figures s1a-s8d: mass spectral and nmr spectral data for ramelteon metabolites
DESCRIPTION
Supplemental Information for: METABOLISM OF RAMELTEON IN HUMAN LIVER MICROSOMES AND CORRELATION WITH THE EFFECT OF FLUVOXAMINE ON RAMELTEON PHARMACOKINETICS by R. Scott Obach and Tim F. Ryder Drug Metabolism and Disposition. - PowerPoint PPT PresentationTRANSCRIPT
Supplemental Information for: METABOLISM OF RAMELTEON IN HUMAN LIVER
MICROSOMES AND CORRELATION WITH THE EFFECT OF FLUVOXAMINE ON RAMELTEON
PHARMACOKINETICSby R. Scott Obach and Tim F. Ryder
Drug Metabolism and Disposition
Figures S1a-S8d: Mass spectral and NMR spectral data for ramelteon metabolites
100 150 200 250 300m/z
0
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0
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Inte
nsity
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1000000
260
282261 305283262153 171 230204190 242 307216
204
161 187169 242133 260205140105 218126187
159204161133 175143117 252 274
NL: 1.39E620091125a-12#3384-3426 RT: 36.65-37.04 AV: 5 F: ITMS + c ESI Full ms [150.00-360.00]
NL: 1.07E620091125a-12#3382-3428 RT: 36.65-37.04 AV: 5 F: ITMS + c ESI d Full ms2 [email protected] [60.00-275.00]
NL: 6.73E520091125a-12#3379-3430 RT: 36.61-37.08 AV: 6 F: ITMS + c ESI d Full ms3 [email protected] [email protected] [50.00-420.00]
MS1
MS2dau 260
MS3dau 204
Figure S-1a; Ramelteon
NH3+
O
m/z 204
O
CH2+
m/z 187
O
m/z 159
+
Ramelteon NMR Assignments
HN
O
CH3
O
0.99,10.1
2.06,28.5
7.79
3.09,36.8
1.93, 1.43,32.8
4.51, 4.42,70.6 3.16,
3.06,27.9
6.89,122.7
6.52,106.5
2.79,2.65,29.9
2.17,1.71,31.2
3.12,41.5
172.8
143.4
132.2
158.9122.2
COSY: Black ArrowsHMBC: Red Arrows
Figure S-1b
1H NMR Spectrum of RamelteonFigure S-1c
COSY NMR Spectrum of RamelteonFigure S-1d
HSQC NMR Spectrum of RamelteonFigure S-1e
HMBC NMR Spectrum of RamelteonFigure S-1f
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1500
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nsity
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276
298277299278224153 250204171 191 260
258
161 204187 230104 133 240 276156159
161204
187 230169
133 240229157 258199112 281159
131141144129 160
NL: 1.21E620091125a-12#2998-3042 RT: 32.51-32.91 AV: 5 F: ITMS + c ESI Full ms [150.00-360.00]
NL: 7.28E520091125a-12#2999-3036 RT: 32.51-32.81 AV: 4 F: ITMS + c ESI d Full ms2 [email protected] [65.00-290.00]
NL: 9.88E420091125a-12#2997-3033 RT: 32.47-32.85 AV: 5 F: ITMS + c ESI d Full ms3 [email protected] [email protected] [60.00-530.00]
NL: 1.71E320091125a-12#2993-3034 RT: 32.50-32.79 AV: 3 F: ITMS + c ESI d Full ms4 [email protected] [email protected] [email protected] [50.00-330.00]
MS1
MS2dau 276
MS3dau 258
MS4dau 159
Figure S-2a; Metabolite A
HN
O
CH3
HO
O
1.94,20.9
3.93,67.1
7.76
3.13,36.5
1.94, 1.48,32.7
4.51, 4.45,70.4 3.20,
3.06,27.7
6.89,122.7
6.52,106.5
5.46
2.79,2.66,29.9
2.19,1.73,31.1
3.09,41.5
COSY: Black Arrows
Figure S-2b; Metabolite A
1H NMR SpectrumFigure S-2c; Metabolite A
COSY NMR Spectrum
Figure S-2d; Metabolite A
HSQC NMR SpectrumFigure S-2e; Metabolite A
HN
O
CH3
HO
O
Figure S-3a; Metabolite B
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nsity
0
50000
100000
150000
258
298
259 276 299153 171 190 260215 230 277195 249202
185 240171159 258213131 223140123104185
157
202159173131 186155 203117
NL: 1.86E520091125a-12#2705-2739 RT: 29.48-29.76 AV: 4 F: ITMS + c ESI Full ms [150.00-360.00]
NL: 1.81E520091125a-12#2705-2736 RT: 29.48-29.76 AV: 4 F: ITMS + c ESI d Full ms2 [email protected] [60.00-270.00]
NL: 7.81E420091125a-12#2704-2741 RT: 29.44-29.81 AV: 5 F: ITMS + c ESI d Full ms3 [email protected] [email protected] [50.00-415.00]
MS1
MS2dau 258
MS3dau 202
HN
O
CH3
O
0.99,9.4
2.07,28.3
7.76
5.94,100.7
6.91,122.6
2.18,1.71,31.2
3.09,41.1
6.53,106.5
1.91, 1.43,32.7
3.09,36.9
3.22,35.9
2.79,2.66,29.8
HO 7.17, 7.09
COSY: Black Arrows
Figure S-3b; Metabolite B
1H NMR Spectrum
Figure S-3c; Metabolite B
1H NMR Spectrum Showing Diastereomeric Mixture
Hydroxyl Proton 1
Hydroxyl Proton 2
Extra coupling of the aromatic doublets Methine
Figure S-3d; Metabolite B
COSY NMR Spectrum
Figure S-3e; Metabolite B
HSQC NMR Spectrum
Figure S-3f; Metabolite B
HN
O
CH3
O
HO
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Inte
nsity
0
100000
200000
300000
400000258
298259153 299260190171 230215202 269255 283
202
185 240171159 258213 223131 140185
157
202159173131 155 267108
NL: 4.13E520091125a-12#2634-2664 RT: 28.63-28.93 AV: 4 F: ITMS + c ESI Full ms [150.00-360.00]
NL: 3.75E520091125a-12#2632-2665 RT: 28.63-28.93 AV: 4 F: ITMS + c ESI d Full ms2 [email protected] [60.00-270.00]
NL: 1.78E520091125a-12#2631-2661 RT: 28.59-28.88 AV: 4 F: ITMS + c ESI d Full ms3 [email protected] [email protected] [50.00-415.00]
Figure S-4a; Metabolite C
MS1
MS2dau 258
MS3dau 202
HN
O
CH3
O
0.98,10.0
2.05,28.2
7.76
4.46,4.20,78.8
7.03,124.8
2.17,1.74,31.0
3.28,41.0
6.59,107.4
2.00, 1.53,32.7
3.10,36.9
5.36,68.9
OH
2.80,2.67,29.9
5.44
COSY: Black Arrows
Figure S-4b; Metabolite C
1H NMR Spectrum
Figure S-4c; Metabolite C
COSY NMR Spectrum
Figure S-4d; Metabolite C
HSQC NMR Spectrum
Figure S-4e; Metabolite C
HN
O
CH3
O
OH
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nsity
0
100000
200000
300000276
277298274153 171 278230190 256238215209 308
185
220
258189177 203159149 229 276140 247110 129 286157 185
170
167142129 183115 186
NL: 3.17E520091125a-12#2482-2517 RT: 27.00-27.32 AV: 4 F: ITMS + c ESI Full ms [150.00-360.00]
NL: 1.41E520091125a-12#2483-2516 RT: 27.00-27.32 AV: 4 F: ITMS + c ESI d Full ms2 [email protected] [65.00-290.00]
NL: 2.57E420091125a-12#2482-2519 RT: 26.97-27.37 AV: 5 F: ITMS + c ESI d Full ms3 [email protected] [email protected] [50.00-385.00]
Figure S-5a; Metabolite D
MS1
MS2dau 276
MS3dau 185
COSY: Black Arrows
HN
O
CH3
O
0.980
2.05
7.76
3.05
1.87, 1.41
4.51, 4.45,
3.20,3.06,
6.43,6.03
2.72,2.62?
2.14,1.65
3.1
HO
Figure S-5b; Metabolite D
1H NMR Spectrum
Figure S-5c; Metabolite D
1H NMR Spectrum Showing the Ratio of Regioisomers
New Aromatic Singlet Together integrate to about 1
Figure S-5d; Metabolite D
COSY NMR Spectrum
Figure S-5e; Metabolite D
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250000258
298259159153 299171 190 260230 274215195 249 290159
185 240202 215160 258230141133110 149159
131
141
129117 160157
NL: 2.72E520091125a-12#2134-2163 RT: 23.19-23.49 AV: 5 F: ITMS + c ESI Full ms [150.00-360.00]
NL: 7.03E420091125a-12#2129-2166 RT: 23.13-23.49 AV: 6 F: ITMS + c ESI d Full ms2 [email protected] [60.00-270.00]
NL: 1.12E420091125a-12#2131-2168 RT: 23.16-23.53 AV: 6 F: ITMS + c ESI d Full ms3 [email protected] [email protected] [50.00-330.00]
Figure S-6a; Metabolites E and F
MS1
MS2dau 258
MS3dau 159
HN
O
CH3
O
0.98
2.06
7.79
3.07
1.88, 1.37
4.54,4.45,
7.02
2.07,1.93
3.13
6.60
2.04, 1.50
3.11
3.16
OH
5.01,4.86
2.50,1.49
5.11, 4.96
COSY: Black Arrows
Figure S-6b; Metabolites E and F
1H NMR Spectrum
Figure S-6c; Metabolites E and F
1H NMR Spectrum Shows Diastereomeric Mixture
Hydroxyl Proton 1
Methine 1Hydroxyl Proton 2
Methine 2
Figure S-6d; Metabolites E and F
COSY NMR Spectrum
Figure S-6f; Metabolites E and F
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nsity
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258
274185 298
259153 190171 275230215 260 299195 249
185
159 202 240215104 258173 229138121 269185
157
170
167142129 183115 186 208 249
NL: 1.32E520091125a-12#2389-2415 RT: 25.91-26.13 AV: 4 F: ITMS + c ESI Full ms [150.00-360.00]
NL: 1.43E520091125a-12#2395-2426 RT: 25.98-26.29 AV: 5 F: ITMS + c ESI d Full ms2 [email protected] [60.00-270.00]
NL: 3.65E420091125a-12#2398-2426 RT: 26.01-26.24 AV: 4 F: ITMS + c ESI d Full ms3 [email protected] [email protected] [50.00-385.00]
Figure S-7a; Metabolite G
MS1
MS2dau 258
MS3dau 185
HN
O
CH3
O
0.95,9.7
2.01,28.1
7.66
4.50,4.45,70.6
6.89,123.1
2.18,1.71,39.9
6.60,107.4
1.89,1.75,38.7
3.06,34.7
3.19,3.27,27.1
2.79,2.66,28.1
HO5.06
COSY: Black Arrows
Figure S-7b; Metabolite G
1H NMR Spectrum
Figure S-7c; Metabolite G
COSY NMR Spectrum
Figure S-7d; Metabolite G
HSQC NMR Spectrum
Figure S-7e; Metabolite G
HN
O
CH3
O
HO
No Methine
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274
258275153 185171 190 296230 291259238151 215 305
256
218
200185157 228104 257171 274238131 145121183
256
238157 200 211155 228171 239199133108 257117
NL: 7.69E420091125a-12#2428-2449 RT: 26.36-26.52 AV: 3 F: ITMS + c ESI Full ms [150.00-360.00]
NL: 4.41E420091125a-12#2432-2452 RT: 26.38-26.53 AV: 3 F: ITMS + c ESI d Full ms2 [email protected] [65.00-285.00]
NL: 1.11E420091125a-12#2429-2455 RT: 26.35-26.57 AV: 4 F: ITMS + c ESI d Full ms3 [email protected] [email protected] [60.00-525.00]
Figure S-8a; Metabolite H
MS1
MS2dau 274
MS3dau 256
HN
O
CH3
O
0.99
2.05
7.83
3.09
2.05, 1.46
4.70,4.65,
3.22
7.44
2.76,2.36
3.37
O
6.85
COSY: Black Arrows
Figure S-8b; Metabolite H
1H NMR Spectrum
Figure S-8c; Metabolite H
COSY NMR Spectrum
Figure S-8d; Metabolite H