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