508
E.S. Spencer et al. / European Journal of Medicinal Chemistry 93 (2015) 501e510
(J ¼ 32.5 Hz), 134.0, 135.3 ppm. 19F NMR (CF3CO2H): ꢁ63.2 ppm.
Spectra in agreement with literature values.
7. Biological assays
4-(isothiocyanatomethyl)benzenesulfonamide (20) [49] was
obtained from 4-aminomethyl benzenesulfonamide hydrochloride
and 1,10-thiocarbonyldiimidazole according to method 4 (white
solid, yield 37.5%) [50]. 1H NMR data matched the reported data
7.1. Isothiocyanates and related compounds
Stock solutions (1 M) of isothiocyanates and related compounds
were prepared in sterile DMSO and stored at 4 ꢂC. Isothiocyanates
were diluted further with DMSO and added to cells so that the final
concentration of DMSO in the media was kept constant at 0.1%.
[49]. 1H NMR (DMSO-d6, 400 MHz):
d
7.87 (d, 2H, J ¼ 8.4 Hz); 7.56
(d, 2H, J ¼ 8.4 Hz); 7.40 (s, 2H, NH2); 5.05 (s, 2H). 13C NMR (DMSO-
d6, 100 MHz):
d 143.9, 138.5, 129.9, 127.6, 126.2, 47.6. HRMS for
C8H8N2NaO2S2 calculated 250.9919, found: 250.9912.
7.2. Recombinant MIF; cloning, expression and purification
5-(isothiocyanatomethyl)benzo[d][1,3]dioxole (21) [34] was
obtained from 1,3-benzodioxol-5-methylamine and 1,10-thio-
carbonyldiimidazole according to method 4 (yellow oil, 68.1%) [50].
1H NMR data matched the reported data [34]. 1H NMR (CDCl3,
Human MIF cDNA was obtained from HeLa cells, ligated into a
pET28
DH5
of kanamycin (50
MIF expression
a
expression vector (Novagen), and transformed into E. coli
(Invitrogen). Resultant clones were selected in the presence
g/mL).
was isopropyl-D-1-
a
400 MHz):
d
6.81e6.75 (m, 3H); 5.98 (s, 2H, CH2); 4.60 (s, 2H,
m
OCH2). 13C NMR (CDCl3, 100 MHz):
d
148.2, 147.7, 127.9, 120.6, 108.5,
induced
with
107.6, 101.4, 48.6). HRMS [M-H]- for C9H6NO2S calculated 192.0125,
found: 192.0120.
thiogalactopyranoside (IPTG) and the cells were harvested by
centrifugation. Cells were lyzed by freeze thawing, dounce ho-
mogenization, and vortex mixing with an equal volume of glass
beads (106 pm; Sigma G-8893). The lysate was clarified by centri-
(1-isothiocyanatoethyl)-benzene (22) [51] was obtained by
reacting 1-phenylethanol with oxalic acid and sodium thiocyanate
according to method 3 [38,39]. IR (neat) 3062, 3029, 2975, 2169,
2928, 2870, 2081, 1603, 1492 cmꢁ1. 1H NMR (CDCl3): 7.37e7.26 (m,
5H), 4.92 (q, 1H, J ¼ 6.8 Hz), 1.68 (d, 3H, J ¼ 6.79) ppm. 13C NMR:
25.0, 57.0, 125.4, 128.2, 128.9, 132.3, 140.2 ppm. Spectra in agree-
ment with literature values.
(1-isothiocyanatopropyl)-benzene (24) [52] was obtained by
reacting 1-phenyl-1-propanol with oxalic acid and sodium thio-
cyanate according to method 3 [38,39]. IR (neat) 3063, 3029, 2967,
2932, 2874, 2075 cmꢁ1. 1H NMR (CDCl3): 7.15e7.35 (m, 5H), 4.62 (t,
1H, J ¼ 8.0 Hz), 1.88 (m, 2H), 0.94 (t, 3H, J ¼ 8.0 Hz) ppm. 13C NMR:
10.5, 32.3, 63.2, 125.9, 128.1, 128.8, 132.0, 138.8 ppm. Spectra in
agreement with literature values.
fugation at 38,000 g for 30 min, followed by filtration with 0.45
and then 0.22 M membrane filters.
mM
m
The clarified lysate was fractionated through a DEAE anion ex-
change column using fast protein liquid chromatography. MIF-
containing fractions were pooled and loaded onto a MONO-Q
HR5/5 column (GE Healthcare). The bound protein was eluted us-
ing Tris-buffered saline (50 mM Tris-Cl, 150 mM NaCl, pH 7.5), and
the fractions containing purified rhMIF were concentrated using a
centrifugal filter device with a 5 kDa molecular-weight cutoff
(Corning Lifesciences). The mass of full length MIF (12,345 Da) was
confirmed by mass spectrometry using a Velos Pro ion trap mass
spectrometer (Thermo Fisher Scientific).
4-(1-isothiocyanatopropyl)phenol (25) [38] was obtained by
reacting 4-(1-hydroxypropyl)phenol (AKos) with oxalic acid and
sodium thiocyanate according to method 3 [38,39]. IR (neat) 2115,
2078, 1721, 1512 cmꢁ1. 1H NMR (CDCl3): 7.26 (m, 2H), 6.84 (m, 2H),
4.62 (dd, 1H, 4.0 Hz, 4.0 Hz), 1.92 (m, 2H), 0.99 (t, 3H, 4.0 Hz) ppm.
13C NMR: 10.6, 32.3, 55.4, 62.9, 114.2, 127.2, 131.1, 131.7, 159.4 ppm.
Spectra in agreement with literature values.
rhMIF was diluted to 0.8
(TBS, 50 mM Tris, 150 mM NaCl, pH 7.4) and 19
transferred to a 96 well plate. Appropriate dilutions of ITCs (1
were added to the rhMIF and incubated at room temperature for
5 min before measurement of tautomerase activity.
m
M with sterile Tris-buffered saline
L aliquots were
L)
m
m
7.3. Cell culture
1-(1-Isothiocyanato-2-methylethyl)-4-methoxybenzene
(26)
was obtained by reacting 1-(4-methoxyphenyl)-1-propanol
(Aldrich) with oxalic acid and sodium thiocyanate according to
method 3 [38,39]. IR (neat) 2049, 1510, 1244 cmꢁ1. 1H NMR (CDCl3):
7.20 (d, 2H, J ¼ 7.2 Hz), 6.90 (d, 2H, J ¼ 6.8 Hz), 4.62 (m, 1H), 3.81 (s,
3H), 1.95 (m, 2H), 1.01 (t, 3H, J ¼ 6.0 Hz) ppm. 13C NMR: 10.6, 32.3,
55.4, 62.9, 114.2, 127.2, 131.1, 131.7, 159.4 ppm.
The Jurkat T-lymphocyte cell line was obtained from American
Type Culture Collection (#TIB-152, Rockville, MD, USA) and main-
tained in RPMI 1640 containing 10% fetal bovine serum, 100 units/
mL penicillin, and 100 mg/mL streptomycin (Invitrogen). Cells were
grown at 37 ꢂC in a humidified atmosphere with 5% CO2. Cells were
resuspended in fresh antibiotic-free medium 1 h before treatment
at a density of 1 ꢀ 106 cells/mL. Following treatment with ITCs for
30 min, 1.5 ꢀ 106 Jurkat cells were collected and resuspended in
1,10-(isothiocyanatomethylene)bisbenzene (27) was obtained
from Aldrich or from reacting diphenylmethanol with oxalic acid
and sodium thiocyanate according to method 3 [38,39]. IR (neat)
60 mL of lysis buffer (40 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM
3055, 3031, 2925, 2162, 2139, 2090 cmꢁ1
.
1H NMR (CDCl3):
EDTA, 1 mM EGTA, 1.6 mg/mL Complete™ protease inhibitors and
7.48e7.23 (m, 10H), 5.99 (s, 1H) ppm.
1% CHAPS). Insoluble material was removed by centrifugation at
1-(2-isothiocyanatoethoxy)-2-methoxybenzene (29) was ob-
tained from 2-(2’-methoxy)phenoxyethyl amine and 1,10-thio-
carbonyldiimidazole according to method 4 (white solid, yield
15,000 g for 4 min. Protein extracts (20 mL) were transferred to a 96
well plate in duplicate for measurement of tautomerase activity.
64.2%) [50]. 1H NMR (CDCl3, 400 MHz):
d
7.01e6.91 (m, 4H); 4.22 (t,
7.4. MIF tautomerase assay
2H, J ¼ 5.6 Hz); 3.90 (t, 2H, J ¼ 4.4 Hz); 3.88 (s, 3H). 13C NMR (CDCl3,
100 MHz):
d
150.3, 147.3, 122.9, 120.9, 116.0, 112.5, 67.9, 56.0, 44.7.
L
-dopachrome methyl ester was prepared by combining 500
mL
HRMS for C10H11NNaO2S calculated 232.0403, found: 232.0407.
6-Isothiocyanato-1-hexanol (32) [53] was prepared from 6-
amino-1-hexanol, carbon disulphide and hydrogen peroxide ac-
cording to method 2 [37]. 1H NMR data matched the reported data
[53]. IR (neat) 3367, 2934, 2859, 2099 cmꢁ1. 1H NMR (CDCl3): 3.65
(t, 2H, J ¼ 6.5 Hz), 3.52 (t, 2H, J ¼ 6.6 Hz), 1.72 (m, 2H), 1.59 (m, 2H)
1.43 (m, 4H) ppm. 13C NMR: 25.0, 26.4, 29.9, 32.4, 45.0, 62.0 ppm.
of a 20 mM sodium periodate stock with 1.5 mL of 20 mM -3,4-
L
dihydroxyphenylalanine methyl ester (in ultrapure H2O) respec-
tively in 8 mL of Bis Tris buffer (50 mM Bis Tris, pH 6.2, 1 mM EDTA).
Components were reacted for 5 min before immediate use in the
tautomerase assay. 180 mL of dopachrome solution was added to
each well and the change in absorbance at 475 nm due to dop-
achrome tautomerization was monitored for 2 min using a Softmax
Pro 190 spectrophotometer (Molecular Devices, Sunnyvale, CA,