Joseph R. Lennox, Ph.D. A Brief Presentation of My Background to the Folks on the ACS Network 2009 Enantiospecific Synthesis of Annulated Nicotine Analogues

Joseph R. Lennox, Ph.D.

A Brief Presentation of My Backgroundto the Folks on the ACS Network

2009

Enantiospecific Synthesis of Annulated Nicotine AnaloguesFrom D-Glutamic Acid

Ion Channel Modulation in Urge Urinary Incontinence:From KATP to Chloride Channels

Toll-Like Receptor 7 (TLR7) Nucleoside Drugs & Prodrugs:From Concept to Delivery and Beyond

Part I Enantiospecific Synthesis of Annulated Nicotine Analogues From D-Glutamic Acid

Part II Ion Channel Modulation in Urge Urinary Incontinence: From KATP to Chloride Channels

Part III Toll-Like Receptor 7 (TLR7) Nucleoside Drugs & Prodrugs: From Concept to Delivery and Beyond

• Basic Principles• Amino Acid Esters => ANA971• 6-Amino Deoxy• 6- Deoxy => ANA975

Ion Channel & TLR Discovery Programs

Department of ChemistryUniversity of California, Berkeley

Examples of Amino Acid Derived Chiral Heterocycles

R *OH

O

NH2

NR

N

N

O

O

HN

O

N

NR

D- and L-Amino Acids

Piperadines

Indolizidines

Anatoxins

Pyrrolidines

Carbapenams

Carbacephems

Sardina & Rapoport....Chem. Rev. 1996, 96, 1825-72.

RN

Tropanes

RN

Tropanes


Tropane and Homotropane Analogs of Nicotine

RN

( )n

via

» Intramolecular Alkylation (Lennox & Turner)

» Heck Chemistry (Turner & Zhai)

» Ring Expansion (Ahn & Lennox)

» Intramolecular Olefin Methathesis (?)

N Me

N

Pyridine ringskewed andperpendicularto pyrrole.

Elmore & Dougherty.... J. Org. Chem. 2000, 65, 742-747.Glennon & Dukat.... Med. Chem. Res. 1996, 6, 465-468.

N

1

23

45

1'

2'3'

4'

5'

6'


Why Nicotine?

Clinical interests in the benefits of nicotinoids have been rising:

• nAChRs widely distributed in human brain

• Antinociceptive effects of (S)-(-)-nicotine known since 1932

• Potential therapeutic indications currently under investigation

Alzheimer’s disease Schizophrenia

Anxiety Tourette’s syndrome

Depression Ulcerative Cholitis

Parkinson’s disease


Effects on Molecular Geometry of Methylene Additions

[2.2.1][3.2.1][4.2.1]


Synthetic Plan

Key Step

Synthetic Strategy [2.2.1] System

NR

CO2Me

N

ClRN

N

D-Glutamic Acid

HO OH

O O

NH2

RN CO2Me

N

Cl

RN CO2Me

N

Cl

RN

N

CO2Me

RN CO2Me

N

Cl

FunctionallyCompatible

Base

SNAr


Preparation of [2.2.1] Cyclization Precursor

HO OH

O O

NH2

1. H2O, rflx2. MeOH, SOCl2

98% NH

O CO2Me

Boc2O, Et3N,DMAP, CH2Cl2

NBoc

O CO2Me

90%

4-chloropyridine,LDA, -78°C

Et2O/Hex/THF3:1:1 (92%) N

CO2Me

OCl

NHBoc

HCl / EtOAc

91%

N•HCl

CO2Me

OCl

NH2•HCl

aq NaHCO3,EtOAc

N CO2Me95%

N

Cl


Tropane Formation

NBoc

CO2Me

N

Cl2.2 eq LDA, THF,-78° to -20°C

variable yields:30 to 50%

N

N

BocCO2Me

2.2 eq LiDMP, THF,-78° to -20°C (55%)

LiOH, dioxane,H2O

NaBH4, HOAc,MeOH, -38°C

NH

CO2Me

N

Cl

cis : trans 72 : 28

85%

Boc2O, Et3N,THF, -15°C to RT

98%

98%

N CO2Me

N

Cl


De-Carboxylation Sequence

N

N

BocO

SN

O

1. (COCl)2, Py, 0°C2. 2-Mercaptopyridine-

N-oxide, CH2Cl2,Et3N

t-BuSH, h,CH2Cl2

H•HClN

N•HCl

HCl, MeOHN

N

Boc 1. NaOH, MeOH2. HCHO, HCO2H

Me•HClN

N•HCl

58% 3 steps

98%

N

N

BocCO2H

3. HCl/Et2O, MeOH 90% 3 steps


Unexpected Results

N

N

Me HN

N

H Me

371K

Protonation of pyrrolidine nitrogen results in energetically stablechiral nitrogen as indicated by NMR experiment.


Variable Temperature 1H NMR in DMSO-d6


Reactivity of Iodopyridine vs. Chloropyridine

NH

CO2Me

N

Cl EtO2CCl, Et3N,THF, 0°C

NCO2Et

CO2Me

N

Cl

NCO2Et

CO2Me

N

I

12 h 93%95%

NaI, EtO2CCl / AcCl(10:1), MeCN, ))))

NCO2Et

CO2Me

N

I

Li2.2 eq LiDMP, THF,-78° to -20°C -LiI


Furan Trapping & Deuterium Quench

N

N

EtO2CCO2Me

NEtO2CCO2Me

N

NCO2Et

CO2Me

N

O

NBoc

CO2R

N

Cl

DD

, -78°C to RT

NBoc

CO2Me

N

Cl2.2 eq LiDMP orLDA, -78°, 2h

then MeOH-d4

31%

[4+2] Cycloaddition

R = Me, CD3

O


Steric Analysis of the Lithium Amide Bases

N

MeMe

H

HN

MeMe

Me

MeMe NSi

MeMe

Me Si

Me

MeVS VS

LiDMP LiTMPLiHMDS

N

NBoc

CO2Me

Cl

can metallatehere...

but notdeprotonatehere

Hindered Bases

Me N

MeMe

Me

H

H

LDA

VS

Part II

Ion Channel Modulation in Urge Urinary Incontinence:From KATP to Chloride Channels

Wyeth-AyerstResearch

Program Goal:

To develop a bladder selective potassium channel opener which will oppose spontaneousdetrusor instability (contractions) without affecting the normal micturition reflex and withoutlowering blood pressure.

Rationale:

Activation of potassium channels will hyperpolarize bladder smooth muscle, bringing the tissue further from the threshold for calcium channel activation thereby inhibitingcontractions.

Therapeutic Objective


Increase K+ conductance in bladder muscle cells

Hyperpolarize cells

Decrease spontaneous contractions

Decreased urinary urgency

Increased bladder capacityIncreased bladder capacity

Objective / Therapeutic Goal


Benzopyrans N-Cyanoguanidines Thioamides

cromakalim pinacidil aprikalim

O

N

OH

MeMe

O

NC N

NH

NH

NCN S

NS NHMe

O

O

NH

CF3

O

HO Me

Tertiary Carbinols

ZD-6169

Pyridines / Pyrimidines

NHN

NO

O

N

N

NH2

O

NH2N

nicorandil minoxidil

Structural Classes of KATPCOs


Benzopyrans Thioamides

Potent bladderrelaxants modestly selective

Cyclobutenedione diamides

N-Cyanoguanidines

Potent bladderrelaxants non-selective

O

N

OH

MeMe

O

NC

S

NS NHMe

ON

NH

NH

NCN

N

NH

NH

NCN

NH

AlkylNH

Aryl

OO

NH

AlkylNH

Aryl

OO

Modification of “First Generation” Antihypertensive KCOs


O

N NH

NC

O

Intended Target:Cromakalim Analog

O

OBr

CO2Me

H2NHN

O

NBr

NH CO2Me

Zn, HCO2H;CuCN

O

HN

Br

CO2Me

O

HN

Br

CO2H

1

LiOH, THF, H2O

Fisher Indole

Synthesis

NitrileSurrogate

IC50(B) 15.1mBSR 7.9GR 0%non-KATP

Mechanism?

X

Serendipity is the Forebearer of an Unexpectedly Novel Lead


HN

Br

CO2H

O

HNOBr

CO2H

Et

IC50(B) = 4.6BSR = 6.1Mech BKInactive in vivoSolubility: Marginal

IC50(B) = 8.6 MBSR = 24.5Mech BKInactive in vivoSolubility: Very Poor

HN

Me

O

CO2LiF3C

Cl

IC50(B) = 0.52BSR = 5.3Mech Cl BlockerActive in vivoSolubility: Acceptable

Summary of Lead Development


O

HN

CO2LiF3C

Compound DIC50(B) = 0.52 µM, BSR = 5.3

GR = 37%» Active in vivo in hypertrophied rat bladder

model and Levin hyperactive rabbit bladder

ClO

HN

CO2LiF3C

Compound DIC50(B) = 0.52 µM, BSR = 5.3

GR = 37%» Active in vivo in hypertrophied rat bladder

model and Levin hyperactive rabbit bladder

Cl

Mechanism of Action • Potent blocker of chloride channels and activator of BK (maxi-K) channels.

Alternative Therapeutic Indications for Chloride Channel Blockers • Asthma, Cardiac Arrhythmia, Cystic Fibrosis, Eye Cataracts, Ischemia, Malaria, Sickle Cell Anemia.

Current Status • Unknown • Pfizer is developing an analog slightly outside the genus of “tricyclic patent” for the treatment of cystic fibrosis.

A Non-KATP Dependent Bladder Selective Biological Lead


AcknowledgementsDepartment of Chemistry

University of California, Berkeley

Chemistry

Mr. Schuyler Antane

Dr. John Butera

Mr. Bradford Hirth

Pharmacology

Dr. Thomas Argentieri

Dr. Dale Hartupee

Dr. Joseph Hinson

Mr. N. Wesley Norton

Mr. Jeffrey Sheldon

Ms. Dawn Warga

Ms. Alexandra Wojdan

Chemistry

Dr. Young-Gil Ahn

Professor Henry Rapoport

Dr. Sean Turner

Molecular Graphics

Mr. Ken Yamaguchi (Bartlett)

Support

Tularik

Part III

Toll-Like Receptor 7 (TLR7) Nucleoside Drugs & Prodrugs: From Concept to Delivery and Beyond

Classical Medicinal Chemistry

Chemical Lead

Structure Activity Relationship

Orally Bioavailable Prodrug

Refined Biological Lead

Clinical Candidate(orally bioavailable if possible)

Prodrug-Based Medicinal Chemistry

Clinical Candidate(not orally bioavailable)

Structure Bioavailability Relationship

Biological Lead

Refined Prodrug With Excellent PK

Clinical Candidate(orally bioavailable)

Non-Orally BioavailableClinical Candidate

FDA Approvalvia an

Accepted OralFormulation

FDA Approval(IV Formulation)

Hospital Stay

Higher InsuranceCosts or Inability

of Patient to ReceiveTreatment Due to

Financial Constraints

Medicinal Chemistry Slightly Redefined

• TLR1 – Triacyl lipopeptides (bacteria)• TLR2 – Peptidoglycan (bacteria)• TLR3 – dsRNA (virus) [e]• TLR4 – LPS (bacteria)• TLR5 – Flagellin (bacteria)• TLR7 – ssRNA (virus) [e]• TLR8 – ssRNA (virus) [e]• TLR9 –CpG DNA (bacteria, virus) [e]

• TLR1 – Triacyl lipopeptides (bacteria)• TLR2 – Peptidoglycan (bacteria)• TLR3 – dsRNA (virus) [e]• TLR4 – LPS (bacteria)• TLR5 – Flagellin (bacteria)• TLR7 – ssRNA (virus) [e]• TLR8 – ssRNA (virus) [e]• TLR9 –CpG DNA (bacteria, virus) [e]

*

Toll-Like Receptors (TLRs): Pattern-Recognition Receptors That Are Attractive Drug Targets

Isatoribine

Me

dia

n L

og

Vira

l Lo

ad

Ch

an

ge

200 mg QD

400 mg QD

600 mg QD

800 mg QD800 mg QD

-0.75

-0.50

-0.25

0.00

0.25

0.50

Once Daily Dosing x 7 Days

1 Day after Last Dose

1 Week after Last Dose

p= 0.001

(Hepatology, vol. 42,p724-731, 2005)

Isatoribine: Human IV Administration Gives Positive Results

HN

N N

S

OHO

O

O

H2N

HO OH

Isatoribine (ANA245)

Prodrug enables a very efficient oral administration

An inactive “masked” TLR agonist prodrug avoids

exposure of gut immune tissue and thus avoids side effects.

Metabolic processes in the body remove the "mask,"

providing beneficial effects of systemic exposure to

TLR agonist.

Prodrug enables a very efficient oral administration

An inactive “masked” TLR agonist prodrug avoids

exposure of gut immune tissue and thus avoids side effects.

Metabolic processes in the body remove the "mask,"

providing beneficial effects of systemic exposure to

TLR agonist.

Anatomy impedes oral administration

“Masking” of ANA245 is Necessary for an Oral TLR-7 Prodrug

HN

N N

S

OHO

O

O

H2N

HO OH

HN

N N

N

OHO

O

H2N

HN

N N

N

OHO

O

H2N

OH

HN

N N

N

OO

O

H2N

H2N

O

HN

N N

N

OO

O

H2N

H2N

O

OH

IncreasedOral Bioavailability

Approx 15% F to60% F (human)

IncreasedOral Bioavailability

Approx 10% F to60% F (human)

Aciclovir(HSV 1 & 2)

Valaciclovir

Ganciclovir(HSV 1 & 2)

Valganciclovir

ANA245 (Isatoribine)

Aqueous Solubility Low

Oral Bioavailability Poor

Amino acid esters of antiviralnucleosides are absorbed byintestinal PEPT1 peptidetransporter.

Amino acid esters of antiviralnucleosides are absorbed byintestinal PEPT1 peptidetransporter.

[1] Czock, D. et al. Clin. Pharmacol. Therapeutics 2002, 72(3), 142-150; [2] Jung, D. et al. Pharmacokinetics & Pharmacodynamics1999, 39, 800-804; [3] Wiltshire, H. et al. Clin. Pharmacokinetics 2005, 44(5), 495-507; 1999, 37(2), 167-176; [4] Brown, F. et al. Clin. Pharmacokinetics 2005, 44(5) 495-507.

Guanosine-Like Nucleosides Notorious for Poor Oral Bioavailability- Amino Acid Ester Prodrugs Improve OBA

HN

N N

S

OO

O

O

H2N

HO OHH2N

O 2 HCl

HN

N N

S

OO

O

O

H2N

HO OHH2N

O

HN

N N

S

OO

O

O

H2N

HO OHH2N

O

HN

N N

S

OO

O

O

H2N

HO OHMeHN

O

1F 8% (monkey)

2F 3% (monkey)

3F 6% (monkey)

ANA971 (Val-245)F 46% (beagle)F 4% (monkey)

%F is based upon levels ofANA245 observed in eitherurine or plasma.

%F is based upon levels ofANA245 observed in eitherurine or plasma.

HN

N N

S

OHO

O

O

H2N

HO OH

Isatoribine (ANA245)

Aqueous solubility >80 mg/mLAqueous solubility >80 mg/mL

5’ Amino Acid Ester Prodrugs Prepared – Aqueous Solubility Enhanced

HN

N N

S

OHO

O

HO OH

O

H2N

HN

N N

S

OHO

O

O

H2N

O O

2,2-DMP, acetoneDMSO

MeSO3H (cat), rt88-94%

N-Boc-(L)-Val-OH,EDC, DMAP, Py-DCE,

0°C to rt, >90%

HN

N N

S

OO

O

O

H2N

O OBocHN

O

HN

N N

S

OO

O

O

H2N

HO OHH2N

O 2 HCl

HCl (g), i-PrOAc (anh), rt

>90%

ANA971Overall Yield 70%

ANA245 4 5

Process Chemistry Issues

(1) All reactions require >85% yield after purification.

(2) No chromatography permitted.

(3) Final compound must have >98% purity with nosingle impurity >1%.

Process Chemistry Issues

(1) All reactions require >85% yield after purification.

(2) No chromatography permitted.

(3) Final compound must have >98% purity with nosingle impurity >1%.

Synthesis and Large Scale Production of ANA971

Cottam, H. et al. J. Med. Chem. 1991, 34, 3006-3010. Lee, et al. Bioorg. Med. Chem. Lett. 1999, 9, 1365-1370.Alargov, et al. Monatshefte für Chemie 1997, 128, 725-732.

N

N N

N

H2N

AcO

N

N N

N

H2N

OHO

Famvir (Famciclovir)

6-Deoxy Aciclovir

HN

N N

N

H2N

HO

O

AcO HO

HN

N N

N

H2N

OHO

O

Aldehyde Oxidase

Esterase

Xanthine Oxidase

Penciclovir

Aciclovir

Acyclovir 22%6-Deoxy Acyclovir 66%

Oral Bioavailability in Rats(active nucleoside)

Penciclovir 1.5%6-Deoxy Penciclovir 9%Dipropionyl 6-Deoxy Penciclovir 27%Diacetyl 6-Deoxy Penciclovir 41%

Hodge, et al. Antimicrobial Agents & Chemotherapy 1989, 33, 1765-1773.Rashidi, et al. Drug Metabolism & Disposition 1997, 25, 805-813.

Krenitsky, et al. Proc. Natl. Acad. Sci. USA 1984, 81, 3209-3212

6-Deoxy “Nucleosoids” Improve Oral Bioavailability

HN

N N

S

OHO

O

O

H2N

HO OH

Ac2O, Et3N, DMAP

MeCN, 0° C to rt89%

HN

N N

S

OAcO

O

O

H2N

AcO OAc

P2S5, Py

rflx, 90%

HN

N N

S

OAcO

S

O

H2N

AcO OAc

RaNi,acetone;H2S/EtOH60%

N

N N

S

OAcO

O

H2N

AcO OAc

K2CO3 (cat)

MeOH, 89%

N

N N

S

OHO

O

H2N

HO OH

ANA245 22 52

54 53

Median Levels Adjusted to 1 mg/kg Doses

1

10

100

1000

0 1 2 3 4

Hours Post-Dose

Pla

sma

Co

nce

ntr

atio

n n

g/m

L

245 from 245 IV 245 from 6d245 IV 245 from 6d245 PO 6d245 from 6d245 IV 6d245 from 6d245 PO

Cottam, H. et al. J. Med. Chem. 1990, 33, 407-415.

Synthesis of 6’-Deoxy ANA245 (6d245)

N

N N

S

OHO

O

H2N

HO OH

TBSCl, Im

DMF, rt 82%

N

N N

S

OTBSO

O

H2N

HO OH

Ac2O, Et3N

MeCN, 92%

N

N N

S

OTBSO

O

H2N

AcO OAc

N

N N

S

OHO

O

H2N

AcO OAc

HF·Py, THF>98%

ANA975

54 55 56

PK Summary for ANA975

• Aqueous solubility 4 mg/mL

• Caco2 permeable

• Rate of conversion excellent

• OBA (monkey) 65% @ 20 mg/kg

• Approximate 80% conversion in humans

PK Summary for ANA975

• Aqueous solubility 4 mg/mL

• Caco2 permeable

• Rate of conversion excellent

• OBA (monkey) 65% @ 20 mg/kg

• Approximate 80% conversion in humans

Intermediate Synthesis of ANA975

HN

N N

S

OHO

O

O

H2N

HO OH

N

N N

S

OHO

O

H2N

AcO OAc

HN

N

O

NH2H2N

7 Steps

12-14%

4 Steps

71%

OLD SYNTHESIS

NEW SYNTHESIS

NH

S O

O

POCl3

DMF, 45%

NH

S O

Cl

OHC H2N NH2·HCl

NH

NaOMe, IPArflx, 44%

N

N NH

SO

H2N

OAcO

AcO OAc

OAc1. BSA, TMSOTf, MeCN,

2.

N

N N

S

OAcO

O

H2N

AcO OAc

Candida Antarctica lipase(Novozyme resin)

Phosphate buffer pH 7.0,acetone, rt, 1-2 days, 98%

N

N N

S

OHO

O

H2N

AcO OAc

ANA975

ANA975

(64-70%)

57

ANA245

58 59 30

53

Process Chemistry of ANA975

J. Chem. Soc. Perkin Trans. I 1992, 973-978.

Santaniello, E. Tetrahedron 2000, 56, 3239-3243.

Acknolwdgements

Medicinal Chemistry Biology Development

Erik Rueden Bob Aust Devron AverettStephen Webber Virginia Banh Lisa BaumanAlan Xiang Simon Fletcher Bradley Kerr Kevin Steffy Renee Lamb

Wei-Cheng LiawJohn Ng

Tingmin Wang

Analytical Chemistry Drug Metabolism

Danhua Chen Darian BartkowskiKimkim Dao Levan DarjaniaXiaoxing Liao Hovik GukasyanYvonne Yao Leo Kirkovsky

Daniel NorrisDan Yuan

Documents

Joseph R. Lennox, Ph.D. A Brief Presentation of My Background to the Folks on the ACS Network 2009 Enantiospecific Synthesis of Annulated Nicotine Analogues