Mik

e S

. Lee

Mile

ston

e D

evel

opm

ent S

ervi

ces

Adv

ance

d C

hem

istr

y D

evel

opm

ent

Nor

th A

mer

ican

Use

rs M

eetin

gA

pril

19-2

0, 2

011

Prin

ceto

n, N

ew J

erse

y

Hig

hly

Su

cce

ssfu

l S

tra

teg

ies f

or

Str

uctu

re I

de

ntifica

tio

n U

sin

g

LC

/MS

: C

urr

en

t A

pp

lica

tio

ns a

nd

Em

erg

ing T

rends

P.O

. Box

178

New

tow

n, P

A 1

8940

-017

8T

el:

267

757

0462

info

@m

ilest

oned

evel

opm

ent.c

om

The

Top

10

Blo

ckbu

ster

Dru

gs –

Yea

rlyW

orld

wid

e S

ales

(M

arch

200

8)

Mil

esto

ne D

evel

opm

ent

Serv

ices

Eig

ht

dru

gs in

bo

ldfa

ce

will

lo

se

exclu

siv

ity b

y 2

01

1.

Sale

s R

ank

Pro

duct

2008

Sal

es*

($B

)

1L

ipit

or –

Pfiz

er13

.2

2P

lavi

x –

BM

S/Sa

nofi

7.4

3N

exiu

m–

Ast

raZ

enec

a7.

1

4A

dvai

r–

GSK

6.9

5R

ispe

rdal

– J&

J4.

7

6Z

ypre

xa–

Lill

y4.

6

7Se

roqu

el–

Ast

raZ

enec

a4.

5

8Si

ngul

air

– M

erck

4.4

9E

ffex

or–

Wye

th4.

0

10A

rane

sp–

Am

gen

3.9

•“G

o”

an

d “

Sto

p”

De

cis

ion

s D

ep

en

de

nt

on

Sa

mp

le

T

hro

ug

hp

ut

an

d E

arly E

limin

atio

n o

f W

ea

k C

an

did

ate

s

Dru

g D

evelo

pm

ent

Overv

iew

–

Pip

elin

e D

ynam

ics

C

LIN

ICA

L

DEV

ELO

PM

EN

T

PR

EC

LIN

ICA

L

DEV

ELO

PM

EN

T

D

RU

G

DIS

CO

VER

YM

AN

UFA

CTU

RIN

G

•T

arge

t ID

•Le

ad ID

•S

cree

ning

•O

ptim

izat

ion

•P

roce

ss R

esea

rch

•F

orm

ulat

ion

•M

etab

olis

m•

Tox

icol

ogy

•S

cale

Up

•P

harm

acok

inet

ics

•D

eliv

ery

•S

afet

y

•P

rodu

ctio

n•

QA

/QC

•Le

gal

Mil

esto

ne D

evel

opm

ent

Serv

ices

Le

e,

M.S

., L

C/M

S A

pp

lica

tio

ns in

Dru

g D

eve

lop

me

nt,

Jo

hn

Wile

y

an

d S

on

s:

Ne

w Y

ork

, N

Y 2

00

2

�Q

ua

lita

tive

-

Ra

pid

str

uctu

re c

on

firm

atio

n

-

Str

uctu

re I

de

ntifica

tio

n

� Q

ua

ntita

tive

-

Se

nsitiv

e,

hig

hly

sp

ecific

an

aly

sis

-

Tra

ce

-mix

ture

an

aly

sis

� M

ech

an

istic A

pp

roa

ch

es

-

Im

mu

ne

re

sp

on

se

-

Re

active

me

tab

olit

es

-

To

xic

im

pu

ritie

s/d

eg

rad

an

ts

• In

dust

ry P

refe

renc

es fo

r Q

uick

Info

rmat

ion

and

Fas

t Dec

isio

n-M

akin

g

Ana

lytic

al F

ocus

– D

evel

opm

ent o

fIn

dust

rial A

pplic

atio

ns Mil

esto

ne D

evel

opm

ent

Serv

ices

Str

uctu

re A

naly

sis

Matr

ix:

Pharm

aceutical A

naly

sis

Pre

fere

nces

Pure

M

ixtu

re

Non-

T

race

T

race

HP

LC

/UV

LC

/MS

MS

UV

IRXra

y

NM

RL

C/N

MR

• T

race

-Mix

ture

Sam

ple

Typ

e is

a P

rimar

y D

river

for

A

ccel

erat

ed D

rug

Dis

cove

ry a

nd D

evel

opm

ent

Mil

esto

ne D

evel

opm

ent

Serv

ices

M.S

. Lee

, LC

/MS

App

lica

tion

s in

Dru

g D

evel

opm

ent,

John

Wil

ey &

Son

s; N

ew Y

ork

2002

Mass S

pectr

om

etr

y A

ppro

aches for

Meta

bolit

e Identification

Mil

esto

ne D

evel

opm

ent

Serv

ices

•B

ase

d o

n t

he

fa

ct

tha

t m

eta

bo

lite

s c

on

tain

va

rio

us s

ub

str

uctu

res o

f

the

ori

gin

al d

rug

mo

lecu

le

–L

ee

et a

l., A

nn

u. R

ep

. M

ed

. C

he

m.

1986,

21, 313-3

21.

–L

ee

an

d Y

ost,

Bio

me

d.

En

v.

Ma

ss S

pe

ctr

om

.1

98

8, 15, 193-2

04.

•F

ull

sca

n m

ass s

pe

ctr

om

etr

y a

llow

s f

or

co

nfirm

atio

n o

f m

ole

cu

lar

we

igh

t

•T

an

de

m m

ass s

pe

ctr

om

etr

y (

MS

/MS

) p

rovid

es r

ap

id id

en

tifica

tio

n o

f

su

bstr

uctu

res

•M

eth

od

olo

gy p

rovid

es a

ra

pid

an

d s

yste

ma

tic a

pp

roa

ch

with

ou

t th

e

use

of

sta

nd

ard

s

Sum

mar

y of

Dru

g M

etab

olite

Iden

tifica

tion

Str

ateg

y Mil

esto

ne D

evel

opm

ent

Serv

ices

•B

uild

the

pict

ure

puzz

le -

ste

pwis

e!–

Mol

ecul

ar w

eigh

t cor

resp

onds

to th

e ou

tsid

e of

the

puzz

le

–D

irect

com

paris

on o

f hom

olog

ous

pict

ures

•T

hink

che

mis

try

–E

xplo

it th

e fa

ct th

at io

niza

tion

is th

e in

itiat

or o

f fra

gmen

tatio

n

–A

cid-

base

che

mis

try

•C

onne

ct th

e pi

eces

–N

o ne

ed to

iden

tify

all f

ragm

ent i

ons

to p

ropo

se s

truc

ture

–A

ddre

ss r

earr

ange

men

ts la

ter

Mil

esto

ne D

evel

opm

ent

Serv

ices

Buspirone

Mil

esto

ne D

evel

opm

ent

Serv

ices

NN

NN

NO O

Substr

uctu

re -

MS

/MS

Pro

duct Io

n

Sp

ectr

um

of B

usp

iro

ne

Mil

esto

ne D

evel

opm

ent

Serv

ices

[M+H]+

386

222

265

291

343

180

198

168

152

14815

0

10912

2

95

Su

bstr

uctu

re T

em

pla

te o

f B

usp

iro

ne

Mil

esto

ne D

evel

opm

ent

Serv

ices

NN

NN

NO O

12

3

45

6

Su

bstr

uctu

re T

em

pla

te o

f B

usp

iro

ne

Mil

esto

ne D

evel

opm

ent

Serv

ices

Sub

stru

ctur

e

For

mul

a

Mas

s

Obs

erve

d (Y

/N)

1

C17

H30

N3O

2

30

8

N

2

C13

H20

NO

2

22

2

Y

3

C

9H14

NO

2

168

Y

4

C

12H

19N

4

219

N

5

C8H

13N

4

165

N

6

C4H

3N2

79

N

Su

bstr

uctu

re T

em

pla

te o

f B

usp

iro

ne

-

Form

ula

s a

nd F

ragm

ents

Mil

esto

ne D

evel

opm

ent

Serv

ices

NN

NN

NO O

222

168

Substr

uctu

re T

em

pla

te -

Usefu

l T

ips

Mil

esto

ne D

evel

opm

ent

Serv

ices

•F

ragm

enta

tion

prod

uces

one

neu

tral

and

one

ion

•N

itrog

en r

ule

–C

ompo

unds

with

odd

num

ber

of n

itrog

en a

tom

s ha

ve o

dd M

Wan

d vi

ce v

ersa

•A

dd tw

o (2

) hy

drog

en a

tom

s (p

roto

natio

n an

dre

arra

ngem

ent)

for

ion

form

ed fr

om h

eter

o-at

omsu

bstr

uctu

re

MS

/MS

Pro

duct Io

n S

pectr

um

-

Meta

bolit

e A

Mil

esto

ne D

evel

opm

ent

Serv

ices[M

+H

]+

402

95

8072

13915

0

122

196

184

165

210

238

281

307

359

Refined S

tructu

re o

f M

eta

bolit

eA

NN

NN

NO O

+OH

Mil

esto

ne D

evel

opm

ent

Serv

ices

Me

tab

olit

eId

en

tifica

tio

n S

um

ma

ry•

Ke

ep

all

inte

rpre

tatio

ns s

imp

le!

-It

era

tive

an

d “

bin

ary

” str

ate

gie

s b

ase

d o

n a

dd

itio

n a

nd

elim

ina

tio

n

rea

ctio

ns fro

pro

ton

atio

n a

nd

fra

gm

en

tatio

n, re

sp

ective

ly

• T

he

mo

st

va

lua

ble

pie

ce

of

da

ta is o

fte

n t

he

co

nfid

en

t a

ssig

nm

en

t o

f

mo

lecu

lar

we

igh

t

•A

dd

itio

n r

ea

ctio

ns c

orr

esp

on

d t

o io

niz

atio

n

-A

na

lyte

fo

rms a

co

mp

lex w

ith

hyd

ron

ium

io

n fo

llow

ed

by h

yd

roly

sis

to

resu

lt in

pro

ton

ate

d m

ole

cu

lar

ion

-M

ole

cula

r w

eig

ht chara

cte

rization d

erived fro

m full

scan m

ass s

pectr

um

•E

limin

atio

n r

ea

ctio

ns c

orr

esp

on

d t

o f

rag

me

nta

tio

n-

Typic

ally

no s

tandard

s a

re a

vaila

ble

; use p

are

nt dru

g a

s a

sta

ndard

- C

onstr

uct pre

dic

ted s

ubstr

uctu

re tem

pla

te o

f th

e p

are

nt dru

g b

ased o

n

fra

gm

enta

tion a

dja

cent

to t

he p

roto

nate

d h

ete

roato

m

-F

ragm

enta

tion tem

pla

te c

onfirm

ed fro

m M

S/M

S p

roduct io

n s

pectr

um

•D

rug

me

tab

olit

es o

fte

n r

eta

in t

he

sa

me

co

re s

tru

ctu

re a

s t

he

pa

ren

t

dru

g - S

imila

r additio

n a

nd e

limin

ation r

eactions (

ioniz

ation a

nd fra

gm

enta

tion)

Mil

esto

ne D

evel

opm

ent

Serv

ices

Worldvie

w 1

540

Mil

esto

ne D

evel

opm

ent

Serv

ices

�In

itial

Met

hod

Dev

elop

men

t-

Sam

ple

prep

arat

ion

- H

PLC

met

hod

deve

lopm

ent

- LC

/MS

/MS

met

hod

deve

lopm

ent

� P

re-V

alid

atio

n� V

alid

atio

n� A

pplic

atio

n

• R

oger

Hay

es, S

cher

ing-

Plo

ugh

Res

earc

h In

stitu

te

(CP

SA

200

3)

New

Analy

tical T

ools

Leads to M

ore

Specia

lized U

nders

tandin

g

Mil

esto

ne D

evel

opm

ent

Serv

ices

Analy

tical F

igure

s o

f M

erit -

Qualit

ative

and Q

uantita

tive A

naly

sis

Sen

sitiv

ity

Sen

sitiv

ity

Sen

sitiv

ity

Acc

urac

y

Pre

cisi

on

Spe

ed

Con

trol

• I

ncre

ased

Per

form

ance

Thr

ough

Pre

cise

and

A

utom

ated

Con

trol

Mil

esto

ne D

evel

opm

ent

Serv

ices

M.S

. Lee

, LC

/MS

App

lica

tion

s in

Dru

g D

evel

opm

ent,

John

Wil

ey &

Son

s; N

ew Y

ork

2002

Accura

te M

ass o

f a P

rocess Im

purity

– T

OF

MS

267.1

082

268.1

168

Th

e M

+H

of

the

ma

in c

om

po

un

d

ha

s t

he

mo

lecu

lar

form

ula

of

C11H

15N

4O

4

Th

e M

+H

of

the

im

pu

rity

ha

s

me

asu

red

ma

ss t

ha

t fits

th

e

mo

lecu

lar

form

ula

of

C11H

15N

4O

4 w

ith

an

err

or

of

-

1.4

3 p

pm

Dat

a co

urte

sy o

f K

en L

ewis

–O

pAns

MS

with 4

0 C

ycle

/s, 5-9

0%

B

Gra

die

nt in

0.6

5m

inP

eak c

apacity o

f >

40

in 3

9 s

ec

in t

he M

S c

hro

mato

gra

m!!!

0.0

50

.10

0.1

50

.20

0.2

50

.30

0.3

50

.40

0.4

50

.50

0.5

5

0.6

0T

ime, m

in

0.0

5.0

e4

1.0

e5

1.5

e5

2.0

e5

2.5

e5

3.0

e5

3.5

e5

4.0

e5

4.5

e5

5.0

e5

5.5

e5

6.0

e5

6.5

e5

7.0

e5

Intensity, cps

0.3

4s 0

.36s

0.3

6s

0.4

2s

0.3

6s

Ate

no

lol

Me

top

rolo

l

Pri

mid

on

e

Ve

rap

am

il

Be

clo

me

tha

so

ne

-

dip

rop

ion

ate

H2O

/AC

N

Flo

w =

1.8

ml/m

in

5-9

0%

B in 0

.5m

in

Sto

p tim

e =

0.6

5m

in

80°C

, A

CR

MS

40H

z

100-1

000D

ab 2

wsaz

Dat

a co

urte

sy o

f S

teve

Fis

cher

–A

gile

nt T

echn

olog

ies

Influence o

f M

S D

ata

Acquis

itio

n

Rate

0.0

50

.10

0.1

50

.20

0.2

50

.30

0.3

50

.40

0.4

50

.50

0.5

50

.60

Tim

e,

min

0.0

5.0

e5

1.0

e6

1.5

e6

2.0

e6

2.5

e6

3.0

e6

3.5

e6

4.0

e6

4.5

e6

5.0

e6

5.5

e6

6.0

e6

6.5

e6

7.0

e6

7.3

e6

Intensity, cps

40

Cycle

/s

20

Cycle

/s

5 C

ycle

/s

0.2

70

0.2

80

0.2

90

0.3

00

Tim

e,

min

40

Cycle

/s

5 C

ycle

/s

Dat

a co

urte

sy o

f S

teve

Fis

cher

–A

gile

nt T

echn

olog

ies

“Ah-

ha”

Mom

ent

Mil

esto

ne D

evel

opm

ent

Serv

ices

“You

�re g

onna

nee

d a

bigg

er b

oat.”

-

Po

lice

Ch

ief

Ma

rtin

Bro

dy,

Ja

ws,

19

75

�S

tand

ard

Met

hods

� T

empl

ate

Str

uctu

re Id

entifi

catio

n� D

atab

ases

� S

cree

ning

� In

tegr

atio

n� M

inia

turiz

atio

n� P

aral

lel P

roce

ssin

g� V

isua

lizat

ion

� A

utom

atio

n

• P

ower

ful T

ools

to D

evis

e, C

onst

ruct

and

Refi

ne

Met

hod

Dev

elop

men

t Str

ateg

ies

Mil

esto

ne D

evel

opm

ent

Serv

ices

M.S

. Lee

, LC

/MS

App

lica

tion

s in

Dru

g D

evel

opm

ent,

John

Wil

ey &

Son

s; N

ew Y

ork

2002