystallised from EtOH or MeOH to

give 4 (3.04 g, 85%) as a pale yellow powder.


1H NMR (500 MHz, CDCl3): δ 7.32 (d, 2H, J = 8.5 Hz), 7.26 (d, 1H, J = 5 Hz), 7.00-6.96

(m, 2H), 6.89 (d, 2H, J = 8.5 Hz), 6.34 (d, 1H, J = 9 Hz), 5.81 (dd, 1H, J = 10, 4.5 Hz), 5.34

and 4.99 (ABq, 2H, J = 14.5 Hz), 5.18 (s, 2H), 4.90 (d, 1H, J = 5 Hz), 3.84 (s, 2H), 3.80 (s,

3H), 3.52 and 3.44 (ABq, 2H, J = 18.5 Hz), 3.11 (q, 4H, J = 7.5 Hz), 1.05 (t, 6H, J = 7.5 Hz).

13C NMR (125 MHz, CDCl3): δ 169.8, 164.4, 161.2, 159.9, 134.6, 130.6, 127.8, 127.5,

126.6, 126.4, 126.0, 125.4, 113.9, 71.9, 68.1, 59.2, 57.4, 55.2, 48.3, 37.1, 25.9 11.5.

FTIR (cm-1

, Neat): 3275, 1754, 1706, 1648, 1517, 1362, 1248, 1177, 1096, 1027, 822.

M.P 166 ºC

[α]D (c = 1.0, CHCl3) = +39.0

ESI-HRMS (m/z) Calcd. for 588.1592 [M - H]- C26H30N5O7S2

-, Found 588.1550.


Compound 6:

Sodium iodide: 0.923 g, 6.16 mmol

Anhydrous acetone: 25 mL

3′-chloro-cephalosporin PMB ester 5: 3.00 g, 6.16 mmol

Sodium diazeniumdiolate 3: 0.955 g, 6.16 mmol

Yield 6: 2.69g, 75% (white powder)


1H NMR (500 MHz, CDCl3): δ 7.36-7.24 (m, 7H), 6.88 (d, 2H, J = 9 Hz), 6.08 (d, 1H, J = 10

Hz), 5.81 (dd, 1H, J = 10, 4.5 Hz), 5.33 and 4.98 (ABq, 2H, J = 14 Hz), 5.17 (s, 2H), 4.88 (d,

1H, J = 5 Hz), 3.79 (s, 3H), 3.67 and 3.62 (ABq, 2H, J = 9 Hz), 3.44 and 3.42 (ABq, 2H, J =

18 Hz), 3.10 (q, 4H, J = 7 Hz), 1.05 (m, 6H, J = 7 Hz).

13C NMR (125 MHz, CDCl3): δ 171.1, 164.6, 161.2, 159.9, 133.6, 130.7, 129.4, 129.2,

127.8, 126.7, 126.4, 125.5, 114.0, 72.0, 68.1, 59.2, 57.5, 55.2, 48.4, 43.3, 26.0, 11.5.

FTIR (cm-1

, Neat): 3284, 1778, 1726, 1660, 1519, 1352, 1228, 1187, 1030, 982, 818, 716,

699, 679.

M.P 126-128 ºC

[α]D (c = 1.0, CH2Cl2) = +76.9

ESI-HRMS (m/z) Calcd. for 584.2179 [M + H]+ C28H34N5O7S, Found 584.2205.


Compound 8:





Yield 8: 0.58 g, 14% (pale yellow powder)

Yield 8a (i.e., 2-isomer): 1.76 g, 42% (pale yellow powder)


1H NMR (500 MHz, CDCl3): δ 8.90 (s, 1H), 8.26 (d, 1H, J = 6 Hz), 7.30 (d, 2H, J = 8.5 Hz),

6.86 (d, 2H, J = 8.5 Hz), 5.78 (q, 1H, J = 5 Hz), 5.32 and 5.27 (ABq, 2H, J = 16.5 Hz), 5.18

(s, 2H), 5.17 and 5.02 (ABq, 2H, J = 13 Hz), 4.95 (d, 1H, J= 5 Hz), 3.78 (s, 3H), 3.56 and

3.51 (ABq, 2H, J = 19 Hz), 3.14 (q, 4H, J = 7 Hz), 1.05 (t, 6H, J = 7 Hz).

13C NMR (125 MHz, CDCl3): δ 166.3, 164.4, 161.5, 160.2, 144.5, 130.0, 126.8, 126.6,

126.3, 114.2, 72.1, 68.5, 59.7, 57.5, 55.5, 50.1, 48.4, 26.5, 11.7.


FTIR (cm-1

, Neat): 3290, 3137, 2973, 2902, 1771, 1702, 1662, 1556, 1378, 1233, 1170,

1094, 1049, 801.

M.P 171 ºC

[α]D (c = 1.0, acetone) = -51.9

ESI-HRMS (m/z) Calcd. for 574.1838 [M - H]- C23H28N9O7S

-, Found 574.1830.

Characterisation Data for Compound 8a:

1H NMR (500 MHz, CDCl3): δ 8.94 (s, 1H), 7.94 (d, 2H, J = 8.5 Hz), 7.29 (d, 2H, J = 8.5

Hz), 6.89 (d, 2H, J = 8.5 Hz), 6.51 (s, 1H), 5.57 (q, 1H, J = 4.5 Hz), 5.35 and 5.28 (ABq, 2H,

J = 16.5 Hz), 5.27 (d, 1H, J = 3.5 Hz), 5.14 (s, 2H), 5.04 (s, 1H), 4.88 and 4.65 (ABq, 2H, J

= 12.5 Hz), 3.80 (s, 3H), 3.11 (q, 4H, J = 7 Hz), 1.06 (t, 6H, J = 7 Hz).

13C NMR (125 MHz, CDCl3): δ 166.7, 164.5, 163.3, 160.0, 144.1, 130.4, 126.6, 124.4,

118.8, 114.1, 104.7, 74.8, 68.2, 60.4, 55.3, 53.2, 49.9. 48.2, 11.4.

FTIR (cm-1

, Neat): 3283, 3136, 2973, 2933, 1769, 1727, 1668, 1513, 1249, 1170, 1030, 990.

M.P 133 ºC

[α]D (c = 1.0, CHCl3) = 260.6


-, Found 574.1799.


Compound 10:





Yield 10: 1.96 g, 66% (off-white powder)


1H NMR (500 MHz, CDCl3): δ 7.36 (m, 2H), 7.32 (d, 2H, J = 8.5 Hz), 7.26 (m, 3H), 6.88 (d,

1H, J = 8.5 Hz), 6.05 (d, 1H, J = 9 Hz), 5.82 and 5.80 (dd, 1H, J = 4.5, 5 Hz), 5.21-5.15 (m,

3H), 4.91 (d, 1H, J = 4.4 Hz), 4.87 (d, 1H, J = 13.5 Hz), 3.80 (s, 3H), 3.68 and 3.62 (ABq,

2H, J = 16 Hz), 3.52 and 3.43 (ABq, 2H, J = 18.5 Hz), 3.46 (t, 4H, J = 7 Hz), 1.91 (t, 4H, J =

7 Hz).

13C NMR (125 MHz, CDCl3): δ 171.0, 164.7, 161.3, 159.9, 133.5, 130.6, 129.4, 129.2,

127.8, 126.8, 126.7, 125.3, 114.0, 71.4, 68.0, 59.1, 57.4, 55.2, 50.7, 43.3, 26.1, 22.8.

FTIR (cm-1

, Neat): 3265, 2965, 2162, 2030, 1756, 1714, 1652, 1612, 1536, 1486, 1446,

1392, 1266, 1244, 1217, 1180, 1013, 986.


M.P 157 ºC

[α]D (c = 1.0, MeOH) = +20.6


-, Found 580.1895

Compound 12:







1H NMR (500 MHz, CDCl3): δ 8.33 (d, 2H, J = 4.5 Hz), 7.39-7.26 (m, 5H), 7.32 (d, 2H, J =

7.5 Hz), 6.88 (d, 2H, J = 7.5 Hz), 6.56 (t, 1H, J = 2 Hz), 6.18 (d, 1H, J = 9 Hz), 5.82 (q, 1H, J

= 5 Hz), 5.24 and 4.95 (ABq, 2H, J = 13.5 Hz), 5.18 (d, 2H, J = 2.5 Hz), 4.91 (d, 1H, J = 4.5

Hz), 3.98 (t, 4H, J = 4.5 Hz), 3.78 (s, 3H), 3.66 and 3.61 (ABq, 2H, J = 16 Hz), 3.53 and 3.40

(ABq, 2H, J = 18.5 Hz), 3.41 (t, 4H, J = 4.5 Hz).

13C NMR (125 MHz, CDCl3): δ 171.0, 164.7, 161.3, 159.9, 157.8, 133.6, 130.7, 129.4,

129.2, 127.7, 126.6, 125.8, 125.7, 113.9, 110.7, 71.8, 68.1, 59.2, 57.4, 55.2, 50.9, 43.3, 42.3,

26.1.


FTIR (cm-1

, Neat): 3286, 3137, 2976, 2908, 2904, 1772, 1702, 1662, 1557, 1411, 1377,

1232, 1049.

[α]D (c = 1.0, MeOH) = +39.5

ESI-HRMS (m/z) Calcd. for 675.2344 [M + H]+ C32H35N8O7S

+, Found 675.2373.

M.P 136 ºC

Compound 14:







1H NMR (500 MHz, CDCl3): δ 7.36-7.24 (m, 6H), 6.93-6.91 (m, 2H), 6.88 (d, 2H, J = 9 Hz),

6.16 (m, 1H), 5.81 (q, 1H, J = 5 Hz), 5.24 (d, 1H, J = 2.5 Hz), 5.21 (s, 2H), 5.17 and 4.96


(ABq, 2H, J = 12 Hz), 4.91 (d, 2H, J = 2.5 Hz), 3.78 (s, 3H), 3.67 and 3.58 (ABq, 2H, J = 16

Hz), 3.53 and 3.40 (ABq, 2H, J = 18.5 Hz), 3.51 (m, 4H), 3.29 (m, 4H).

13C NMR (125 MHz, CDCl3): δ 171.3, 164.9, 161.5, 160.2, 150.4, 133.8, 130.9, 129.6,

129.5, 129.4, 127.9, 126.9, 126.0, 121.0, 116.8, 114.2, 72.0, 68.4, 59.4, 57.7, 55.4, 51.1, 48.4,

43.5, 26.1.

FTIR (cm-1

, Neat): 3389, 3286, 3197, 3030, 2897, 1756, 1647, 1607, 1546, 1492, 1448,

1384, 1351, 1224, 1004.

[α]D (c = 1.0, MeOH) = +54.3


+, Found 673.2409.

M.P 106 ºC


(c) General Procedure B: Deprotection of cephalosporin-3′-diazeniumdiolate PMB esters

The general procedure is exemplified with cephalosporin-3′-diazeniumdiolate PMB ester 4 to

produce 15:

The PMB-protected cephalosporin-3′-diazeniumdiolate 4 (1.00 g, 1.69 mmol) and anhydrous

anisole (0.55 g, 5.09 mmol) were stirred at 0 ⁰C for 30 mins with neat trifluoroacetic acid (4.0

mL). The mixture was poured slowly onto crushed ice (~ 100 g) and upon melting the

aqueous mixture was extracted with CH2Cl2 (3 x 70 mL). The combined organic extracts

were dried over anhydrous MgSO4 and concentrated under reduced pressure. The residue was

purified by silica gel column chromatography (EtOAc:MeOH:H2O, 85:15:0.5) to provide the

cephalosporin-3′-diazeniumdiolate free acid 15 (0.285 g, 36%) as a pale yellow powder.


1H NMR (500 MHz, CD3OD): δ 7.26 (s, 1H, J = 1 Hz), 6.96-6.92 (m, 2H), 5.73 (d, 1H, J =

5, Hz), 5.29 and 5.07 (ABq, 2H, J = 12.9 Hz), 5.05 (d, 1H, J= 4.8 Hz), 3.83 and 3.79 (ABq,

2H, J = 15 Hz), 3.62 and 3.53 (ABq, 2H, J = 18 Hz), 3.16 (q, 4H, J = 7.0 Hz), 1.04 (t, 6H, J

= 7.0 Hz).

13C NMR (125 MHz, CD3OD): δ 173.3, 166.1, 164.5, 137.4, 130.2, 128.4, 127.7, 126.9,

125.8, 73.0, 60.8, 59.0, 49.0, 37.1, 27.1, 11.7.

FTIR (cm-1

, Neat): 3275, 2156, 1754, 1661, 1656, 1522, 1320, 1235, 1065, 995.

M.P 87 °C

[α]D (c = 1.0, MeOH) = +84.4

ESI-HRMS (m/z) Calcd. for 468.1017 [M - H]- C18H22N5O6S2

-, Found 468.0996.


Compound 1:

Cephalosporin-3′-diazeniumdiolate PMB ester 6: 1.5 g, 2.57 mmol

Anhydrous anisole: 0.416 g, 3.85 mmol

Trifluoroacetic acid: 7.0 mL



1H NMR (500 MHz, CD3OD): δ 7.35-7.26 (m, 5H), 5.75 (d, 1H, J = 4.8, Hz), 5.36 and 5.07

(ABq, 2H, J = 12.9 Hz), 5.05 (d, 1H, J= 4.8 Hz), 5.03 (m, 1H), 3.68-3.45 (m, 4H), 3.19 (q,

4H, J = 7.0 Hz), 1.07 (t, 6H, J = 7.0 Hz).


13C NMR (125 MHz, CD3OD): δ 175.4, 167.3, 166.6, 137.2, 132.1, 131.0, 130.4, 128.8,

123.0, 74.6, 61.5, 59.8, 50.1, 44.0, 27.7, 12.6.

FTIR (cm-1

, Neat): 3287, 1780, 1663, 1505, 1337, 1223, 998, 618.

M.P 84-86 °C

[]D (c = 1.0, CH2Cl2) = +19.4

ESI-HRMS (m/z) Calcd. for 462.1453 (C20H24N5O6S) [M – H]-, Found 462.1465.

Compound 16:





Yield 16: 0.16 g, 81% (white powder)


1H NMR (500 MHz, CD3OD: δ 9.40 (d, 1H, J = 6 Hz), 9.11 (s, 1H), 5.72 (d, 2H, J = 8 Hz),

5.29 (s, 2H), 5.22 and 4.99 (ABq, 2H, J = 22 Hz), 5.01 (s, 1H), 3.52 and 3.49 (ABq, 2H, J =

19 Hz), 3.10 (q, 4H, J = 7 Hz), 0.95 (t, 6H, J = 7 Hz).

13C NMR (125 MHz, CD3OD): δ 167.4, 165.8, 164.4, 146.0, 128.1, 127.5, 72.9, 60.6, 58.7,

50.3, 49.3, 27.1, 11.7.

FTIR (cm-1

, Neat): 3282, 3136, 2975, 2875, 1773, 1703, 1662, 1559, 1414, 1227, 1170,

1026, 800.

M.P 156 ºC.

[α]D (c = 1.0, MeOH) = +117.3


-, Found 454.1266.

Compound 16a:

Cephalosporin-3′-diazeniumdiolate PMB ester 8a: 0.5 g, 0.87 mmol




Yield 16a: 0.33 g, 83% (white powder)

Characterisation Data for Compound 16a:

1H NMR (500 MHz, CD3OD): δ 9.20 (s, 1H), 6.68 (s, 1H), 5.55 (d, 1H, J = 4 Hz), 5.40 (s,

2H), 5.30 (d, 1H, J = 4 Hz), 5.06 and 4.81 (ABq, 2H, J = 13 Hz), 3.11 (q, 4H, J = 7 Hz), 1.05

(t, 6H, J = 7 Hz).

13C NMR (125 MHz, CD3OD): δ 168.8, 166.1, 163.8, 144.8, 123.1, 120.5, 74.8, 60.7, 53.2,

49.9, 49.2, 48.2, 10.6.

FTIR (cm-1

, Neat): 3281, 3137, 2968, 2902, 1771, 1705, 1701, 1556, 1229, 1168, 1052, 802.

[α]D (c = 1.0, MeOH) = +117.0


-, Found 454.1240.

M.P 156 ºC.

Compound 17:







1H NMR (500 MHz, CD3OD): δ 7.30-7.22 (m, 5H), 5.72 (d, 1H, J = 7.5 Hz), 5.23 and 4.97

(ABq, 2H, J = 22.5 Hz), 5.07 (d, 1H, J = 8 Hz), 3.64-3.50 (m, 4H), 3.50 (t, 4H, J = 7 Hz),

1.93 (t, 4H, J = 7 Hz).

13C NMR (125 MHz, CD3OD): δ 174.5, 166.2, 164.5, 136.4, 130.4, 129.5, 127.9, 127.6,

72.5, 60.7, 59.1, 51.5, 43.1, 27.2, 23.7.

FTIR (cm-1

, Neat): 3296, 1756, 1729, 1642, 1530, 1469, 1429, 1388, 1318, 1141, 1028, 944.

[α]D (c = 1.0, MeOH) = 122.8

ESI-HRMS (m/z) Calcd. for 484.1261 [M + Na]+ C20H23N5NaO6S

+, Found 484.1281.

M.P 146 ºC.

Compound 18:





Yield 18: 0.23 g, 93%

Notes: After stirring at 0 ⁰C for 30 min the reaction mixture was poured slowly onto crushed

ice (~ 100 g). Upon melting, the aqueous mixture was filtered through a sintered funnel. The

gummy residue was triturated with diethyl ether to give brown coloured solid. The solid was

washed several times with ether and upon drying yielded a tan coloured solid.


1H NMR (500 MHz, CD3OD): δ 8.37 (s, 2H), 7.34-7.23 (m, 5H), 6.65 (s, 1H), 5.84 (s, 1H),

5.24 and 4.97 (ABq, 2H, J = 7.8 Hz), 5.14 (s, 1H), 3.97 (s, 4H), 3.69-3.41 (m, 4H), 3.46 (s,

4H).

13C NMR (125 MHz, CD3OD): δ 172.1, 166.4, 164.0, 163.0, 159.5, 137.5, 130.9, 129.9,

128.0, 126.7, 111.9, 72.8, 61.5, 58.2, 52.0, 43.3, 42.3, 26.1.

FTIR (cm-1

, Neat): 3286, 3137, 2976, 2908, 2904, 1772, 1702, 1662, 1557, 1411, 1232,

1049.

[α]D (c = 1.0, MeOH) = +36.6


+, Found 555.1799.

M.P 116 ºC


Compound 19:




Yield 19: 0.276 g, 67%

Notes: After stirring at 0 ⁰C for 1/2 h the reaction mixture was poured slowly onto crushed

ice (~ 100 g). Upon melting, the aqueous mixture was filtered through a sintered funnel. The

gummy residue was triturated with diethyl ether to give a brown coloured solid. The solid

was washed several times with ether and upon drying yielded a tan coloured powder.


1H NMR (500 MHz, CD3COCD3): δ 8.05 (d, 1H, J = 8.5 Hz), 7.36-7.23 (m, 8H), 7.02 (d,

2H, J = 7.5 Hz), 6.84 (t, 1H, J = 7.5), 5.85 (q, 1H, J = 5 Hz), 5.25 and 5.01 (ABq, 2H, J =

13.5 Hz), 5.14 (d, 1H, J = 4.5 Hz), 3.70 and 3.58 (m, 4H), 3.55 (m, 4H), 3.35 (m, 4H).

13C NMR (125 MHz, CD3COCD3): δ 171.6, 165.8, 163.1, 151.5, 136.5, 130.0, 129.8, 129.1,

127.4, 125.9, 120.7, 117.2, 72.2, 60.3, 58.5, 51.6, 48.7, 42.9, 26.7.


FTIR (cm-1

, Neat): 3397, 3286, 3197, 3028, 2897, 1755, 1647, 1607, 1548, 1492, 1448,

1383, 1351, 1225, 1004, 985.

[α]D (c = 1.0, MeOH) = +57.7


+, Found 553.1847.

M.P 96 ºC.

(d) General Procedure C: Potassium salt formation from cephalosporin-3′-diazeniumdiolate

free acids

The general procedure is exemplified with cephalosporin-3′-diazeniumdiolate free acid 15 to

produce 20:


Cephalosporin-3′-diazeniumdiolate free acid 15 (0.20 g, 0.42 mmol) was suspended in H2O

(1.0 ml) at 0 oC to which was then added an ice-cold aqueous solution of KOH (0.023 g in

100 µL H2O, 0.42 mmol). A pale yellow solution was rapidly formed and stirring was

continued for a further 20 minutes at 0 oC. The aqueous solution was washed with CH2Cl2 (2

x 2 mL) and then frozen and freeze-dried to provide the potassium cephalosporin-3′-

diazeniumdiolate carboxylate salt 20 (0. 21 g, 99 %) as a pale yellow powder.


1H NMR (500 MHz, D2O): δ 7.21 (d, 1H, J = 10 Hz), 6.89-6.88 (s, 2H), 5.49 (d, 1H, J = 4.5,

Hz), 5.08 and 4.85 (ABq, 2H, J = 12.6 Hz), 4.96 (d, 1H, J= 4.8 Hz), 3.81 and 3.74 (ABq, 2H,

J = 15 Hz), 3.46 and 3.25 (ABq, 2H, J = 18 Hz), 2.96 (q, 4H, J = 7.0 Hz), 0.85 (t, 6H, J = 7.0

Hz).

13C NMR (125 MHz, D2O): δ 174.4, 168.3, 164.7, 135.8, 132.9, 127.6, 127.5, 125.9, 115.4,

73.9, 59.4, 57.7, 49.0, 36.2, 25.5, 10.7.

FTIR (cm-1

, Neat): 3395, 3326, 3210, 2883, 2816, 1600, 1369, 1233, 1034.

[α]D (c = 1.0, MeOH) = +90.9

ESI-HRMS (m/z) Calcd. for 508.0721 [M + H]+ C18H23N5O6S2K

+, Found 508.0742.


Compound 21:

Cephalosporin-3′-diazeniumdiolate free acid 1: 0.463 g, 1.0 mmol

H2O: 1.0 mL

KOH: 0.056 g in 100 L H2O, 1.0 mmol



1H NMR (500 MHz, CD3OD): δ 7.30-7.20 (m, 5H,), 5.65 (d, 1H, J = 4.8, Hz), 5.35 and 4.85

(ABq, 2H, J = 12.0 Hz), 4.98 (d, 1H, J = 4.8, Hz), 3.60 and 3.55 (ABq, 2H, J = 14Hz), 3.57

and 3.37 (ABq, 2H, J = 18 Hz), 3.15 (q, 4H, J = 7.0 Hz), 1.04 (t, 6H, J = 7.0 Hz).

13C NMR (125 MHz, CD3OD): δ 174.6, 168.7, 165.3, 136.5, 134.5, 130.2, 129.6, 128.0,

116.9, 74.7, 60.4, 58.9, 49.4, 43.1, 26.8, 11.8.

IR (cm-1

, Neat): 3395, 1765, 1609, 1495, 1345, 1248, 997, 679


M.P 35-37 °C

[]D (c = 1.0, CH2Cl2) = + 19.0

ESI-HRMS (m/z) Calcd. for 502.1157 C20H25KN5O6S [M + H]+, Found 502.1163.

Compound 23:


H2O: 2.0 mL

KOH: 0.06 g in 100 L H2O, 1.08 mmol



1H NMR (500 MHz, D2O): δ 7.35-7.2 (m, 5H), 5.53 (d, 1H, J = 4 Hz), 5.05 and 4.72 (ABq,

2H, J = 12 Hz), 4.99 (d, 1H, J = 4 Hz), 3.62-3.58 (m, 2H), 3.50 and 3.30 (ABq, 2H, J = 13

Hz), 3.4 (m, 4H), 1.82 (m, 4H).

13C NMR (125 MHz, D2O): δ 175.5, 168.4, 164.9, 135.0, 132.3, 129.3, 129.1, 127.9, 116.5,

72.9, 59.2, 58.0, 51.6, 42.3, 25.8, 22.5.


FTIR (cm-1

, Neat): 3399, 3283, 3194, 2976, 2980, 2897, 1758, 1648, 1609, 1542, 1492,

1451, 1386, 1351, 1224, 1001.

[α]D (c = 1.0, MeOH) = -390.3

ESI-HRMS (m/z) Calcd. for 500.1001 [M + H]+ C20H23KN5O6S

+, Found 500.1015.

Compound 24:


H2O: 2.0 mL

KOH: 0.01 g in 40 L H2O, 0.18 mmol




1H NMR (500 MHz, CD3OD): δ 8.33 (s, 2H), 7.39-7.26 (m, 5H), 7.32 (d, 1H, J = 7.5 Hz),

6.56 (t, 1H, J = 2 Hz), 5.63 (d, 1H, J = 5 Hz), 5.24 and 4.95 (ABq, 2H, J = 13.5 Hz), 5.18 (d,

2H, J = 2.5 Hz), 4.91 (d, 1H, J = 4.5 Hz), 3.98 (m, 4H, J = 4.5 Hz), 3.66 and 3.61 (ABq, 2H,

J = 16 Hz), 3.53 and 3.40 (ABq, 2H, J = 18.5 Hz), 3.41 (t, 4H, J = 4.5 Hz).

13C NMR (125 MHz, CD3OD): δ 174.8 165.0, 162.8 159.2, 136.5, 130.1, 129.9, 127.0,

118.2, 112.0, 74.3, 60.5, 59.2, 51.9, 43.3, 42.3, 26.1.

FTIR (cm-1

, Neat): 3394, 3282, 3190, 3034, 2980, 2894, 1756, 1645, 1609, 1543, 1492,

1448, 1352, 1224, 994.

[α]D (c = 1.0, MeOH) = -190.6

ESI-HRMS (m/z) Calcd. for 593.1328 [M + H]+ C24H26KN8O6S

+, Found 593.1366.


4. Amperometric Measurements of NO Release from 1, 15-19.

Nitric oxide (NO) was detected amperometrically using an Apollo 4000 free radical analyser

(World Precision Instruments) fitted with an NO specific probe (ISO-NOP). The NO probe,

which was freshly calibrated using a solution of S-nitroso-N-acetylpenicillamine (SNAP)

according to the manufacturer’s instructions, was immersed in a vial containing 10 mL Tris

buffer pH 7.0 (100 mM) with continuous stirring at room temperature. The various reagents

were added successively while monitoring NO levels in real-time. The -lactamase used was

a commercial penicillinase from Bacillus cereus purchased from Sigma.

5. Microtitre Plate Biofilm Dispersion Assays

P. aeruginosa PAO1 wild type biofilms were grown from a 1:200 dilution of a P. aeruginosa

overnight culture in LB in 1 mL M9 minimal medium (containing 48 mM Na2HPO4, 22 mM

KH2PO4, 9 mM NaCl, 19 mM NH4Cl, 2 mM MgSO4, 20 mM glucose, 100 µM CaCl2, pH

7.2) in tissue-culture treated 24-well plates (BD) incubated at 37 °C with shaking at 180 rpm.

After an initial 5 h incubation, sub-inhibitory imipenem (Sigma) was added to wells to a final

concentration of 0.3 µg/mL and the plates incubated for a further 1 h. The 6 h-old biofilms

were then treated with test solutions of compounds in less than 1 min per plate and the plates

incubated for a further 15 min at 37 °C with shaking. After removing the supernatant the

remaining biofilms were washed once with PBS (1.0 mL) before adding crystal violet stain

(1.0 mL; 0.2% crystal violet, 1.9% ethanol and 0.08% ammonium oxalate in PBS). The plates

were incubated on the bench for 20 min before washing the wells with PBS (2 x 1.0 mL).

Crystal violet bound to the biofilm on the well surfaces was redissolved by adding 100%

ethanol (1.0 mL) and quantified by measuring OD550 of the homogenized suspension. OD


measurements of control wells where no bacteria were added at the beginning of the

experiment were subtracted from all values (i.e. OD550 = 0.18).

6. References

1 K. R. A. Abdellatif, Y. Dong, Q-H. Chen, M. A. Chowdhury and E. E. Knaus. Bioorg.

Med. Chem. 2007, 15, 6796.

2 J. E. Saavedra, P. J. Shami, L. Y. Wang, K. M. Davies, M. N. Booth, M. L. Citro, and

L. K. Keefer. J. Med. Chem. 2000, 43, 261.

3 J. E. Saavedra, M. N. Booth, J. A. Hrabie, K. M. Davies, L. K. Keefer. J. Org. Chem.

1999, 64, 5124.


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Synthesis of Cephalosporin- -Diazeniumdiolates: Biofilm ...Synthesis of Cephalosporin-3′-Diazeniumdiolates: Biofilm Dispersing NO-Donor Prodrugs Activated by -Lactamase Nageshwar