Journal of Medicinal Chemistry
Article
solid was filtered and washed with EtOAc. The solid was transferred to
an Erlenmeyer flask and suspended in EtOAc (20 mL). The
suspension was refluxed for 5 min. Upon cooling, the suspension
was then filtered and the solid was dried under vacuum to give product
(CH2O), 81.65 (CMe3), 81.71(CMe3), 101.03 (CHarom), 111.88
(CHarom), 112.30 (CHarom), 113.65 (Carom), 122.50 (Carom), 124.38
(Carom), 128.51 (CHarom), 129.03 (CHarom), 137.23 (CHCCO), 150.38
(Carom), 154.36 (Carom), 161.16 (Carom), 161.30 (CCOO), 171.74
(CH2CO), 172.28 (CH2CO, broad), 172.48 (CH2CO).
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4 (1.96 g, 75%) as a yellow solid. H NMR (400 MHz, DMSO-d6): δ
2.94 (s, NCH3, 6H), 6.66−6.83 (m, ArH, 4H), 7.48−7.63 (m, ArH,
3H), 8.00 (s, CCHAr, 1H). HR ESI-MS (m/z): calcd, 282.1130;
found 282.1186 [M + H]+.
[4,10-Bis-carboxymethyl-7-({3-[3-(4-dimethylaminophenyl)-
2-oxo-2H-chromen-7-yloxy]propylcarbamoyl}methyl)-
1,4,7,10-tetraazacyclododec-1-yl]acetic Acid (9). A solution of 8
(0.052 g, 0.058 mmol) in TFA (2 mL) was stirred at room
temperature for 24 h. TFA was removed under reduced pressure and
the residue dissolved in water (2 mL). The slightly cloudy solution was
filtered and the solvent was evaporated to give 9·TFA as a bright
yellow solid (0.048 g, quantitative). 1H NMR (400 MHz, D2O): δ 1.82
(m, CH2CH2CH2, 2H), 3.20 (s, NCH3, 6H), 2.7−4.3 (b, CH2
macrocycle, CH2 acetic arms, OCH2, NCH2, 28H), 6.57 (d, J = 2.3
Hz, ArH, 1H), 6.66 (dd, J = 2.3 Hz, J = 9.7 Hz, ArH, 1H), 7.27 (d, J =
8.8 Hz ArH, 1H), 7.55 (d, AB system, J = 8.9 Hz ArH, 2H), 7.62 (d,
AB system, J = 8.9 Hz, ArH, 2H), 7.68 (s, CCH, 1H). ESI-MS (m/
z): calcd, 725.35; found, 725.33 [M + H]+.
{3-[3-(4-Dimethylaminophenyl)-2-oxo-2H-chromen-7-
yloxy]propyl}carbamic Acid tert-Butyl Ester (5). To a solution of
4 (0.155 g, 0.55 mmol) and (3-bromopropyl)carbamic acid tert-butyl
ester (0.164 mg, 0.69 mmol) in dry acetonitrile (5 mL) was added
cesium carbonate (0.349 mg, 1.07 mmol). The reaction mixture was
stirred at 70 °C and monitored by TLC (MeOH/DCM 2/98, Rf =
0.76) until completion (∼20 h). The solvent was removed under
reduced pressure, and the residue was triturated and then decanted,
first with water (2 × 10 mL) and subsequently with diethyl ether (2 ×
5 mL). The residual solid was dried under reduced pressure to yield
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compound 5 (0.186 g, 79%) as a yellow-orange solid. H NMR (400
Gadolinium(III) [4,10-Bis-carboxymethyl-7-({3-[3-(4-dime-
t h y l a m i n o p h e n y l ) - 2 - o x o - 2 H - c h r o m e n - 7 - y l o x y ] -
propylcarbamoyl}methyl)-1,4,7,10-tetraazacyclododec-1-yl]-
acetate (10). To a solution of 9 (0.042 g, 0.058 mmol) in water (10
mL) was added a solution of GdCl3 in water (5.3 mM, 12.5 mL, 0.067
mmol) while maintaining the pH at 6.0 by addition of NaOH(aq), 1 M.
The solution was stirred at 50 °C for 12 h. The pH was raised to 9.0.
The solution was filtered through a 0.2 μm filter, and the pH was
lowered to 7.0 by adding HCl. The solution was loaded on a Hypersep
C-18 cartridge, and the stationary phase was washed extensively with
water. The product was recovered after gradient elution with MeOH/
H2O from 1:9 to 3:2. The fractions containing the product were
evaporated under reduced pressure to give 10 (0.039 g, 68%) as a
yellow solid. HR-ESI-MS (m/z): calcd 880.2520; found 880.2501 [M
+ H]+. A correct Gd isotope pattern was observed.
Animal Handling. All animal experiments were performed in
accordance with guidelines approved by the Institutional Animal Care
and Use Committee of Case Western Reserve University (Protocols
2010-0006 and 2010-0007). Two-month-old Swiss-Webster R/J mice
were purchased from Harlan Laboratories, Indianapolis, IN. Two-
month-old C57BL/6 mice and C3Fe.SWV-Mbpshi/J shiverer mice
were obtained from The Jackson Laboratory, Bar Harbor, ME.
Sprague−Dawley rats were purchased from Harlan Laboratories,
Haslett, MI.
Preparation of Frozen Sections. Mice were deeply anesthetized
with isoflurane and perfused via the ascending aorta with 1× PBS
followed by 4% paraformaldehyde in PBS. Brains were removed and
incubated for 24 h in 4% paraformaldehyde in PBS at 4 °C and then in
30% sucrose at 4 °C until submerged. Frozen sections were used for
fluorescence microscopy. For preparation of fresh frozen sections, the
cryoprotected tissues were first frozen in OCT on dry ice before axial
sectioning (20 μm) with a cryostat at −20 °C.
In Vitro Staining. Frozen sections or floating sections mounted
on Superfrost slides (Fisher Scientific) were incubated with an
aqueous solution of compound 10 (100 μM) for 20 min at room
temperature. Excess compound 10 was removed by briefly rinsing the
sections in PBS before coverslipping with Vectashield mounting
medium (Vector Laboratories; Burlingame, CA). Sections were then
examined using epifluorescence microscopy with a Leica DM5000B
microscope equipped with an HCX PL FLUOTAR 1.25×/0.04
objective and using the A4 filter (360/40 nm band-pass excitation, 400
nm dichromatic mirror, 470/40 nm band-pass suppression). In some
cases, tissue sections were double stained with antimyelin basic protein
(MBP) monoclonal antibody (MAb) (see below), in which case the
coverslipping and Vectrashield were omitted.
MHz, CDCl3): δ 1.43 (s, CCH3, 9H), 1.99 (m, CH2CH2CH2, 2H),
2.97 (s, NCH3, 6H), 3.32 (m, NCH2, 2H), 4.03 (t, OCH2, J = 5.99 Hz,
2H), 4.88 (t b, NH, 1H), 6.71−6.82 (m, ArH, 4H), 7.35 (d, ArH, J =
8.49, 1H), 7.58−7.63 (m, ArCH2, and CCHAr, 3H). 13C NMR (100
MHz, CDCl3): δ 28.29 (CCH3), 29.29 (CH2CH2CH2), 37.59
(NCH2), 40.23 (NCH3), 66.05 (OCH2), 79.11 (CCH3), 100.71
(CH), 111.84 (CH), 112.53 (CH), 113.69 (C), 122.46 (C), 124.57
(C), 128.24 (CH), 129.01 (CH), 136.90 (CH), 150.34 (C), 154.42
(C), 155.89 (CO), 160.89 (C), 161.13 (CO). ESI-MS (m/z): calcd,
439.22; found 439.12 [M + H]+.
7-(3-Aminopropoxy)-3-(4-dimethylaminophenyl)chromen-
2-one (6). Compound 5 (0.25 g, 0.57 mmol) was dissolved in
trifluoroacetic acid and stirred at room temperature for 12 h. The
slightly cloudy solution was filtered to remove any insoluble impurity
before the solvent was evaporated under reduced pressure. The residue
was dissolved in dichloromethane (25 mL) and extracted with 0.2 M
NaOH(aq) (3 × 25 mL) to yield 6 (106 mg, 93%) as a glassy yellow
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solid. H NMR (400 MHz, CD3OD): δ 2.19 (dt, CH2CH2CH2, J =
7.3, 5.8 Hz, 2H), 3.18 (t, NCH2, J = 7.3 Hz, 2H), 3.27 (s, NCH3, 6H),
4.2 (t, OCH2, J = 5.8 Hz, 2H), 6.91−6.96 (m, ArH, 2H), 7.57−7.62
(m, ArH, 3H), 7.85−7.90 (m, ArH, 2H), 8.04 (s, CCHAr, 1H). 13C
NMR (150 MHz, 60 °C, DMSO-d6): δ 26.38 (CH2CH2CH2), 35.84
(NCH3), 42.62 (NCH2), 65.44 (OCH2), 100.66 (CH), 112.68 (CH),
113.06 (CH), 116.34 (2 × C), 122.31 (C), 129.13 (CH), 129.31 (CH),
139.30 (CH), 146.13 (C), 154.30 (C), 159.68 (CO), 161.02 (CO).
ESI-MS (m/z): calcd, 339.17; found 339.19 [M + H]+.
[4,10-Bis-tert-butoxycarbonylmethyl-7-({3-[3-(4-dimethyla-
minophenyl)-2-oxo-2H-chromen-7-yloxy]propylcarbamoyl}-
methyl)-1,4,7,10-tetraazacyclododec-1-yl]acetic Acid tert-
Butyl Ester (8). A solution of 6 (0.075 g, 0.222 mmol), DOTA-
tris-(t-Bu) ester (7, 0.102 g, 0.177 mmol), HOBT (0.041 g, 0.222
mmol), HBTU (0.101 g, 0.266 mmol), and DIPEA (0.046 g, 0.355
mmol) in dry DMF (10 mL) was stirred at room temperature for 20 h
(TLC MeOH/DCM, 1/9, Rf = 0.66). The solvent was removed under
reduced pressure, and the residue was triturated with water and then
decanted (2 × 10 mL). The yellow residue was dissolved in DCM (15
mL) and the solution dried over Na2SO4. The solvent was removed
under reduced pressure and the residue dissolved in a minimum
amount of DCM and purified by preparative TLC (MeOH/DCM, 12/
88). After isolation, the product was recovered by rinsing the silica
with the same eluent. Evaporation of the solvent gave 8 (0.104 g, 66%)
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as a yellow solid. H NMR (400 MHz, CDCl3): δ 1.44 (s, b, CCH3,
18H), 1.46 (s, CCH3, 9H), 2.04 (m, CH2CH2CH2, 2H), 3.00 (s,
NCH3, 6H), 3.40 (m, NCH2, 2H), 4.06 (t, OCH2, J = 6.3 Hz, 2H),
1.5−3.7 (b, CH2 macrocycle, CH2 acetic arms, 24 H), 6.62 (t, NH, J =
6.0 Hz, 1H), 6.74−6.78 (m, ArH, 3H), 6.87 (dd, ArH, J = 2.42, 8.62
Hz, 1H), 7.40 (d, ArH, J = 7.4 Hz, 1H), 7.62 (m, ArH, 2H), 7.67 (s,
CCH, 1H). 13C NMR (100 MHz, CDCl3): δ 27.81 (CCH3), 27.84
(CCH3), 28.73 (CH2CH2CH2), 36.48 (CONHCH2), 40.27 (NCH3),
48.13 (NCH2, ring, broad), 52.41, (NCH2, ring, broad), 55.45
(NCH2CO, broad), 55.57 (NCH2CO), 55.68 (NCH2CO), 66.36
Immunohistochemistry. For immunohistochemistry, sections
mounted on slides were incubated in a solution containing anti-
MBPMAb primary antibody (rat anti-MBP, 1:300; Chemicon,
Temecula, CA) diluted in 1% normal donkey serum overnight at 4
°C. Following three rinses with PBS, sections were incubated in
donkey anti-rabbit rhodamine red X-conjugated secondary antibody or
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dx.doi.org/10.1021/jm201010e | J. Med. Chem. 2012, 55, 94−105