Enabling Tools in Drug Discovery

Microwave-Assisted Organic Synthesis &

Automated Flash Chromatography

Shahnaz Ghassemi, Ph.D.

Boston, MA05/26/05

2

NH

NO

S

OOH

O

NH2OH

S

O

O

NH2NNF3C

N+

N

R1

R2

N

N

H3CO

H3CO

NH2

N

N

O

O

O

CH 3SO 3H

NH

NO

S

OOH

O

NH2OH

O

O

S

O

O

N

NN

N

OH

Discovery in Chemistry

Enabling Tools

Strategy

NHCH3.HCl

O

O

S

O

O

NHCH3.HCl

N

NN

N

OH

O

O

S

O

O

N

NN

N

OH

Serendipity

N

N

H3CO

H3CO

NH2

N

N

O

O

O

CH3SO3H

Knowledge

3

MAOS as Enabling Tool in Drug Discovery

Growing Interest in MSOS

ShortensReaction Time

ReproducibleResults

Increases ProductPurity

4

MAOS Reaction Optimization Selecting Initial Time and Temperature

• Temperature

• Time

Arrhenius Equation

K = A e–DG/RT

• SolventPolar solvents will heat rapidlyDMF, NMP, DMSO, ROH, H2O

Non polar solvent will heat poorly

Toluene, Dioxane, THF, DCM

• Increase ionic concentration

• Add polar co-solvent

5

Presentation Overview

Sulfamide

• Microwave-Assisted Synthesis

Normal and reversed phase Flash Chromatography

1,3,5-Substituted triazine

• Solid Phase Microwave Assisted Synthesis

• Rapid Purification of Compounds Synthesized by MAOS

Microwave-Assisted Sulfamide Synthesis

7

Sulfamoyl Synthesis

• The sulfamide compounds are noted for theirpotent antibacterial activity

O NH

SO2N

R1 R

IPka comparable

to carboxylic acid ~ 4S

OO

NR1

NR

R3

H

Crop Protection AgentsAnti-cancer

Artificial sweetnersPTB-1b inhibitors may have role in controlling blood sugar in diabetes

8

Literature Procedure of Sulfahydantion

O

NH2

OClH

4-MeOC6H4CHO

NaOAc, NaCNBH3

O

NH

O

OMe

Overnight reaction

One-day reactionfollowed by neutralization and extractions

O

N SO2

NH

MeO

NaOMe,MeOH

Two-day reactionfollowed by extractions and chromatography

O

N

O

OMeSO2NH2

H2NSO2Cl

Fernaando Albericio, Lois M. Bryman, Javier Garcia, Enrique l. Michelotti, Ernesto Nicolas, and Colin M. Tice, J. comb. Chem. 2001, 3, 290-300

Total time for Conventional synthesis: 4 days

9

Modifications in Sulfahydantion Synthesis Procedure

• Microwave irradiation used to shorten reaction time

• Automated flash chromatography used for fast isolation of product

10

Instrumentation for MAOS

11

Microwave-Assisted Sulfahydantion Synthesis

Microwave 2 min.90 ºC

Microwave 3 min.80 ºC

-CNBH3

DIEA, MeOH

O

NH2

OClH

4-MeOC6H4CHO O

NH

O

OMe

O

N

O

OMeSO2NH2

NaOMe / MeOH

Microwave

5 min / 100 ºCO

N SO2

NH

MeO Total time for MAOS: 15 min

H2NSO2ClMicrowave 80 ºC

4 min.

12

Microwave-Assisted Reductive AminationRacemization Study

The extent of racemization in the two-step one pot microwave-assisted reductive amination was studied by comparing 1H-NMR (500 MHz) of the LL and DL diastereomers

+

NHBoc

O

NH

O

Et

LL

NHBoc

O

NH

O

Et

DL

• No racemization was detected

13

Microwave-Assisted Amine Synthesis

O

ONH

NH

OO

O

NHO

O

NH

O

CF3

O

NHO

O

O

NH

O

ClCl

NHO

O

OMe

NH

CF3

O

OBrNHO

ONH

Cl

O

O

Cl

Method A 84% 68%83%79%

75%88%88%89%85%Method A

Method A: 2 eq. 1 M NaCNBH4

Method B 79%74% 85% 63%

73%78%80%84%79%Method B

Method B: 2 eq. Si-CNBH4

Method C: 2 eq. PS-CNBH4

Method B 80%72% 73% 59%

65%81%77%80%80%Method B

14

Sulfahydantion Compounds Isolated yields

NS

BrO ONH

O

67%

N

O

S NH

Cl

Cl

O O

74%

ON

S NHO

O

70%

NS

CF3O ONH

O

87%

O

N

O

SNH

OO

65%

N

ClCl

SOO

NH

O

68%

HNH

O

OO

N

SNH

O

O

79%

HNH

O

OO

N

SNH

O

O

79%

Conventional method of synthesis

4 days

Microwave-assisted organic synthesis

1 hour

15

Microwave-Assisted Acyclic Unsymmetrical Sulfamides Synthesis

SOO

NO

O

R1

NR

R3

R3-OHPh3PDEAD

μW 80 °C1-4 min.

Amine/Aniline

μW 80 °C5 min

SOO

NO

O

R1

NR

H

SCl

OO

NCO DCM0° C

SCl

O O

NH O

OOH

μW 100 °C5 min

Si-Pheyl-SOH3SOO

NR1

NR

R3

H

Total time for MAOS: 15 min

Abdaoui, M.; Dewyter, G.: Toupet, L.: Montero, J.-L. Tettrahedron 2000, 56, 2427-2435.

16

Acyclic Unsymmetrical Sulfamides

* Shahnaz Ghassemi, Kristin Fuchs, Molecular Diversity , vol.9, issue 4, 2005

S OO

NHNN

SO O

NH2NH

SO

O

NH2NH

SO

O

NH2N

SO

O

NH2N

SO O

NH2N

N

SOO

NH N

N

S

OO

NHN

S O

O

NH

NN

# Products % Yield M+ + Na 1H NMR

15 90 209.11 4.19 (s, 2H), 4.2 (s, 2H), 7.257 (m, 6H)

16 75 195.032.48 (s, 2H), 6.93(t, 1H), 7.05 (m, 2H), 7.16 (m, 2H), 9.22 (s, 1H)

17 75 209.053.17 (s, 3H), 5.02 (s, 2H), 7.36 (m, 5H)

18 61 235.63 0.92 (t, 2H), 3.37 (t, 2H), 4.19 (s, 2H), 6.89 (s, 2H), 7.16 (m, 4H)

19 91 264.022.51 (bs, 2H), 3.11 (t, 4H), 3.21 (t, 4H), 6.83 (m, 1H), 6.98 (d, 2H), 7.389 (t, 2H)

20 71 325.382.52 (m, 1H), 2.79 (t, 2H), 3.44 (t, 2H ), 4.20 (d, 2H) 4.29 (s, 2H), 7.33 (m, 9H)

21 92 353.993.12 (t, 4H), 3.06 (t, 4H), 4.19 (s, 2H), 4.68 (s, 1H), 6.832 (m, 3H), 7.22 (m, 7H)

22 89 354.42 1.92 (m, 2H), 2.68 (m, 4H), 3.23 (m, 2H), 3.49 (m,2H), 4.54 (s, 2H),5.68(bs, 1H), 7.1-7.5 (m, 8H)

17

Review in Literature Procedure

R

R1

NH

O

LG

SOO

NH2NH2

R O

R1

NS

NH2

O OLG

Hydrolysis of Isocyanates

SO

O

ClNH

OH

OSO

OCl NH2H OH

O

H2OOH

R R1

N

H

S

Cl

OO

NH O

O CH3 CH3

CH3

SCl

OO

NCO

R

R1

NH

O

LG

NS

N

O O

BocR1

R

H

Sulfamoylchloride

R

O

R1 NS

NH

OO

SO

O

NR1

NR

R3

H

1- Mitsunbu2- Boc-removal

Andrew D. Campbell, Alan M. Birch, AstraZeneca Research and Development, Synlett 2005: 0834-0838

TransamidationCyclitive cleavage

18

One Step Sulfamoylation & Cyclization

NCH3H3CN

CH3 CH3

N

NCH3CH3

N

160 ºC15 Min.

SOO

NH2NH2

N

N

+ Yield 2-65%

O

NHR O CH3

R1

S

O

N NH

O OR

R1

O

N OS NH2

O O

R

R1

S

O

N NH

O OR

R1

Microwave 180 ºC3 Min.

• Alternative non-nucleophilic strong base have examined

Andrew D. Campbell, Alan M. Birch, AstraZeneca Research and Development, Synlett 2005: 0834-0838

Microwave-Assisted Solid Phase Synthesis

20

Growing Interest in Microwave-Assisted Solid Phase Synthesis

Reduces SolvationProblem

significantly Shortens Reaction Time

Reduces Degradationof the Polymer Support

Yu, H. M.; Chen, S. T.; Wang, K. T. J. Org. Chem., 1992, 57, 4781Larhed, M.; Lindeberg, G.; Hallberg, A. Tetrahedron Lett., 1996, 37, 8219Hoel A. M. L.; Nielsen, J. Tetrahedron Lett., 1999, 40, 3941

21

Literature Reported Procedure 1,3,5-Substituted Triazine

Total time: 18 hours

Khersonsky, M. S.; Chang, Y-T., J.Comb. Chem. 2004, 6, 474-477

N

N

N

S

SHR1

NHR2

NH2R2, NMP/ n-BuOH, DIEA120 ºC 3 h

m-CPBA

4 h. room T N

N

N

S

SHR1

NHR2O

O

SHSHN

N

N

ClCl

R1

+THF, DIEA

65 °C 3 hN

N

N

S

SHR1

Cl

μW 80 ºC 10 min

μW, 130 ºC, 10 min.

μW, 100 ºC6 min.

NHR3R4

90 ºC 8 h

NN

NNHR2

R1

NR3

R4

μW, 130 ºC, 10 min.Total time: 18 hours

40 min

PS-thiophenol is used in solid phase preparation of highly pure trisubstituted triazine library

PS SH

22

Enhancing Triazine Purity

Piperidine

TLC: Crude productNN

N N

N

N

H

H

NH

• MAOS+ Flash = Overall 1 hr. • purity >99% isolated yield 64%

Product

Automated Flash Chromatography in Rapid Isolation of Organic

Product

24

Conventional Workup & PurificationOf Reductive Amination Product

1. Solvent is evaporated

2. The residue is suspended in 5% aq NaOH and extracted with EtOAc (x2)

3. Organic extract is dried over MgSO4

4. Concentrated under reduced pressure

5. Crude product is taken up in EtOAc and extracted with 5% HCl (x 2)

6. The combined aqueous extracts is neutralized with solid K2CO3

7. Cooled to <5 ºC in an ice bath, and treated with 50% aq NaOH

8. The aqueous layer was extracted with EtOAc (x 3)

9. The EtOAc extracts is combined, dried over MgSO4 and concentrated

25 2

Flash Workup & Purification

2. Samplet inserted onto Flash cartridge

1. Reaction mixture transferred onto Samplet

3. Cartridge loaded into barrel

4. Automated Flash chromatography was run

Samplet

SP Automated Purification System

26

DIEAMeOH

OMe

HON

OMe

HO

OCNBH+

ONH2O

R.T.overnight

TLC conditionEtOAC / Hexane 4:6

RF = 0.55

RF = 0.23Weak Solvent n-Hexane

Strong Solvent Ethyl acetate

Cartridge FLASH 25+S

Flow rate 25 ml/min

UV Wavelength Collection 254 nm

Monitor 254 nm

Collection Mode Threshold

Calculated: 285.13Found: M+1 285.99M+1+Na 307.95

Product

Flash Purification of Amine

27

C18 Flash Cartridge Performance

Consistent resolution, retentionand same peak shape

NH

N N

NH

O

O

NH2

O2N

O

O

Br

H

O

OO

NH2

OEt

ClH

C18 Flash 12

+ M

After 25 time

injection

28

C18 Flash Purification in Suzuki Reaction

• Water can be used as solvent in microwave-assisted Suzuki reaction– Reaction is completed in minutes

*N. E. Leadbeater and M. Marco, Org. Lett., 2002, 4, 2973

+Pd(OAc)2, Na2CO3

TBAB, H2OMicrowave R

XR

B(OH)2

• The product is isolated through extraction followed by chromatography

29

NH2

Reagents:Phenylboronic Acid (1.3 mmol, 156 mg)Na2CO3 (3.0 mmol, 324 mg)TBAB (1.0 mmol, 328 mg)Pd(OAc)2 (0.004 mmol, 1 mg)4-iodoanaline (1.1 mmol, 237 mg)+ 2.0 mL of waterAll reagents were combined and irradiated by microwave for 2.5 minutes at 160°C. The pure product was isolated by C18 flash chromatography without any work-up. Isolated yield: 91.8%

C18 Flash Purification in Rapid Purification of Suzuki Reaction

Pd(OAc)2

Na2CO3

TBAB, H2OMicrowave 2.5 min.

160 ºC

+INH2 (HO)2B

Method C18 25 S Weak Solvent Water

Sample 5 20 05 a Strong Solvent Methanol

User shahnaz Cartridge FLASH 25+S

Project suzuki Flowrate 25mL/min

Date 2005-May-20 UV Wavelength Collection 254 nm

Gradient Start End Length

Eq 10% 10% 2 CV

1 10% 20% 1 CV

2 20% 100% 8 CV

3 100% 100% 3 CV

NH2

91.8% Isolated yield

30

Microwave-Assisted Friedel-Crafts Alkylation

4-(3,4-dichlorophenyl)-3,4-dihydro-1-(2H)Naphthalenone

O H

Cl

Cl

O

ClCl

+AlCl3

60 °C 6 hours

μW 140 °C 2 min.

88.3% Isolated yield

Conclusion

32

The Real Story of the Turtle and the Rabbit

33

The Real Story of the Turtle and the Rabbit

34

How to Win

?

The Real Story of the Turtle and the Rabbit

35

Secret Weapon!

36

The Real Story of the Turtle and the Rabbit

37

SUMMARY

• MAOS is a versatile tool that speeds up the chemistry development process– Both in solution and solid phase organic synthesis

• Automated Flash Chromatography can eliminates the workup and purification bottleneck

• Simplification and enhancement of drug discovery process

38

Discovery Chemistry Group in Charlottesville

Kristin FuchsRebecca PrevisDr. John Gupton (University of Richmond, VA)