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Z. Bai et al. / European Journal of Medicinal Chemistry 159 (2018) 339e356
J ¼ 5.5 Hz, 2H), 3.67e3.62 (m, 2H), 3.17 (d, J ¼ 5.1 Hz, 3H). 13C NMR
4.76 (s, 2H), 4.74 (s, 1H), 3.71 (m, 2H), 3.43 (m, 2H). 13C NMR (CDCl3,
ppm):
215.2, 204.8, 139.5, 132.7, 130.3, 58.1. IR (KBr, cmꢀ1):
(CDCl3, ppm):
d
212.5, 209.2, 198.8, 62.3, 57.8, 41.4. IR (KBr, cmꢀ1):
d
2077 (s), 2001 (vs), 1978 (s), 1944 (s), 1198 (m). ESI-HRMS (m/z):
2084(s), 1998(vs), 1952(s), 1944(vs), 1248 (m), 745 (m). ESI-HRMS
(m/z): Calcd. for C14H12O5NS2MnNa [MþNa]þ: 415.9435; found
415.9462.
Calcd. for C8H8O5NS2MnNa [MþNa]þ: 339.9522; found 339.9536.
Complex 10 Yield: 44%. 1H NMR (CDCl3):
d
5.71 (s, 1H), 5.24 (s,
207.6, 199.2,
2H), 4.25 (s, 2H), 3.13 (s, 3H). 13C NMR (CDCl3, ppm):
d
120.3, 58.7, 43.2. IR (KBr, cmꢀ1): 2078 (s), 1995 (vs), 1977 (vs), 1940
(vs), 1657 (m). ESI-HRMS (m/z): Calcd. for C9H8O4NS2MnNa
[MþNa]þ: 335.9773; found 335.9712.
4.3. CO-releasing tests with myoglobin assay
The release of CO from the metal carbonyl complexes was
studied spectrophotometrically by measuring the conversion of
deoxy-myoglobin (deoxy-Mb) to carbonyl myoglobin (Mb-CO). The
amount of Mb-CO formed was quantified by measuring the
absorbance at 540 nm. A stock solution of myoglobin (lyophilised
Complex 11 Yield: 46%. 1H NMR (CDCl3):
d 6.04 (m, 2H), 5.34 (m,
4H), 3.87 (d, J ¼ 6.0 Hz, 4H). 13C NMR (CDCl3, ppm):
d 212.5, 209.2,
205.6, 141.2, 57.2. IR (KBr, cmꢀ1): 2084(m), 1995(vs), 1974(s),
1929(vs), 1652(m). ESI-HRMS (m/z): Calcd. for C11H10O4NS2MnNa
[MþNa]þ: 361.9029; found 361.9062.
horse heart) (60 mM final concentration) was prepared fresh by
Complex 12 Yield: 38%. 1H NMR (CDCl3):
d
3.71 (q, J ¼ 7.1 Hz, 4H),
dissolving the protein in phosphate buffered saline (PBS, 0.1 M,
pH ¼ 7.4). Sodium dithionite (0.1%) was added to convert the
myoglobin stock to deoxy-Mb. A 2 mL quantity of this solution was
assayed to obtain a deoxy-Mb spectrum and then bubbled with CO
to get an Mb-CO spectrum. Each complex was dissolved in water
and added to deoxy-Mb in the cuvette, mixed using a pipette and
1.26 (t, J ¼ 7.2 Hz, 6H). 13C NMR (CDCl3, ppm):
d
208.7, 201.4, 53.2,
14.1. IR (KBr, cmꢀ1): 2084(s), 1987(vs), 1946(vs), 1938(vs), 1450 (m).
ESI-HRMS (m/z): Calcd. for C9H10O4NS2MnNa [MþNa]þ: 337.9429;
found 337.9483.
Complex 13 Yield: 48%. 1H NMR (CDCl3):
d
3.47 (s, 4H), 2.11 (m,
2H), 0.94 (m,12H). 13C NMR (CDCl3, ppm):
d
211.8, 63.4, 24.1. IR (KBr,
then overlaid with 500 mL light mineral oil to prevent CO escaping
cmꢀ1): 2086(s), 1996(vs), 1952(s), 1945(vs), 1382(m). ESI-HRMS (m/
or the myoglobin being oxygenated. This is the standard procedure;
other experiments have been undertaken using different concen-
trations of myoglobin.
In these experiments, the absorption peak of deoxy-Mb at
560 nm was replaced by the two peaks of Mb-CO at 540 and
578 nm. The concentration of myoglobin in the stock solution was
calculated from the absorption maximum of the Mb-CO solution at
540 nm.
z): Calcd. for C13H19O4NS2Mn [MþH]þ: 372.0736; found 372.0763.
Complex 14 Yield: 37%. 1H NMR (CDCl3):
d
3.25 (t, J ¼ 6.7 Hz, 4H),
1.63 (m, 4H), 1.46 (m, 4H), 1.04 (t, J ¼ 7.6 Hz, 6H). 13C NMR (CDCl3,
ppm):
d
212.5, 199.6, 55.3, 31.6, 21.4, 15.2. IR (KBr, cmꢀ1): 2084 (s),
2001(vs), 1947(s), 1928(s), 1464(m), 1448(m). ESI-HRMS (m/z):
Calcd. for C13H19O4NS2Mn [MþH]þ: 372.0836; found 372.0810.
Complex 15 Yield: 46%. 1H NMR (CDCl3):
d 3.71 (m, 2H), 3.28 (s,
3H), 3.54 (t, J ¼ 6.8 Hz, 2H), 3.11 (s, 2H), 1.02 (t, J ¼ 7.4 Hz, 3H). 13
C
NMR (CDCl3, ppm):
d
215.5, 198.1, 78.3, 55.6, 53.4, 15.7. IR (KBr,
4.4. H2S measurement
cmꢀ1): 2078(s), 1978(vs), 1952(s), 1942(vs), 1446(m), 739 (s). ESI-
HRMS (m/z): Calcd. for C10H13O5NS2MnH [MþH]þ: 345.9816;
found 345.9810.
A 5 mM solution of Na2S in sodium phosphate buffer (20 mM,
pH 7.4) was prepared (Na2S.9H2O, 120.20 mg in 100 mL volumetric
flask) and used as the stock solution. Aliquots of 50, 100, 200, 400,
Complex 16 Yield: 41%. 1H NMR (CDCl3):
d
7.14e7.37 (m, 5H),
201.9, 197.4, 132.7,
4.85 (s, 2H), 3.09 (s, 3H). 13C NMR (CDCl3, ppm):
d
600, 800,1000,1500
50 mL volumetric flask and dissolved in sodium phosphate buffer to
obtain the standard solutions in 5, 10, 20, 40, 60, 80, 100, 150 M,
respectively. 1 mL aliquot of the respective solution was reacted
with the methylene blue (MBþ) cocktail: 30 mM FeCl3 (200
L) in
1.2 M HCl, 20 mM of N,N-dimethyl-1,4- phenylenediamine sulfate
(200 L) in 7.2 M HCl, 1%w/v of Zn(OAc)2 (100 L) in H2O at room
mL of the Na2S stock solution were added into a
131.6, 61.4, 46.7. IR (KBr, cmꢀ1): 2086(s), 1995(vs), 1977(s), 1929(vs),
1450(m), 729 (m). ESI-HRMS (m/z): Calcd. for C13H11O4NS2Mn
[MþH]þ: 363.0010; found 363.0057.
m
Complex 17 Yield: 43%. 1H NMR (CDCl3):
d
7.22e7.44 (m, 10H),
m
4.83 (s, 4H). 13C NMR (CDCl3, ppm):
d 213.4, 141.7, 138.6, 135.4, 56.2.
IR (KBr, cmꢀ1): 2083(s), 2005(vs), 1987(s), 1944(vs), 1452(m), 750
(m). ESI-HRMS (m/z): Calcd. for C19H14O4NS2MnNa [MþNa]þ:
461.9442; found 461.9472.
m
m
temperature for at least 15 min (each reaction was performed in
triplicate). The absorbance of methylene blue was measured at
lmax ¼ 670 nm in UVeVis spectrophotometer (Lambda 25). The
Na2S calibration curve was obtained.
Complex 18 Yield: 51%. 1H NMR (CDCl3):
d
7.18e7.44 (m, 5H),
3.51 (s, 1H). 13C NMR (CDCl3, ppm):
d 209.3, 144.3, 142.7, 137.8, 46.1.
IR (KBr, cmꢀ1): 2084(s), 1996(vs), 1978(s), 1946(vs), 1450(m),
753(m). ESI-HRMS (m/z): Calcd. for C12H8O4NS2MnK [MþK]þ:
387.0212; found 387.0213.
The reaction was initiated by adding 15
mL of stock solution of
the complex or ligand (60 M) into pH 7.4 phosphate buffer (30 mL)
m
containing accelerator for TECP (1.0 mM). Then 2.0 mL of reaction
aliquots were periodically taken and transferred to colorimetric
Complex 19 Yield: 41%. 1H NMR (CDCl3):
d
7.11e7.59 (m, 4H),
3.46 (s, 3H). 13C NMR (CDCl3, ppm):
d
221.6, 204.5, 151.7, 138.2,
cuvette containing zinc acetate (1% w/v, 200
1,4-phenylenediamine sulfate (20 mM, 400
ferric chloride (30 mM, 400 L) in 1.2 M HCl. The absorbance
mL) and N, N-dimethyl-
133.6, 45.4. IR (KBr, cmꢀ1): 2078(s), 1979(vs), 1947(s), 1940(vs),
1093 (m). ESI-HRMS (m/z): Calcd. for C12H8O4ClNS2Mn [MþH]þ:
383.8964; found 383.8979.
mL) in 7.2 M HCl and
m
(670 nm) of the resulted solution was determined 15 min thereafter
using an UVeVis spectrometer (Lambda 25). The H2S concentration
of each sample was calculated against a calibration curve of Na2S.
H2S amount released from the compounds was measured every
3 min, then described the curve about H2S releasing amount vs
time.
Complex 20 Yield: 36%. 1H NMR (CDCl3):
d
7.28e7.56 (m, 4H),
218.3, 212.4, 144.6, 135.2,
133.8, 47.2. IR (KBr, cmꢀ1): 2082(s), 1998(vs), 1949(vs), 1943(vs),
3.43 (s, 3H). 13C NMR (CDCl3, ppm):
d
1091 (m). ESI-HRMS (m/z): Calcd. for
[MþNa]þ: 405.8783; found 405.8792.
Complex 21 Yield: 41%. 1H NMR (CDCl3):
C
12H7O4ClNS2MnNa
d
6.82e7.16 (m, 4H),
4.78 (s, 2H), 3.44 (s, 3H), 3.05 (s, 3H). 13C NMR (CDCl3, ppm):
d
213.6,
4.5. Cytotoxicity assays
204.7, 138.1, 126.3,133.6. IR (KBr, cmꢀ1): 2078(s), 1999(vs), 1967(vs),
1946(vs), 1252(m), 792(m). ESI-HRMS (m/z): Calcd. for
All cell lines were purchased from cell resources Center for
Shanghai Life Science Institute of Chinese Academy of Sciences
(China). The cells were cultivated at 37 ꢁC, 10% CO2, 100% humidity
C
14H12O5NS2MnNa [MþNa]þ: 415.9435; found 415.9421.
Complex 22 Yield: 42%. 1H NMR (CDCl3):
d 7.13e7.28 (m, 5H),