Building Blocks for Big Pharma Contract Research Experience 2001-2005

This presentation is dedicated to Theodora Greene. Her book “Protective Groups in Organic Synthesis” provided me with the idea to use thiols to

liberate pyrrole-3-carboxylic acids; an idea that resulted in a step forward in my career.

Building Blocks for Pharma Synopsis

• Executed and supervised the production of 250 high-quality, unique building blocks and scaffolds for Big Pharma clients

• Responsible for planning and logistics to meet monthly quota (= # of compounds x complexity)

• Feasibility studies on proposed building blocks and direct reporting to clients

• Responsible for quality of deliverables and accompanying documentation

• Supervised team of Ph.D. and M.S. chemists

Pyrrole-3-carboxylic Acids, 2002

Pyrrole-3-carboxylates are prepared in high yield by a Michael addition of

an enaminoester with a nitroolefin, followed by intra-molecular acid-catalyzed

condensation:

Saponification should provide the building block…….

Pyrrole-3-carboxylic Acids Synthetic scheme, increase diversity along the way

Feasibility study showed

R1: (substituted)-alkyl

R2: alkyl, benzyl

R3: alkyl, aryl, heteroaryl

R3 is the most effective source of diversity

Pyrrole-3-Carboxylic Acids Diversity and Logistics

•Three points of diversity: alkylating agent (R1), amine (R2), and aldehyde

(R3)

•Find balance between diversity (client) and monthly quota (contract, cash

flow)

•Pyramid approach; introduce diversity late:

•Use commercially available diversity points

•Maximize throughput by scale-up of a limited # of labor-intensive

ketoesters

•Randomize diversity points to maximize use of (synthesized) stock

•Keep Mw< 300

Pyrrole-3-Carboxylic Acids Diversity and Logistics

But most importantly:

•Develop a general procedure for expediency, accept lower

yields of outliers

•Avoid chromatography (loss of time, CRO responsible for

cost of materials)

Pyrrole-3-carboxylic Acids Custom Ketoester/Enaminoester Synthesis

Generate ketoester dianion with NaH and n-BuLi; quench with

alkylating agent. 100-300 g Batches prepared; purification by vacuum distillation.

Leaving group X determines outcome!

Pyrrole-3-carboxylic Acids Ketoester/Enaminoester Synthesis

A standard protocol for the synthesis of enaminoesters was developed by simply mixing

and rfx in THF/HOAc; the crude was carried forward:

Some representative examples:

Pyrrole-3-carboxylic Acids Henry reaction/Knoevenagel; synthesis of nitro olefins

Selected examples

Purified by distillation (R3 = alkyl) or crystallization (R3 = aryl)

(Commercial)

Pyrrole-3-carboxylic Acids Production strategy

Most building blocks designed around cheap, commercial ketoesters

Ketoester scale-up

One chemist

Scale-up nitroolefins

Team

} Commercial amine Complete building block

Individual chemist

Future use

Pyrrole-3-carboxylic Acids Deprotection methylesters

Esters resist deprotection by saponification or with hard nucleophiles

Acid-catalyzed hydrolysis leads to unstoppable decarboxylation to “useless” pyrroles

Pyrrole-3-carboxylic Acids Not quite an aromatic system…..

A pyrrole-3-carboxylate behaves as a vinylogous carbamate; resists

nucleophiles such as hydroxyl ion

A pyrrole-3-carboxylic acid, once formed, behaves as an

enamine, is protonated, and immediately decarboxylates

Basic conditions:

Acidic conditions:

Pyrrole-3-carboxylic Acids Sulfur nucleophiles to the rescue

Keinan et al. described the alkoxydecarboxylation of ketoesters

and malonates by treatment with thiols and cesium carbonate

in hot DMF.

Thiophenols are most effective: JOC 51, 1986, 3165

Pyrrole-3-carboxylic Acids Sulfur nucleophiles to the rescue

•Pyrrole-3-carboxylate is a soft leaving group, responds

to soft nucleophiles

•Initial success with thiophenol, but what about the stench?

•How to effectively remove excess thiophenol, a weak acid,

•From the acidic product?

Pyrrole-3-carboxylic acids

Use 4-Aminothiophenol for demethylation

•Commercially available (TCI Japan)

•$40 for 25 g (2002)

•No stench (faint licorice-like smell)

•Non-volatile

•Low-melting solid (a disadvantage)

•Better nucleophile that thiophenol

•Basic aminogroup would allow removal

by acid wash

Pyrrole-3-carboxylic acids Loss of CO2 needs to be controlled

Loss of CO2 during deprotection with a sulfur nucleophile

is promoted by:

•Higher rxn temperature: > 110 oC

•Extended reaction times: > 12 h

•Higher concentration: > 0. 25 M

•Low pH: < 2 @ drop of product from extraction water

Standard conditions during production:

•2 eq. 4-aminothiophenol

•5 eq. K2CO3

•0.15 M in DMF at 100-105 oC

•Monitor with LCMS/ELSD, use in-process judgment

•Drop or extract product from aqueous at pH = 3-4

Graphic Presentation Optimal Conditions

90oC 110oC

No reaction - CO2

Tim

e

- CO2

con

c

- CO2

0.25 M

0.1M

Waste Low conversion

- CO2

pH

2

Temp

Pyrrole-3-carboxylic acids Results from production

•70+ Pyrrole-3-carboxylic acids of 95%+ purity* were

delivered in 8 months (3 FTE’s)

•Deliverables in 5-50+ g amounts

•Observations: •Increase of steric hindrance (R1 or R3) leads to faster decarboxylation. In one

case upon storage at ambient

•“Greasy” substituents allow extraction of pyrrole-3-carboxylic acids from

basic aqueous layer. Acid purified by silica plug

* Purity per LCMS/ESLD x NMR purity (for residual solvents)

Pyrazoles and Isoxazoles, 2003

•The continuation of the collaboration depended on solid proposals

for the rapid synthesis of 30-60 building blocks in 5-50 g amounts

•Building blocks, acids or amines, needed to possess “medical chemistry”

characteristics: ideally low molecular weight heterocycles

•The synthesis needed to demonstrate versatility: introduce

maximum diversity by a general procedure to secure expediency

•Proposed to bank on experience with enaminoesters, starting materials

for pyrazole-3-carboxylic acids and isoxazole-3-carboxylic acids

•Design accepted by Client for production

Azole Template Series; A Need for Consistency and Diversity

•Multiple chemists w/ minimal supervision

•Flexible staffing (easy procedures), influx

Moscow colleagues

•Few early intermediates

•Diverse, commercial building blocks

•Minimize outliers

Diversity

Azole Synthesis Mechanistic Intermezzo

For isoxazoles, the more nucleophilic N-atom of H2NOH attacks

Pyrazoles and Isoxazoles Enaminoesters; acylation

Near quantitative yields over two steps; no need for purification.

Selected examples for commercial acid chlorides:

Pyrazoles and Isoxazoles Pyrazoles were designed by combining:

•R2 = aryl with hydrazine (R3 = H) or methyl hydrazine (R3 = Me)

•R2 = alkyl with substituted hydrazines, R3 = alkyl or aryl

Simple rfx of acylated enaminoester with hydrazine or hydroxylamine in THF

w/ HOAccat provided a high-yielding standard protocol. Two examples:

Specific example proposed by Client

HCl avoids catalyst poisoning

Pyrazoles and Isoxazoles Alternate route, more diversity

An alternative entry to enaminoesters

Access to 5-substituted pyrazole and

isoxazole-4-carboxylic acids

Steric hindrance and lower nucleophilicity require harsher conditions for ring closure

For example:

Pyrazoles and Isoxazoles The simplest isoxazoles

Trifluoromethyl-substituted Pyrazoles Developed by ongoing R&D during production to counter production attrition

and secure # of deliverables

Enaminoesters are cleanly acylated by TFA-anhydride providing access

to 3-trifluoromethylated pyrazoles:

Cu-catalysis provided an unprecedented (2002) regioselective N-arylation which was

confirmed with 2D-NMR

Pyrazole and Isoxazole-3-carboxylic Acids Isolation building blocks, loss of CO2 not an issue

Pyrazole and Isoxazole-3-carboxylic Acids Deliverables

•Delivered 51 building blocks, 20-25 g, >95% purity* in

8 months w/ 2.5 FTE’s

•Slower production pace, but much higher diversity than

pyrrole-3-carboxylic acid production:

additional chemistries per building block

more custom ketoesters

re-synthesis of fluorine-substituted building blocks

to meet post-delivery by Client

* Purity per LCMS/ESLD x NMR purity (for residual solvents)

Palladium Catalyzed Amination (2004) Old Compound, Modern Approach

Denatured ethanol with 3% methanol gave methoxy-substituted quinolines

as major product

100 g negotiated as add. delivery

Old chemistry from 1982 notebook

Reflux w/, and distillation of large excess of piperazine

resulted in condenser and distillation head clogged

with sublimate.

Residual piperazine complicated purification.

Palladium Catalysis New elegant chemistry

Tri-hydrochloride, dihydrate

80% over two steps

Ligand for less active C-Cl bond

A high-yielding palladium-catalyzed amination provides

solution to a practical scale-up problem.

Dihalopyridines in Negishi Chemistry Client’s Proposal (2005):

Dibromopyridine:

•Difficult to make

•Instable

•Poor regioselectivity in palladium catalysis

Even after separation, assignment of regio isomers is not trivial;

a regioselective coupling would avoid this problem.

Alternative Halopyridines for Negishi Reaction; Known, Stable, and Easily Accessible

A literature example hints towards regioselective Negishi

coupling at the 2-position of 2,4-dichloropyridine

Negishi Differentation by NMR

Assignment based on higher

reactivity of C-I bond

NMR fingerprint different from B;

confident assignment of 2-substituted

regioisomer

Careful choice of nucleophiles allowed delivery of two

pure and characterized building blocks.

Acknowledgement

I want to express my gratitude to my

colleagues at CRL that made it a great place to work and achieve.

In particular I want to thank Gene and Gala Vaisberg for showing that the deep

American plunge pays off if you work hard.