View
226
Download
2
Category
Preview:
Citation preview
Metabolite Identification and Characterization
Chandra Prakash, Ph. D.Pfizer Global Research and Development, Groton, CT
Outlines Introduction Metabolism Reactions LC-MS strategies for metabolite identification Triple Stage Quadrupole (TSQ) LC/MS/MS Ion Traps (LCQ and LTQ) QTOF
Analytical techniques combined with mass spectrometry for characterization of metabolites Derivatization H/D exchange
LC-NMR
Fate of Drugs in Living Organisms
Affinity
Receptor Pharmacological Activity
Biotransformation
Phase IMetabolites Excreted
Reactive intermediates Conjugated metabolites
Phase IIBioactivation
Adducts ExcretedToxicity/Adverse drug reaction
Detoxification
DrugDrug
Why Identify Metabolites?
Most of the drugs are eliminated from the body by metabolism: Detoxification process-This is good.
The metabolites modulate the efficacy of drugs in the treatment of disease.
The metabolites may possess pharmacological activity.
The metabolites may be toxic: Bioactivation- bad. Pharmaceutical industries are mandated by
regulatory agencies to identify metabolites of NCE. Metabolites may provide new leads.
Xenobiotic Metabolism
Phase I (Activation/Detoxification) Polar reactive groups introduced products most often more polar and less lipophilic more water soluble
Phase II (Detoxification) Covalent "conjugation" to endogenous substances reactions most often abolish biological activity and
add to polarity very water soluble
Phase I Metabolism
Hydroxylation- aliphatic, aromatic Epoxidation- aliphatic, aromatic O-, N-, S- Dealkylation Oxidative Deamination N-, S-, P- Oxidation Reduction Hydrolysis Aromatization
Phase II Metabolism
Glycoside Conjugation Glucuronide other sugars
Sulfate Conjugation Methylation (O-, S-, N-) Acylation Amino acid Conjugation Glutathione Conjugation
Knowledge of Basic Organic ChemistryKnowledge of Drug Metabolism and
Basic Metabolic ReactionsKnowledge of Concepts of Mass
SpectrometryInterpretation of Mass Spectra for
Structural ElucidationInterpretation of NMR Spectra for
Structural Elucidation
Identifying Metabolites-Prerequisite
Identifying Metabolites-Basic Needs
Need the molecular weight Need product ions to get structure information Need product ion spectrum of the parent drug
and metabolites Use the nitrogen rule
Techniques for the identification of metabolites
LC-API MS/MSSingle Stage Quadrupole (SSQ) LC/MSTriple Stage Quadrupole (TSQ) LC/MS/MSIon Traps (LCQ and LTQ) QTOF
Analytical Techniques combined with MSDerivatizationEnzymatic hydrolysisH/D exchange
LC/NMR
LC/MS
LC Ion Source Mass Analyzer
Atomospheric Pressure
Chemical Ionization(APCI)
Electrospray(ESI)
Single QuadrupoleTriple QuadrupoleIon Trap (LCQ)Q-TOF
General Rules for Choosing Polarity of Ion Detection and pH
Positive ion Detection Basic samples Decrease pH
Acetic acid pH (3-5) Formic acid pH (2-3) TFA pH (1-2)
pH at least 2 units below pKa of samples
General Rules for Choosing Polarity of Ion Detection and pH
Negative ion Detection Acidic samples Increase pH
Ammonium hydroxide
pH at least 2 units above pKa of samples
Quadrupole
IonSource
Q0 Q1 Detector
IonSource
Q0 Q1 Q2 Q3Ar
Detector
Single stage quadrupole (SSQ)
Triple stage quadrupole (TSQ)
Advantages of a TSQ MS
Renders selectivity due to mass separation at two stages.
Helps to rapidly identify metabolites in matrices without purification.
MASS SPECTRUM Mass Spectrometers Do Not Measure Mass. It is
plot of the mass-to-charge ratios (m/z) vs. the % relative intensities of the ions, where base peak is the most abundant ion in the spectrum
If single charge, z=1 and m/z = m Three types of ions in a mass spectrum;
Intact molecule one or more chargesMolecular mass
Fragment ionsStructure information Background ionsfrom non-analyte species
Natural Isotopic Abundance of Common Elements
Element
Carbon
Hydrogen
Oxygen
Nitrogen
Chlorine
Sulfur
Isotope Mass12C13C1H2H
16O18O14N15N35Cl37Cl32S33S34S
%98.91.1
99.980.0299.80.2
99.60.4
75.824.2
95.30.764.20
Mass
Element Nominal Mass Exact Mass
C 12 12.0000
H 1 1.0078
O 16 15.9949
N 14 14.0031
Average Mass
12.011
1.00797
15.9994
14.003
Cl 35 34.968935.45
S 32 31.97232.06
Average vs. Exact Mass
Average mass results from occurrence of isotopes. (See below) This is what we weigh
Exact mass results from non-integer masses of sub-atomic particles. This is what the Mass Spec sees Deviation of exact from nominal is the
Mass Defect
Examples (C,H,O,N compounds)
XanomelineC14H23N3OS
CaffeineC8H10N4O2
ZiprasidoneC21H21N4OSCl
Compound Integer Avg. Mass Exact Mass
Examples (C,H,O,N compounds)
XanomelineC14H23N3OS
CaffeineC8H10N4O2
ZiprasidoneC21H21N4OSCl
194.1785 194.0802
281.4057 281.1556
412.9197 412.1120
194
281
412
Compound Integer Avg. Mass Exact Mass
Nitrogen Rule
oddEvenoddOdd number of nitrogens (1, 3, 5)
EvenoddEvenEven number of nitrogens (0, 2, 4)
M+ (EI)[M+H]+ or [M-H]-M .W.Compound
Mass value
.
F, W. McLafferty and F. Turecek, Interpretation of Mass Spectra, Fourth Edition (1993)
Mass Changes Associated with Phase I Metabolism
Hydroxylation- aliphatic, aromatic Lose H & add OH = (M+16)
Epoxidation- aliphatic, aromatic Lose H & add OH = (M+16)
O-, N-, S- Dealkylation Add H & lose alkyl (R) = (M+1-R)
N-, S- Oxidation Add O = (M+16)
Reduction Add 2H = (M+2)
Mass Changes Associated with Phase II Metabolism
Glucuronide Conjugation Lose H & add glucuronic acid (M-1+176) = (M+176)
Sulfate Conjugation Lose H & add HSO3- (M-1+81) = (M+80)
Methylation (O-, S-, N-) Lose H & add CH3 (M-1+15) = (M+14)
N-acetylation Lose H & add CH3CO- (M-1+43) = (M+42)
Amino acid Conjugation Lose H2O & add amino acid (M-18+R)
Glutathione Conjugation Various addition of SG-amino acid (M+306-X; X=leaving group)
LC/MS/MS Techniques for the Identification of Metabolites
Q1 or Full Scan
Q1 Q2 Q3OR
N2
M1 M2
M3
Similar to an LC/MS total ion chromatogram.
Fromcolumn
All ions Scanned
m/z
Only Q1 operational (LC/MS mode)
Full Scan MS of MicrosomalIncubation of Compound X
m/z250 300 350 400 450 500
0
20
40
60
80
100
Rel
ativ
e A
bu
nd
ance
404
387 425426.336.1
295.2 359.2279.2 315.1 489.1473.241.4
RT:
HLM Control
250 300 350 400 450 500m/z
0
20
40
60
80
100
Rel
ativ
e A
bu
nd
ance
394 410
428380
Background subtracted
396414
250 300 350 400 450 500m/z0
20
40
60
100
Rel
ativ
e A
bu
nd
ance
410
394
428380
329.1 376.1347.1295.2 458.4279.4 489.0313.1
HLM + Substrate
396414404
80
Problem Set:Full Scan MS of Metabolites of Compound X (MW 394)Determine possible additions of functionality of metabolites
250 300 350 400 450 500m/z
0
20
40
60
80
100
Rel
ativ
e A
bu
nd
ance
394410
428
380
396
414
Q1 Q2 Q3ORFromcolumn
Product Ion Spectrum
One ion selected(parent ion)
N2
Fragmentation All ions scanned(product ions)
50 500
421
375162
78
m/z
Reli
nten
sity
(%)
A product ion spectrum
CID Product Ion Spectrum of Compound Y
100 200 300 400m/z
100
75
50
25
0
Rel
ativ
e In
tens
ity (%
)
121
147 167
177
469
N
N O
177
167
147-OMe
+H
MH+
Q1 Q2 Q3ORFromcolumn
Precursor Ion Scan
Scanned over a mass range
N2
Fragmentation(all ions fragmented)
Ions with specificproduct ions traced
M1 M2
M3
m/z
Precursor ion scan
Precursor ion experiment yields a spectrum of all parent ions which have the same product ion in their spectrum
Q1 Q2 Q3ORFromcolumn
Neutral Loss Scan
N2
M1M2
M3
m/z
Neutral loss scan
All ions selected Fragmented scanned at the same rate as Q1but with a mass offset
Mass offset corresponds to the mass of neutral fragment loss during fragmentation
Neutral loss experiment yields a spectrum of all parent ions which lose a selected neutral loss fragment
O
N
S
OO
NH2
-81
M+H = 213
132+
81
132
RE
L. I
NT
EN
SIT
Y (%
)
m/z
213
136
RE
L. I
NT
EN
SIT
Y (%
)
m/z
213
230
77 149
Full Scan MS
Product ion MS/MS
O
N
S
OO
NH2
MW=212
Interpreting Product Ion MS/MS Spectrum
Systematic Approach for the Identification of Metabolites by LC/MS/MS
GET A Q1 SCAN OF THE COMPOUND IN QUESTION
OBTAIN A PRODUCT ION SPECTRUM OF THE COMPOUND: INTERPRET THE SPECTRUM
IDENTIFY MAJOR FRAGMENT ION AND NEUTRAL LOSS
IDENTIFY MAJOR FRAGMENT ION AND NEUTRAL LOSS
RUN PRODUCT ION SCANS FOR ALL POSSIBLE METABOLITES IDENTIFIED FROM STEP 4 PLUS EXPECTED METABOLITES
INTERPRET THE SPECTRA AND ASSIGN STRUCTURES OF METABOLITES
Example
N
NH
CH3O
*
CID Product Ion Spectrum of a Parent Drug
100 200 300 400m/z
100
75
50
25
0
Rel
ativ
e In
tens
ity (%
)
121
147 167
177
469
N
N O
177
167
147-OMe
+H
MH+
Identify parent scan ions?
HPLC-RAD and TIC Chromatograms for Biliary Metabolites of CJ-11,972
100
75
50
25
0
Rel
. Int
. (%
)
RAD Chromatogram
Time (min)10.0 20.0 30.0 40.0
100
75
50
25
0
Rel
. Int
. (%
)
Par167 Chromatogram
III III
IV+VVI
VIII
VII
IX
XX! XII
(Parents of m/z 167)
CID Product Ion Spectra of Metabolites 485-A and 485-B
50 100 150 200 250 300 350 400 450 500m/z
100
75
50
25
0
Rel
ativ
e In
ten
sity
(%)
73105
121
133163167
175
193
485
485-ARet. Time 29.9 min
O
OH
N
N
167
73193
163-HCHO
50 100 150 200 250 300 350 400 450 500m/z
100
75
50
25
0
Rel
ativ
e In
ten
sity
(%)
57105119132
147
165
177
183
485
485-BRet. Time 36.6 min
N
N O
OH
183
177
147
-HCHO57
CID Product Ion Spectra of Metabolites 455-A and 455-B
NN OH
163
293
276-NH3
167
-tert-butyl 106
50 100 150 200 250 300 350 400 450m/z
100
75
50
25
0Rel
ativ
e In
tens
ity (%
)
82
96
106
163
167
181 276
293
455
455-ARet. Time 28.1 min
O
N
N
43163
167
133
121
50 100 150 200 250 300 350 400 450m/z
100
75
50
25
04356
82
96
121133152
163
167
181 455
455-BRet. Time 37.7 min
Rel
ativ
e In
tens
ity (%
) -CH2O
-C3H6
ION TRAP MS
ION TRAP MS
Sensitivity Ion accumulation
(10-1000 times better sensitivity than quadrupole MS) Specificity
Multistage MS capabilities (MSn)
Speed Can complete an entire scan in 100 ms
Data Dependent Acquisition Acquire MW information and MSn spectra in the same
run High value/Cost Ratio
MS 2
MS 3
Rf
trapping scanning
MS 4
Triple Quad vs LCQ (MS/MS)
Q-TOF
Why use a Q-TOF ?Sensitivity
- detection of low level metabolites in complex matrices in vivo
Exact mass (high resolution mass measurement)
- added confidence in confirming expected metabolites (confirm elemental composition for metabolites with the same nominal mass)
LC/MS/MS
- confirmation of metabolites (compare MS/MS spectra)
- data dependent MS -- MS/MS (time saving, High throughput)
MS operation : Quadrupole MS transmits TOF detects all ions transmitted : full scan mass spectrum
MS/MS operation: Precursor ion selection in quadrupole, collision induced dissociation (CID) in hexapole collision cell product ion detection in TOF: MS/MS spectrum
Operating principle of the Q-TOF mass analyzer
Instrument Capabilities
High Resolution MS and MSMS 20,000 resolution Peak Width of 0.025 at 500 amu
High Resolution= High Selectivity Able to easily separate masses that differ in 0.1 amu
easily
TOF allows fast scan speeds without sacrificing sensitivity or scan ranges in MS or MS/MS modes
High Resolution MS and MSMS 20,000 resolution Peak Width of 0.025 at 500 amu
High Resolution= High Selectivity Able to easily separate masses that differ in 0.1 amu
easily
TOF allows fast scan speeds without sacrificing sensitivity or scan ranges in MS or MS/MS modes
Mass
Element Nominal Mass Exact Mass
C 12 12.0000
H 1 1.0078
O 16 15.9949
N 14 14.0031
Average Mass
12.011
1.00797
15.9994
14.003
Cl 35 34.968935.45
S 32 31.97232.06
Biotransformation of Ziprasidone
Question: Can we differentiate the structures of these metabolite with m/z 429 by TOF?Previous assignment: S-oxides or S-methyl (+ 16) .
m/z 413
M10; m/z 429 M9; m/z 429C21H22N4O2SCl C22H26N4OSCl
NO
HCl
N NN
S
NO
HCl
N NN
S
NO
HCl
N NNH
O SCH3
C21H22N4OSCl
425 426 427 428 429 430 431 432 433 434 m/z0
100 429.1520
431.1516430.1529
432.1510
M9
425 426 427 428 429 430 431 432 433 434 m/z0
100429.1151
431.1140430.1193
432.1145
M10
27.50 28.00 28.50 29.00 29.50 30.00 30.50 31.00 Time0
100M9 M10
Selected Ion Chromatogram and Full ScanMS of M9 and M10
Mass Measurements of M9 and M10
Metab Cal. Mass Obs. Mass+/-mDa +/-ppm Mol. Formula
M9 429.1516 429.1520 0.4 0.9 C22H26N4OSCl
M10 429.1152 429.1151 -0.1 -0.3 C21H22N4O2SCl
Parent 413.1203 413.1205 0.2 0.4 C21H22N4OSCl
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 m/z0
100194.0384
150.0386280.1222
196.0369
282.1195 429.1516
NH
OCl
N NSCH3NH
NH
OCl
CH2+
m/z 194.0373(5.3 ppm)HN SCH3
+
NH
OCl
N NH2+
m/z 150.0377(5.7 ppm)
m/z 280.1217(1.9 ppm)
m/z 429.1516(0.9 ppm)
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 m/z0
100 194.0383
99.0923152.0179
175.0336
196.0370
232.0922 429.1175
NH
OCl
N NSN
O
NH
OCl
CH2+N NH
+H2C
m/z 429.1152(-0.3 ppm)
m/z 194.0373(5.3 ppm)
m/z 99.0922(0.8 ppm)
MS/MS Spectra of Metabolites M9 and M10
N OH N OH
O
N
N
Two Isobaric metabolites of Compound X
R2
BA
241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259m/z0
100
%
242.0783
243.0818
249.1486
Mass corrected and centroid Exact mass: 242.0793 -1.0 mda; ppm error= -3.9 Formula= C12H11NOF3
TOF MS Spectrum of Structure ATOF MS Spectrum of Structure A
240 242 244 246 248 250 252 254 256 258 260 262m/z0
100
%
242.0432
244.0486
243.0458
Mass corrected and centroid Exact mass: 242.0429 0.3mda; ppm error= 1.2 Formula= C11H7NO2F3
TOF MS Spectrum of Structure BTOF MS Spectrum of Structure B
Analytical techniques combined with mass spectrometry for
characterization of metabolites
When Derivatization is Useful
Metabolite is unstable Metabolite is very polar Metabolite is volatile To characterize the functional group To prove MS fragmentation To improve sensitivity when metabolite
is available in only trace amounts
Derivatizations of Hydroxyl Groups
ROH or ArOH ArOCOCH3CH3COCl
(CH3CO)2O
ArOH ArOCH3CH2N2
ArOH +
N
SO2Cl
N
SO2OAr
-Methylation
-Dansylation
ROCOCH3 or -Acetylation
-Silylation
ROH or ArOHMTBSTFA
ArOBDMSROBDMS or
MTBSTFA; Tert-butyldimethylsilyl-N-methyltrifluoroacetamide
Derivatizations of Amines
RNH2CH3COCl
(CH3CO)2O
+
N
SO2Cl
N
SO2NHR -Dansylation
RNHCOCH3
-Acetylation
RNH2 RNHCOCF3MBTFA
RNH2
MBTFA; N-methylbis(trifluoroacetamide)
Derivatization of Carboxyl Group
RCOOHCH3OH/HCl RCOOCH3
-Methylation
RCOOHCH2N2 RCOOCH3
-Triphenylphosphonium Derivative
RCOOH + H2NCH2CH2P(Ph)3+
RCO HNCH2CH2P(Ph)3+
-Reduction
RCOOH RCH2OHLiAlH4
Functional group-specific chemistry
OR3N
R2S=O
TiCl3
TiCl3
R3N
R2S
RCNHNH2
HFAAR
N
NCF3
CF3
Hexafluoroacetylacetone
CID product ion spectra of glucuronide conjugates
100 200 300 400 500m/z
100
75
50
25
0
Rel
ativ
e In
tens
ity (
%)
147162
176
308
326
344484
502
520MH+
(a)
m/z100 200 300 400 500
100
75
50
25
0
Rel
ativ
e In
tens
ity (
%)
147162
176
308
326
344
502
520MH+
(b)
N
CH3
OH
HO
glucuronide
176
-glu.
-H2O
151
344326-H2O308
-H2O
OH
OH
CID product ion spectrum of glucuronide conjugates after methylation
100 200 300 400 500
m/z
100
75
50
25
0
Rel
ativ
e In
tens
ity (%
)
135
161
173
190
322
340
512
530
548
MH +
N
OH
CH 3
OH
-O O
HO HO
H 3 COOC
HO
OCH 3
340 -H2O
322 -H2O 190
161 -H2O
-H2O
151 -glu.
m/z
- H2O
- 2H2O
Peak I
N
OH
CH 3
OH
H 3 CO
O- O
OH OH
COOCH 3
HO
366 176
-H2O -methyl glu.
165
147 -H2O
-methyl glu.
100 200 300 400 500
100
75
50
25
0
Rel
ativ
e In
tens
ity (%
)
121
147 165
176
202 322
340 366 512
530
548 MH +
- H2O
- 2H2O
Peak II
CID Product Ion Spectrum of Sunepitron
100 200 300 400m/z
100
75
50
25
0
Rel
ativ
e In
ten
sity
(%)
56 7081
93
108
122
136
195
209
235
287
330MH+
N
NN
N
N
O
O
+H
122136
209195235
-2H
+H
+H+H
C. Prakash, et al. Drug Metab Disp. (1997) 26, 206-218
CID product ion spectrum of M18
m/z50 100 150 200 250 300
100
75
50
25
0
69
86
93
104
151
195209
235
252
277
294
Rel
ativ
e In
tens
ity (%
)
MH+
2
N
NN
O
O
252
209
R=COCH3R=C(=NH)NH
R
Mass Spectra of M18 Before and After Deuterium Exchange
200 210 220 230 240 250 260 270 280 290 300 310 320m/z
0
20
40
60
80
100
Rel
ativ
e A
bu
nd
ance
298
299297
200 210 220 230 240 250 260 270 280 290 300 310 320m/z
0
20
40
60
80
100
Rel
ativ
e A
bu
nd
ance
294
295
MD+
MH+
CID product ion spectrum of M18 after treatment with HFAA
100 200 300 400
50
38
25
12
0
Rel
ativ
e In
tens
ity (%
)
81
90
108 136 195
209 258
272 299 365
466
N
NN
N
N
O
CF3
CF3
209
258(b)
MH+
O
N
NN
N
N
O
O
CF3
CF3
N
NN
NH
NH2
O
O
CF3COCH2COCF3
Reduced polarityDiagnostic massincrease
Identification of Drug MetabolitesLC-NMR
LC-NMR (Continuous flow or stopped flow) Fast Reportedly sensitive (50 - 200 ng) Amenable to automation Negate the need for isolation Sample Stability Cleaner Spectra
ADVANTAGES
Disadvantages and Limitation of LC-NMR
Sensitivity Nearly eliminates quantitative application
The Chromatograph Solvent Suppression Expensive deuterated mobile phase and
buffers Shimming problems introduced by LC-
gradient methods
What information does NMR provide
Each proton (or carbon atom) in a molecule typically has a different resonant frequency (chemical shift) Thus, NMR spectrum is a fingerprint of a molecule Chemical shifts are governed by nuclear environment,
e.g. CH3O, CH3CN, CH3 NH2 Adjacent NMR-active nuclei 91-4 bonds apart) in a
molecule may couple to one another Coupling constants can be identified by inspection of 1D
spectra
Activation of Acetaminophen by Cytochrome P450 to N-acetyl-p-benzoquinonimine (NAPQI) and subsequent conjugation with Glutathione
N
O
O
CH3 HN
O
O
CH3 HN
OH
O
CH 3
N
O
O
CH3 N
OH
O
CH3 HN
OH
O
CH 3
H
SG
H
SG
SG
SG
GSH
NAPQI
GSH
NAPQI
H+
H+
H+
3' ADDUCT
2' ADDUCT
H+
NH
CH3
O
OH
P450
Proposed Reaction Product of N-acetyl-pbenzoquinone imine(NAPQI) with Glutathione
N
O
O
CH3
GSH
ONAPQI
H+
GSHN
O
CH3
IPSO ADDUCT
CID Product Ion Mass Spectra of Reaction Mixture
1H NMR Data Obtained on The Reaction Mixture
Characterization of Metabolites of Compound X
R1NOR
CH 3
C H 3C H 3
HPLC Radiochromatograms of Compound X Metabolites
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (min)
0.0
0.2
0.4
0.6
Inte
nsity
ParentM22
M23
M3/M4
M24
M26
CID Product Ion Spectrum of compound X
MH+
100
0
50
75
25
100 200 300 400 480
165
326147
469192 335270 41357 367 423
m/z
N
OR
147
165
326
MH+=469
R1
9 8 7 6 5 4 3 2 1 ppm
6.87.07.27.47.67.88.08.28.48.68.8 ppm
Proton double presat 278K d1=10sec CP-533,536 LC peak at 57.41 minutes (m/z=471) injection # 636
CID mass and NMR spectra of M4
100 200 300 400 500m/z
0
100165
145
467324
192
335
342
421
485
N
O
OH
-H2O
-H2O
145
165
324
342
MH+
MH+=485MD+=488
12345678 ppm
100 180 260 340 420 500m/z
0
100165
161
133
483340
295266
409437
N
O
O
161
165
340
MH+
CID mass and NMR spectra of M24
123456789 ppm
MeOD impurities
Proton double presat 278K CP-533,536 metabolite LC peak at 45.30 minutes (m/z=485) injection # 622
aldehyderesonance
2 Methylgroups
-NCH2
-OCH2
MH+= 483MD+= 485
RR1
Conclusions
Combination of LC/MS/MS with other analytical approaches is a powerful tool for solving difficult problems encountered in the analysis of drug metabolites.
SOME REFERENCES
Biochemistry of reactions by Bernard Testa. Biotransformation of Xenobiotics - Andrew Parkinson
- in Casarett and Doulls Toxicology, 5th edition. Drug Biotransformations - Neal Castagnoli - in
Burgers medicinal chemistry 4th edition. Drug Metabolism -Bernard Testa- in Burgers
Medicinal Chemistry, 5th edition. Metabolism of Heterocycles by L. A. Damani in
Comprehensive Heterocyclic Chemistry
Recommended