The Amine Functional Group as a Structural Alert - Delaware Valley Drug … · 2019-09-06 · 2014, 27, 1808-20. N N Cl Cl Cl O N H N R R = Rimonabant N R OH CN N R CN N R N R N R

The Amine Functional Group as a Structural AlertAmit S. Kalgutkar

Medicine Design, Worldwide Research and Development,Pfizer Inc., 1 Portland St, Cambridge, MA [email protected]

Page

2

Advantages of a basic amine motif– Aqueous solubility

• Dipole-dipole interactions between N-H (and C-N) bonds with H2O molecules• Basic amine motif provides a convenient handle for salt preparation

– Distribution • Higher steady state distribution volumes (Vdss) noted with bases relative to anionic or

neutral compounds of comparable lipophilicity (Vdss increases with ↑ in Lipophilicity (LogD7.4))

– High tissue affinity due to electrostatic interactions between bases and anionic phospholipids in membranes

• Inclusion of a basic amine group has been used as a tactic to increase elimination half-life in drug discovery

– t1/2 = 0.693 x Vdss/CL Smith DA, Beaumont K, Maurer TS, Di L J. Med. Chem. 2015, 58, 5691-8.Smith DA, Beaumont K, Maurer TS, Di L J. Med. Chem. 2018, 61, 4273-82.

Promiscuous nature of basic amines• Presence of a 2° or 3°basic center [pKa(B) >

6, cLogP > 1] connected by a 2–5 atom linker to an aromatic ring is a key recognizable molecular/structural attribute of “promiscuous” compounds

– Bioprint® (Cerep) analysis of > 2400 drugs against 141-safety-related targets

• High hit rates of basic amines for aminergic GPCRs

Peters JU, Hert J, Bissantz C et al. Drug Disc Today 2012, 17, 325-35.Lee ECY, Steeno G, Wassermann AM, Zhang L et al. Bioorg. Med. Chem. Lett. 2017, 27, 653-7.

• 1°amines are less promiscuous (in vitro and in vivo) compared to 2° or 3°amines

– 938 basic amines (89% aliphatic) compounds: pKa(B) ~ 5.4, M.W range ~ 200–600

– 1100 neutral compounds (pKa(B) < 5.4 and pKa(A) > 7) used for comparison

– Promiscuous activity > 70% @ 10 µM

4

Concerns around metabolism-driven toxicity with basic amines

• Formation of electrophilic reactive metabolite (RM) as a causative factor in immune-mediated Idiosyncratic Adverse Drug Reactions (IADRs)

• Covalent protein modification by RM• Haptenization (immunogen)

• 68 drugs recalled or associated with a black box warning (BBW) in the US or European markets from 1965–present

– 78–86% positive for structural alert (SA) presence

• SA = functional group that can form a RM– 62–69% RM positive

Stefan AD, Walker DP, Bauman J, Price DA, Baillie TA, Kalgutkar AS, Aleo MD Chem. Res. Toxicol. 2011, 24, 1345-410.

R2NR1

R1, R2 = H or CH3, CH2-X

vs. N

Nn(H2C)N R

R or R1 = alkyl, aromatic,

heteroaromatic, etc.

n = 1-3

R1

R2

O

NR1

R2 = H, alkyl, aromatic,

heteroaromatic, etc.

Are basic amine functionalities SAs?Acyclic vs. cyclic basic amines

Problem Statement

5

PageBioactivation potential of acyclic basic amines

• N-Deamination is a common metabolic fate (e.g., antimigraine drug sumatriptan)

– Stable metabolites are amine, carboxylic acid, and alcohol derivatives

– CYP- or MAO-mediated

R = NH

S OO O

Sumatriptan

NH

N

R

[O]NH

N

R

NH

O

R

NH

O

R OH

NH

OH

RIminium

[O]

[H]HN

MAO-A

AldehydeAmine

Alcohol

Carboxylic Acid

Breck GD, Trager WF Science, 1971, 173, 544-46. Carrington HC, Crowther AF, Davey DG et al. Nature, 1951, 168, 1080.

HN

NO

NH

O N

Iminium

N N

O

LidocaineImidazolidinone metabolite of lidocaine in humans

• The process of N-dealkylation, in theory, will yield electrophilic iminium and aldehyde species

– No evidence for iminium/aldehyde adducts with nucleophiles (e.g., –CN, amines or GSH)

• Intramolecular trapping is possible (e.g., anesthetic lidocaine)

• Multiple acyclic basic amine drugs in market (e.g., SSRIs) with good safety records

Reactivity towards protein NuH(s) seems unlikely

O

CF3

HN

Fluoxetine

ClCl

HN

Sertraline

6666

PageBioactivation potential of cyclic basic amines

• In the case of some cyclic 2◦ and 3◦

basic amines:

– RMs (Iminium and aldehyde) can be trapped with –CN, amines, and even glutathione (GSH))

– Can lead to liver microsomal covalent binding (CB) in a NADPH-dependent fashion

– Some iminium ions are detected as stable metabolites

N

(CH2)n

R

NR

NR

CN

Iminium

NR

OHNH OR

O NH2

Aminoaldehyde

NHNRO

HSNH

NH

O

COOHO

COOHH2N

S

HN

HN

O

COOHHNR

[O]

CN

Cyclic Amines

Kalgutkar AS Chem. Res. Toxicol. 2017, 30, 220-38.Bolleddula J, DeMent K, Driscoll JP et al. Curr. Drug Metab. 2014, 46, 379-419.Masic LP Curr. Drug Metab. 2011, 12, 35-50.

GSH

• These observations imply:– That cyclic basic amine motifs should be considered as a SA since electrophilic

protein-reactive iminium/aldehyde species are formed during metabolism

7

PageHistorical context around iminium ion trapping with cyanide

N

Phencyclidine

NN

N

(S)-Nicotine

N

N

N N

MPTP

Nguyen TL, Gruenke LD, Castagnoli N Jr J. Med. Chem. 1976, 19, 1168-69. Ziegler R, Ho B, Castagnoli N Jr J. Med. Chem. 1981, 24, 1133-38.Ward D, Kalir A, Trevor A et al. J. Med. Chem. 1982, 25, 491-92.Peterson LA, Caldera PS et al. J. Med. Chem. 1985, 28, 1432-36.

• CN trapping employed to probe metabolism-dependent neurotoxicity of amine-based xenobiotics (including drugs)

– E.g., phencyclidine (PCP), (S)-nicotine, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), methapyrilene

• CN conjugate formation led to a proposed role of iminium ion in toxicity (e.g., protein CB associated with iminium ions)

S

NN

N

Methapyrilene

S

NN

N

-CN adduct - Yes -CN adduct - Yes

-CN adduct - Yes-CN adduct - No

8

PageEvolution of iminium ion trapping methods (KCN, K14CN, and/or K13C15N) from the early studies on neurotoxic amines

Cl

NN N N

N

O

O

Nefazodone

S

N Cl

NN

ProchlorperazineN

N

Mianserin

NH

NClN

N

Clozapine

NN

O

O

O

OCl

Cl

N

N

H

Ketoconazole

Mostly prop. compounds; few controls (e.g., flecainidine)

Older marketed antipsychotics (e.g., prochlorperazine) (14C-incorporation relative to nicotine)

999

PageCyclic basic amine bioactivation as a causative factor in IADRs

• Toxicological outcome from in vitro cyclic amine bioactivation is not clear!

– Example 1: Oxidative bioactivation of a homomorpholine ring

• GSH and amine conjugates detected• No cyano conjugates detected

– Lack of iminium ion– Equilibrium favors carbinolamine /

aldehyde

R

ON

O

AZX

CYP

R

ON

OOH

N

F3C

NH

N

O

N

R =

R

OHN

OO

GSH

R

OHN

OHN S

OHN

HO

O

AZX-Cys-Gly

- Glu, - H2O

CH3ONH2

R

OHN

ONO

Lenz EM, Martin S, Schmidt R et al. Chem. Res. Toxicol. 2014, 27, 968-980.

• Studies conducted in HLM/NAPDH• No toxicity disclosed

1010

PageCyclic basic amine bioactivation as a causative factor in IADRs

• Example 2: Anti-obesity drug Rimonabant (Acomplia®); introduced in 2006 and withdrawn in 2008 due to drug-related psychiatric disorders (anxiety and depression)

– High level of CB to HLM (NADPH-dependent)

• CB ↓ ~40% with –CN co-incubation – Iminium species (trapped with –CN)

• CB ↓ ~30% with CH3ONH2 co-incubation – A leucine/enkephalinamide (peptide)

adduct detected in HLM

Foster AJ, Prime LH, Gustafsson F et al. Chem. Res. Toxicol. 2013, 26, 124-35.Thorsell A, Isin EM, Jurva U Chem. Res. Toxicol. 2014, 27, 1808-20.

N N

Cl

Cl

Cl

O

NH

NR

R =

Rimonabant

NR

NR

OH

CNN

RCN

NR

NRN

R

HOO

O

peptide-NH2

Npeptide

*

*

* *

* = position of 14C labels

- 2 e - H

CYP

carbinolamine iminium

enamine

dihydropyridinium• Studies conducted in HLM/NAPDH

• No IADRs (e.g., DILI) reported for rimonabant

11

PageCyclic basic amine bioactivation as a causative factor in IADRs

• Example 3: Tetracyclic antidepressant mianserin, associated with immune-mediated DILI/agranulocytosis (rare 0.01-0.1%)

– Mianserin-specific antibodies in human serum– Cytotoxicity to human leukocytes exposed to

HLM/NADPH incubations with mianserin

– High level of CB to HLM (NADPH-dependent)• Detection of CN conjugates of mianserin and its

major human metabolites• SAR studies – no iminium = no cytotoxicity

N

NR2

Mianserin: R1 = H; R2

= CH38-Hydroxymianserin: R1

= OH; R2 = CH3

N-Desmethylmianserin: R1 = H; R2

= H

R1

N

NR2

R1Iminium ions

CYP-CN

N

NR2

R1

NC

• Recently, GSH conjugates of mianserin quinone-imine species in HLM/NADPH have been reported N

NR

OR = CH3R = H

GSH-EEN

NR

OHR2 = CH3

R2 = H

SG-EE

N

N

N

• Cannot form iminium• Devoid of cytotoxicity

Kalgutkar AS Chem. Res. Toxicol. 2017, 30, 220-38.

Which RM (if any) is responsible for the IADRs?

12

PageStructure-toxicity relationships: Mianserin vs. Mirtazapine

• Mirtazapine (Rameron®) is a 6-aza analog of mianserin

– Marketed in the USA since 1996– Little to no incidence of agranulocytosis and/or

DILI with mirtazapine use

NN

N

Mirtazapine hydroxylation

N-demethylationN-oxidation

Quat-glucuronide Metabolism of mirtazapine in humans (in vivo)• No metabolites detected that could be linked to iminium

• 8-Hydroxymirtazapine (quinone-imine precursor) detected as a major metabolite in human

A comparison of bioactivation attributes of mianserin and mirtazapine in a side-by-side fashion is needed to further assess the role of iminium species in IADRs

Delbressine LP, Moonen ME, Kaspersen FM et al. Clin. Drug Invest. 1998, 15, 45-55.

N

N

Mianserin

NN

N

Mirtazapine

Dose = 45 mg QDDose = 60 mg QD

13

PageCyclic basic amine bioactivation as a causative factor in IADRs

• Example 4: Zwitterion piperidine derivative was toxic to monkeys

– Elevations in liver enzymes and liver necrosis in single dose exploratory tox.

• Rats and dogs devoid of toxicity

– High degree of oxidative metabolism and CB in monkey liver microsomes (relative to rat, dog, and human)

• CB ↓ with co-incubation with CN (but not GSH or methoxylamine)

• CN adduct characterized in monkey liver microsomes

NR1

R2

O

HO

CYP

NR1

R2

O

HO

NR1

R2

O

HOCYP

NR1

R2

O

HO

ONR1

R2

O

HOHO

NR1

R2

O

HOHO

NC

KCN

Baillie TA Chem. Res. Toxicol. 2008, 21, 129-37.

Detected in liver microsomes

Only e.g. (I could find) where iminium species was linked to in vivo toxicity

14

PageCyclic amine bioactivation as a causative factor for mutagenicity

• From a drug discovery perspective:

– All examples (at least the ones disclosed in the public domain) are related to genotoxicity driven through metabolism of the cyclic amine motif

• Example 1: A2a/A1 receptor antagonist (1)

– Mutagenic in Salmonella Ames test – CB to calf-thymus DNA

• Required aroclor1254-induced rat S9 liver fraction/NADPH

– Methoxylamine and –CN attenuated the DNA CB potential

– Analogs 2 and 3 are not mutagenic

Lim HK, Chen J, Sensenhauser C, Cook K et al. Chem. Res. Toxciol. 2011, 24, 1012-30.

N

NH2N

O

N

CYPN N

HO

NH

O

N

NC

NH

NO

KCN CH3ONH2

Covalent Modification of DNA*

* = 3H

1

N

NH2N

O

N2

N

NH2N

O

3N

15

PageCyclic amine bioactivation as a causative factor for mutagenicity

• Example 2: CP-809,101

– Selective and potent 5-HT2Cagonist

– Mutagenic in SalmonellaAmes test

– CB to calf-thymus DNA• Required rat S9 liver

fraction/NADPH)– Methoxylamine attenuated

the DNA CB potential and mutagenic response of CP-809,101

HNN N

N

OCl CYP

CP-809,101

N

N

OCl

OH

HNN N

H

N

O

Cl

O

Quinone-methide

Cl

OH

SGGSH

HNN N

N

OCl CYP

HNN

OH

NH2

N

O

CH3ONH2NH2

N N

N

OCl

NO

Amino aldehydeRegiochemistry of oxidation is arbitrary

CP-809,101

Kalgutkar AS, Dalvie DK, et al. Drug Metab. Dispos. 2007, 35, 848-58.Kalgutkar AS, Bauman JN et al. Bioorg. Med. Chem. Lett. 2009, 19, 1559-63.

Cannot form quinone-methide, Minimal piperidine ring scissionMethyl group is a metabolic soft spot

HNN N

N

O

FF

HNN N

N

O

FF

16

PageWhat can we conclude from these two examples?

• Why is S9/NADPH-dependent mutagenic response not seen more often in the Salmonella Ames test with cyclic basic amines?

• What is so special about the two examples? Some thoughts…– Shape (Flatness of the molecules)?

• Able to intercalate (fit) in the DNA groove?• CYP1A2/3A4 involvement in the metabolism of CP-

809,101 has been characterized– CYP1A2 prefers “flat” molecules– CYP1A1/1A2 expression is significantly enhanced in

rat liver upon Aroclor1254 treatment

N

NH2N

O

N

N

N

N OHN

Cl

1717

PageNeurotoxic potential of some cyclic 3°amines: the MPTP story

• The MPTP finding cannot be ignored (N-alkyl-4-aryl-1,2,3,6-tetrahydropyridine is a SA by itself)

– Contaminant in “street version” of analgesic meperidine – Selective destruction of nigrostriatal neurons– Symptoms similar to idiopathic Parkinson’s Disease (PD)– Requires metabolic bioactivation in brain by MAO-B for

neurotoxic response• N-Methyl-4-phenylpyridinium (MPP+) metabolite

– Ultimate neurotoxin, potent Inhibitor of Mitochondrial Respiration, causes apoptosis and cell Death

• Note –CN trapping proved important in elucidating metabolism of MPTP to MPP+

Kalgutkar AS, Dalvie DK, Castagnoli N Jr, Taylor TJ. Chem. Res. Toxicol. 2001, 14, 1139-62. Peterson LA, Caldera PS, Trevor A et al. J. Med. Chem. 1985, 28, 1432-36.Chiba K, Peterson LA, Castagnoli KP et al. Drug Metab. Dispos. 1985, 13, 342-47.

N

MPTP

N N

BrainMAO-B

MPDP+ MPP+

Brain[O]

N CN

CN

18

PageMetabolism-driven neurotoxicity with cyclic 3°amines

• Tetrahydropyridine-based anti-convulsants in clinical trails– Suspended from development due to

Parkinsonism-like syndrome as adverse effects in primates and/or human

• Can be detected in tox. assessments in mice and/or monkeys

– MPP+ - like derivatives detected as urinary metabolites

• Must watch for N-Alkyl-4-aryltetrahydropyridine (piperidinol), especially ones metabolized to MPP+-like compounds

Kalgutkar AS, Dalvie DK, Castagnoli N Jr, Taylor TJ. Chem. Res. Toxicol. 2001, 14, 1139-62.

F

F

NR

OH

O

NR

OH

OO

F

F

R =

N

O

F

RN

O

F

R

R =

19

PageMarketed drugs containing functionalities capable of generating iminium/aldehyde reactive species• Numerous marketed drugs with cyclic basic amine motif (daily doses – low to high)

– Select examples from the most prescribed/sold drugs (2009), with varying daily doses

• How many “blockbuster” drugs will form –CN or CH3ONH2 conjugates in HLM/NADPH incubations?• HT “RM” trapping assays seldom utilize “safe” drugs

SN

N

N

NH

O

Cl

Ziprasidone

NN SO O

O

N

HN

O

NN

Sildenafil

O

O

O

N

Donepezil

Cl

NN

Meclizine

NH

NN

N

Olanzapine

S

HNN N

OF OH

O

Ciprofloxacin

Cl

NN

Aripiperazole

Cl

O NH

O

Dose = 5.0-23 mg Dose = 25-100 mg

Dose = 10-15 mg

Dose = 500-1500 mg Dose = 10 mg Dose = 25-80 mg

O N

N

Risperidone

F N

N

O

Dose = 12-50 mg

Dose = 40-60 mg

N

N

NHN

HN

O

N

N

ImatinibDose = 400-800 mg

ON

F N O

O

HN

O

Linezolid

Dose = 600-800 mg

20

PageThe list is growing with recent approvals……

• Numerous drugs (~ 43) with cyclic basic amine motif approved from 2009–2018– Select examples (non-oncology/oncology)

• How many of these drugs will form –CN or CH3ONH2 conjugates in HLM/NADPH incubations?• HT “RM” trapping assays seldom utilize “safe” drugs

O

Cl

NH H

Asenapine5-HT2A/2C/6/7 receptors

(Atypical antipsychotic)

N

NH

S

VortioxetineSerotonin modulator

(Antidepressant)

Prucalopride

Cl

H2N O

NH

O N O

5-HT4 receptor agonist

(impaired motility associated with chronic constipation)

VilazodoneSerotonin reuptake inhibitor

(Major depressive disorder)

HN

NN

N

OO

NH2

NiraparibPARP inhibitor

NN

O NH2

NH

AlectinibALK inhibitorO

N

N NH

NO

CobimetinibMEK inhibitorI F

NH

F

F N

O

OH NH

H

CeritinibALK inhibitor

HN

O HN

N

N

Cl

HN

SO

O

NO

ON

N

HN

F BrVandetanibVEGFR, EGFR, RET-TK inhibitor

HNN

NHN

N

N N O

N

RibociclibCDK4/6 inhibitor

21

PageA literature assessment revealed….

Kadi AA, Amer SM, Darwish HW et al. RSC Adv. 2017, 7, 36279–87.

Liu X, Lu Y, Guan X et al. Biochem. Pharmacol. 2015, 97, 111-21.

F

NH

O

N

OHO

F

NO

O

NO

Foretinib (GSK1363089)

CH3ONH2 adduct

CN adduct GSH adducts (of quinone-imine) also detected

Li AC, Yu E, Ring SC et al. Rapid Commun. Mass Spectrom. 2014, 28, 123-34.

NN

NH

O

NH

N

N

NImatinib

ADRS –DILI, transaminases, bilirubin (3-5%)

Multiple CN adducts

Daily dose = 400–800 mg

CN adduct

N

NO

O

NO

Gefitinib

HN

F

Cl

CH3ONH2 adduct

ADRS – rash (44%), DILI (2-4%)Daily dose = 250 mg

• Note the risk-benefit profile– Many oncology drugs (even the ones not containing cyclic basic amine motif) are associated with

IADRs including DILI

All RM Trapping studies conducted in HLM/NADPH

22

PageA literature assessment revealed….

Kenny JR, Mukadam S, Zhang C et al. Drug-drug interaction potential of marketed oncology drugs: in vitro assessment of time-dependent cytochrome P450 inhibition, reactive metabolite formation and drug-drug interaction prediction. Pharm. Res. 2012, 29, 1960-76.

These don’t contain cyclic basic amine motif

ClNH

O

N

SNH

NN

N NOH

Trapping studies in HLM

23

PageLets think logically! MPTP is neurotoxic (but not hepatotoxic) WHY?

• The metabolic fate of MPTP differs in the liver (relative to brain)

– N-Demethylation and N-oxidation are the principal metabolic pathways in liver

– MPDP+ (generated in the liver) is efficiently detoxified to a lactam metabolite by cytosolic Aldehyde Oxidase (AO)

– MPP+ formation noted exclusively in mitochondrial fractions (inhibited by MAOB inhibitor and –CN)

• MPP+ formation markedly ↓ in mitochondrial/cytosol combination

• MPP+ formation markedly ↓ in liver S9 fraction

Yoshihara S, Ohta S Arch. Biochem. Biophys. 1998, 360, 93-98.Wu E, Shinka T, Caldera-Munoz P et al. Chem. Res. Toxicol. 1988, 1, 186-94.Weissman J, Trevor A, Chiba K, Peterson LA et al. J. Med. Chem. 1985, 28, 997-1001.

Ph

N

MPTP

Ph

N

MAO-B

MPDP+

Ph

NH

Ph

NO

CYP FMO

Ph

N

Microsomalmetabolite

Detoxification

Ph

N O

AO

Ph

N

Ph

N O

Detoxification

Consider [O] of iminiums by AO as a detoxification pathway

2424

PageExamples of AO detoxification of iminium species

• Aldehyde Oxidase (AO) is an iminium oxidase (H transfer mechanism; kH/kD ~ 3)

– Not expressed in liver microsomes– Considerable species difference in

expression (Human and monkey >> rat; dog has no AO activity)

– S9 or hepatocytes is needed for assessment of AO role

Brandange S, Lindblom L Biochem. Biophys. Res. Commun. 1979, 91, 991-96.Ruenitz PC, Thomas HG Arch. Biochem. Biophys. 1985, 239, 270-272.Zheng J, Xin Y, Zhang J, Subramanian R et al. Drug Metab. Dispos. 2018, 46, 237-47.Hosogi J, Ohashi R, Maeda H et al. Drug Metab. Pharmacokinet. 2017, 32, 255-64.

N

NH

Nicotine

CYP

N

NH AO

N

NH O

Cotinine-CN

N

NH CN

~80% of nicotine dose is converted to cotinine in humans

NO

R CYP NO

R

AON

O

RO

CNN

O

RCN

NH

O

N

N

NHNR =

Momelotinib (JAK1/2 inhibitor)

Major human metabolite (circulation/excreta)

NH

NR

KW2449 (multikinase inhibitor)

MAO-B

NH

NR

AO

CN NH

NR

CN

NH

NR

O

N

HN

O

R =

M1 (10x ↑ plasma concn. than parent)

25

PageLactam formation with cyclic amines (evidence from human mass balance studies)

• Imatinib– Forms iminium species in vitro

(HLM)• Inferred from multiple CN

conjugates

• However, major human metabolites (circulation/excreta) are:

NN

NH

O

NH

N

N

N

N-Oxide

HydroxylationN-OxideLactam

Lactam

N-Demethylation

Gschwind HP, Pfaar U, Waldmeier F et al. Drug Metab. Dispos. 2005, 33, 1503-12.

N NH2

O

ClF

ClN

N

HN

Crizotinib

N NH2

O

ClF

ClN

N

HNO

• Crizotinib– The major circulating

metabolite (active) in humans is the corresponding lactam

Johnson TR, Goulet TW, Smith EB et al. Xenobiotica 2015, 45, 45-59.

26

PageConcluding remarks• Cyclic amines are bioactivated to iminium and aldehyde intermediates

• On occasions, aldehyde intermediates can covalently adduct DNA (Salmonella Ames)– Assessed (and potentially resolved) in preclinical drug discovery

• Evidence linking cyclic amine bioactivation to IADR (e.g., DILI) is weak– Iminium, aldehydes vs. RMs (e.g., quinone-imines) elsewhere in the molecule– What is the in vivo surrogate for –CN?

• Should cyclic amines be subjected to “RM trapping assays” (e.g., -CN, CH3ONH2, GSH)?– If you chose to do so and unearth a +ve signal, what does this mean?– Consider a general in vitro metabolism study in hepatocytes (humans and animals)

» Examine the detoxification of iminiums to lactams (by Aldehyde Oxidase)» Examine the detoxification of aldehydes to acids (aldehyde dehydrogenase) and/or alcohols

(reductases)

27

PageA case study

8 9 10 11 12 13 14 15 16 17 18 19Time (min)

020406080

100

Rel

ativ

e Ab

sorb

ance 13.26

11.53

12.3211.0710.48

m/z+ = 402 (parent)NR

NR

HOm/z+ = 418

m/z+ = 416

m/z+ = 374

m/z+ = 578

m/z+ = 521

HPLC chromatogram (λ 340 nm) of a human hepatocyte incubation (4 hr) with m/z+ 402

NR N

HR

Om/z+ 402

CYP NR

OHGSH

NH

R

OHS

HNNH

O

HO2C

OCO2H

NH2

H2O, -Glu

S

HN

NH

O

CO2H

HN

R

m/z+ 578

- GlyS

HN

OH

OHN

R

m/z+ 521

• Thiol conjugation – major metabolic fate• Liability addressed in follow-on compounds

• ~ 3 months (RM –ve, potency, PK, etc.) to find a suitable replacement