High Throughput Synthesis High Throughput Synthesis Of Analytically Pure Compounds Of Analytically Pure Compounds

Within Flow ReactorsWithin Flow Reactors

Paul WattsThe University of Hull

CPAC Workshop, Rome, March 20th 2006

Why Miniaturise Synthesis?Why Miniaturise Synthesis?• Improving the efficiency of the drug discovery process

• Enhanced methodology for fine chemical synthesis• Process control P. Watts et al., Drug Discovery Today, 2003, 8, 586

Production TechnologyProduction Technology

Scale-up:Re-optimised at each stage Costly and time consuming

Scale-out

Large-Scale Production

Pilot Plant

Lab-Scale

Large-ScaleProduction

Lab-Scale

Scale-out:Numbering-up/replicationCost effective and flexible

Micro ReactorsMicro Reactors• Defined as a series of

interconnecting channels formed in a planar surface

• Channel dimensions of 10-300 µm• Various pumping techniques

available• Hydrodynamic flow• Electroosmotic flow

• Fabricated from polymers, metals, quartz, silicon or glass

• Why glass?• Mechanically strong• Chemically resistant• Optically transparent

D

C

B

A

Tetrahedron, 2002, 58, 4735Angew. Chem. Int. Ed., 2004, 43, 406OPRD, 2004, 8, 422

Micro Reactor ConstructionMicro Reactor Construction• Reactor holders:

• Standard chips are suitable for both EOF and hydrodynamic pumping

• LioniX/ChemTriX

• Electroosmotic flow:• Application of voltage

induced flow

• Hydrodynamic flow:• Syringe pump connected

using standard fittings

• β-Amino ketones are of synthetic importance as they feature in an array of pharmaceutical compounds

Reaction Conditions: 1.0 M in anhydrous MeCN• Applied fields in the range of 100 to 200 V cm-1 (Reaction length = 4.4 cm)

AzaAza--Michael AdditionMichael Addition

Z

ZN

R1

R2A B C

R

R

R1

NH

R2

99.99PyrrolidineAcrylonitrile

100.0PyrrolidineMethyl vinyl ketone

100.0DiethylamineAcrylonitrile

100.0DiethylamineMethyl vinyl ketone

Conversion (%)ProductMichael DonorMichael acceptor

N

O

N

N

N

O

N

N

O

NH

N

O

Unoptimised

Optimised

Synthesis of Synthesis of Dihydropyrazolines Dihydropyrazolines • Pyrazolines are useful as antibacterial, antifungal, insecticidal and antiparasitic agents

Reaction Conditions: 1.0 M in absolute Ethanol • Applied fields in the range of 100 to 200 V cm-1 (Reaction length = 4.4 cm)

A B CO

R R1

N

R R1

R2NNH2N

R2

100.0

100.0

100.0

100.0

Conversion (%)Productα,β−Unsaturated ketone O N NH

O

Br

N

Br

NH

ON NH

O

Br

N

Br

NH

O

N NH

Unoptimised

Optimised

Fine Chemical SynthesisFine Chemical Synthesis• New methodology for fine chemical synthesis

• Enhanced yields of more pure products etc• But always require purification • Generally batch work up required

KnoevenagelKnoevenagel ReactionReaction• Solution phase Knoevenagel reaction• 1:1 Ratio of reagents (0.5 M) in MeCN• EOF

• 100 % conversion• Reaction very ‘atom efficient’

A

B

C

D

O

R R

O

Me2NH

CHOO

R R

O

• BUT product contaminated with base!!• Traditional solvent extraction needed• This clearly reduces the advantages of flow reactors

Functionally Intelligent ReactorsFunctionally Intelligent Reactors

R

R2 H

OR1

R R1

HR2R and R1 = COOCH3, COCH3, CNR2 = Ph, CH3

N

NHSi

+

• Fabricate micro reactors which enable catalysts and/or supported reagents to be spatially positioned

• Quantitative conversion to analytically pure product

Key Result Key Result -- ReproducibilityReproducibility• Supported reagents deteriorate with

time in batch reactions as a result of physical damage and/or loss

• Reagents last longer in micro reactions as they suffer less damage in flow reactors

• Throughput ca. 10 mg/hr/channel

0

20

40

60

80

100

0 5 10 15 20

Time/hr

Con

vers

ion/

%

1s t Us e 2nd Us e 3rd Us e 4th Us e

Run No. Conversion (%)

1 98.3

2 98.5

3 98.3

4 98.3

5 98.4

6 99.2

7 99.1

8 99.1

9 100.0

10 99.6

11 99.3

12 100.0

13 100.0

14 99.2

Mean = 99.1 %, % RSD = 0.65

OPRD, 2004, 8, 942Tetrahedron, 2004, 60, 8421

KnoevenagelKnoevenagel Reactions Reactions More ExamplesMore Examples

Aldehyde Activated Methylene

Conversion(%)

Yield (%)

Benzaldehyde Ethylcyanoacetate 99.98 99.70 4-Bromobenzaldehyde Ethylcyanoacetate 99.96 99.35 Methyl-4-formyl benzoate Ethylcyanoacetate 100.0 99.80 3,5-Dimethoxybenzaldehyde Ethylcyanoacetate 99.86 99.89 4-Benzyloxybenzaldehyde Ethylcyanoacetate 99.99 99.67 Benzaldehyde Malononitrile 99.98 99.40 4-Bromobenzaldehyde Malononitrile 99.89 99.79 Methyl-4-formyl benzoate Malononitrile 100.0 98.84 3,5-Dimethoxybenzaldehyde Malononitrile 99.87 99.17 4-Benzyloxybenzaldehyde Malononitrile 100.0 99.73

C. Wiles et al., Tetrahedron, 2004, 60, 8421

DithianeDithiane SynthesisSynthesis• Acid catalysed reactions

• Acetal synthesis and deprotection• 1 Equivalent of aldehyde and propanedithiol

• ca. 100 % conversion• > 99 % purity

Amberlyst catalyst

H

O

HS SH

R

H

R

SS

• But product still contaminated with residual thiol• Can a scavenger be incorporated to remove this?

DithianeDithiane Synthesis with ScavengerSynthesis with Scavenger

99.9299.99 (0.005)a61.44-Bromobenzaldehyde

99.9199.99 (0.003)61.74-Chlorobenzaldehyde

99.9499.99 (0.002)65.44-Cyanobenzaldehyde

99.9299.99 (0.001)67.92-Furaldehyde

99.8299.99 (0.008)60.4Methyl-4-formyl benzoate

99.9099.99 (0.003)58.92,4-Dihydrobenzaldehyde

99.2299.99 (0.002)61.14-Benzyloxybenzaldehyde

99.97 99.99 (0.001)70.04-Methylbenzaldehyde

99.9799.99 (0.001)63.04-Biphenylcarboxaldehyde

Flow Rate/µl min-1 Yield/%Conversion/%Aldehyde

Amberlyst CuSO4 on silica

H

O

HS SH

R

H

R

SS

Blue CuSO4 turns yellow Self-indicating scavenger

a Numbers in parentheses represent % RSD

Synthesis of Synthesis of ThioketalsThioketalsO

HS SH

MeCN

CH3 CH3

SS

Kinetically less favourable than acetalisation

Flow Reactor Results:

• % RSD = 0.004• n = 6• Flow rate = 40.4 µl min-1

• Conversion = 99.94 %

Batch (24 hrs)

HS SH

O

SS

Flow Reactor

SS

ThioketalisationThioketalisation ResultsResults

R R1

OHS

R

SSSH

A-15, MeCNR1

99.9599.99 (0.004)40.94-Nitroacetophenone

99.9099.99 (0.0004)41.94-Hydroxyacetophenone

99.9399.99 (0.001)40.34-Aminoacetophenone

99.9199.99 (0.001)43.0Cyclopentanone

99.9699.99 (0.001)41.62-Methylcyclohexanone

99.9199.99 (0.001)42.04-Methylacetophenone

99.5799.99 (0.004) a41.5Acetophenone

41.6

42.2

Flow Rate/µl min-1

99.9099.99 (0.005)Butyrophenone

99.6299.99 (0.003)Cyclohexanone

Yield/%Conversion/%Ketone

a Numbers in parentheses represent % RSD

Chemoselective Thioacetalisation Chemoselective Thioacetalisation Question: What would happen if the compound to be protected

contained both a ketone and an aldehyde?

Chemoselective protection can be achieved by using reagents such as boron trifluoride etherate

We proposed that by simply optimising the residence time within the reactor, product selectivity could be achieved• Remove product before second thiolation occurs

O

O

H3C

H

4-AcetylbenzaldehydeO

H3C

S

S

Desired productCH3

S

S

S

S

Stirred Reactor Stirred Reactor vs.vs. Flow reactorFlow reactor

O

S

S

HS SH

H

O

O

O

S

S

S

S

S

S

Stirred Reactor 24 hr Flow reactor (65 µl min-1)

Un-reacted

FlowBatch

Di-Mono-Reactor Type

Using 1 eq. 1,3-propanedithiol

TwoTwo--Step SynthesisStep Synthesis• Spatially position different ‘incompatible’ reagents

(e.g. acid and base)• Model reaction:

N N

H

H3CO OCH3

H

O

H

O

N NBase

H+MeOH

Step One:

Step Two:

MultiMulti--Step Results: Ethyl Step Results: Ethyl cyanoacetatecyanoacetate

N

O

O

HR

H3CO OCH3

N

O

O

R

A-15

Silica-supported piperazine

99.50.0213 g99.990.553,5-Dimethoxybenzaldehyde

99.30.0284 g99.990.894-Methylbenzaldehyde

99.70.0238 g99.990.75Nitrothiophenecarboxaldehyde

99.10.0219 g99.990.484-Benzyloxybenzaldehyde

99.70.0253 g100.00.65Methyl-4-formyl benzoate

99.80.0298 g99.990.842-Naphthaldehyde

99.70.0284 g99.990.844-Cyanobenzaldehyde

99.60.0277 g99.990.654-Chlorobenzaldehyde

99.80.0338 g99.990.804-Bromobenzaldehyde

99.40.0150 g99.990.50Benzaldehyde

Actual Yield(g)

Flow Rate(µl min-1)

Yield(%)

Conversion(%)

Aldehyde

Protection of Alcohols: Protection of Alcohols: TetrahydropyranylTetrahydropyranyl Ethers (THP)Ethers (THP)How are they synthesised?

Reaction Conditions:

• 1.2 to 2.0 eq. 3,4-dihydro-2H-pyran (DHP)• Catalysed by:

• p-Toluenesulfonic acid, pyridinium p-toluenesulfonate, zirconium tetrachloride, bromodimethylsulfonium bromide

• Stirred at room temperature:• Yields 55-97 %

• Purified by column chromatography:• To remove unreacted alcohol, acid catalyst and excess DHP

OHO

H+ O

O

Batch InvestigationBatch Investigation

Reaction Conditions:

1.0 M Benzyl alcohol/3,4-dihydro-2H-pyran in MeCN

• 5 x 10-3 mmol Acid catalyst (5 x 10-3 mol %)Stirred at room temperature

• Analysed every 30 min

Disadvantages:

Incomplete conversion after 24 hrs• Deprotection occurred

O

OH

O

O

OH

MeCN

OO

O

1.442.454.058.752.7Conversion (%)

3210.50.02Time (Hrs)58.7 % Conversion to THP Ether

(30 min)

Synthesis of THP Ethers in a Synthesis of THP Ethers in a Flow ReactorFlow Reactor

OOOHO

SO3H

Reaction conditions:

• 1.0 M Benzyl alcohol and 3,4-dihydro-2H-pyran in MeCN• 5 mg Silica-supported sulfonic acid• 1.00 µl min-1 100% conversion• Products analysed after 10 min runs

100.062.958.0Conversion (%)

1.002.404.50Flow Rate (µl min-1)

167250333Applied Field (V cm-1)

Cleavage of THP ethersCleavage of THP ethersOO

OHH+

MeOH

OO

• Efficient removal of THP ethers• Acid catalysed alcoholysis or hydrolysis

• Equilibrium dependant

Traditional Approach:

• Bismuth triflate, A-15, p-TsOH, Iron sulfate • Reaction times range 5 min to 24 hr

Flow Reaction Conditions:

• 1.0 M Benzyloxy-tetrahydropyran in MeOH• 5 mg of Silica-supported sulfonic acid • 1.00 µl min-1 (300 V cm-1)• The ‘protecting group’ can be removed in vacuo

OH

O OCH3

OxidationsOxidations• Selective oxidation of primary alcohols to aldehydes

Flow reactor

Reaction Conditions:

• Pressure-driven flow• 0.01 M benzyl alcohol in DCM

Can the product selectivity be controlled by varying the residence time?

H

O

OH

O

OH

3 cm

Silica-supported Jones Reagent

Flow Reactor ResultsFlow Reactor ResultsChromatogram PlotFile: c:\saturnws\data\cw\pdra\2nd year\feb 2005\cw438\cw438.9a07-02-05.smsSample: cw438.9a Operator: hpScan Range: 1 - 371 Time Range: 0.00 - 5.97 min. Date: 07/02/05 10:31

50 100 15 0 200 250 3 00m/z

0%

25%

50%

75%

100%

51 65

79

9 1

108

12 8 1 55 167 197

Spe ct 14.642 min. Scan: 2 88 Chan: 1 Ion: 145 us RIC : 1 616863BP 91 (639 137=10 0%) cw43 8.9a07-02-05.sms

1 2 3 4 5minutes

0 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

MCounts RIC all cw4 38.9a07-02-05.sms

Chromatogram PlotFile: c:\... \data\cw\pdra\2nd year\feb 2005\cw438\cw438.19a 09-02-05.smsSample: cw438.19a Operator: hpScan Range: 1 - 373 Time Range: 0.00 - 5.98 min. Date: 09/02/05 11:17

50 100 15 0 200 250 3 00m/z

0%

25%

50%

75%

100%

50

77

105

122

145 209

Spe ct 15.497 min. Scan: 3 43 Chan: 1 Ion: 1152 us RIC: 266603BP 10 5 (51 736=10 0%) cw43 8.19a 09-02-05.SMS

2 3 4 5minutes

0

5 0

10 0

15 0

20 0

25 0

kCounts RIC all cw4 38.19a 09-02-05.SMS

2 .57 1 m in A rea : 3 59 86 5.184 m in A re a: 13 3

5 .4 95 m in A

Chromatogram PlotFile: c:\saturnws\data\cw\pdra\2nd year\feb 2005\cw438\cw438a 11-02-05.smsSample: Operator: Scan Range: 1 - 371 Time Range: 0.00 - 6.00 min. Date: 11/02/05 08:50

50 100 15 0 200 250 3 00m/z

0%

25%

50%

75%

100%

51

77

90

105

12 8 1 5518 3

Spe ct 14.153 min. Scan: 2 57 Chan: 1 Ion: 197 us RIC : 1 298573BP 10 5 (59 4923=1 00%) cw4 38a 11-02-05.SMS

1 2 3 4 5minutes

0 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

MCounts RIC all cw4 38a 11-02-05.SMS

2. 66 9 min A rea : 3 373 42 .72 4 m in A rea : 1 01 002.8 23 m in Are a: 16 90 92.9 16 m in Are a: 10 15 13 .08 2 m in A rea : 1 17 51

4 .15 9 m in Area : 2 60 65 44

5 .45 6 min A

BATCH FAST FLOW SLOW FLOW

1000≤50Benzyl alcohol0100650Benzyl alcohol

Carboxylic AcidAldehydeFlow Rate/µl min-1AlcoholConversion/% (n = 10)

Note: These flow rates are too fast for EOF pumping!!

More Examples: Selective OxidationsMore Examples: Selective Oxidations

• No Chromium residues are released into the environment• No purification required• The use of a co-oxidant enables the supported oxidant to

be recycled

1000504-Chlorobenzyl alcohol

1000504-Bromobenzyl alcohol

1000503,5-Dimethoxybenzyl alcohol

Conversion/%

Carboxylic Acid

1000504-Cyanobenzyl alcohol

01006504-Cyanobenzyl alcohol

01006504-Chlorobenzyl alcohol

01006504-Bromobenzyl alcohol

01006503,5-Dimethoxybenzyl alcohol

AldehydeFlow Rate/µl min-1Alcohol

ConclusionsConclusions• Micro reactors allow the rapid optimisation of reactions

• High-throughput synthesis• Combinatorial and library synthesis

• Immobilised reagents allow the synthesis of analytically pure compounds

• Micro reactors are suitable for a wide range of reactions• Micro reactors generate products in:

• Higher purity• Higher conversion• Higher selectivity

• In situ formation of reagents• In situ purification of products

P. Watts and S. J. Haswell, Chem. Soc. Rev., 2005, 34, 235

Research Workers and Research Workers and CollaboratorsCollaborators

• Mairead Kelly• Gareth Wild• Tamsila Nayyar• Julian Hooper• Linda Woodcock• Haider Al-Lawati• Ben Wahab

• EPSRC• Sanofi-Aventis

• Dr. Charlotte Wiles• Dr. Nikzad Nikbin• Dr. Ping He• Dr. Victoria Ryabova• Dr. Vinod George• Dr. Leanne Marle

• LioniX• Astra Zeneca• Novartis