Tides Las Vegas, May 17-20,2009 The Best of Both Worlds A Case Study in Harnessing Solid- and Liquid-Phase Peptide Synthesis

Transcription

1 Tides Las Vegas, May 17-20,2009 The Best of Both Worlds A Case Study in arnessing Solid- and Liquid-Phase Peptide Synthesis Matthieu Giraud, Ph.D./ Director / R&D Peptides / Lonza

2 LIQUID PASE SLID PASE

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4 Erectile Dysfunction Market Worldwide USA Total 150 MM men with ED 1 30 MM men with ED 1 millions of people Severe Moderate 18 MM Men Seeking Treatment for ED MM Men Seeking Treatment for ED 2 Mild 618,000 new cases added each year Sources: 1 MMAS J Urol, 1994; 2 Chew et al, Int J Impot Res slide 4

5 Bremelanotide Mechanism of Action Bremelanotide Selective erve Signal Visual & Tactile Stimulation GMP X cgmp cgmp Viagra Levitra Cialis PDE-5 Vasodilation & Erection slide 5

6 Bremelanotide PT141 - Structure C slide 6

7 Liquid Phase Strategy & Challenges 14 synthesis steps Two main Fragments C Boc-Asp(Bzl)-is(Dnp)-(D)Phe- 2TFA.Arg-Trp(For)-Lys(Z)-Me slide 7

8 Solid Phase Strategy & Challenges S Full length elongation Cyclization Selective side chain protection Cost of Goods (Excess of reagents, Solvent, ) slide 8

9 Boc-Trp(For)-Lys(Z)-Me Product losses Work-up slide 9

10 TFA. -Trp(For)-Lys(Z)-Me Sticky gum Drying ptimization slide 10

11 Boc-Arg-Trp(For)-Lys(Z)-Me For stability Emulsion Development of an emulsion free and fast extractive work-up process slide 11

12 Factors to Success - Liquid Phase Approach Avoid isolation of all intermediates Long history expertise slide 12

13 SPPS Route Cl CTC 1) Fmoc-Lys(Alloc)-, DIEA, DCM 2) DIEA, Me CTC S CTC cycles peptide lenghtening: 1) 20% piperidine in MP 2) Fmoc-AA- 1) Allyl/Alloc cleavage 2) Cyclisartion 3) Fmoc-le- 4) 20% piperidine in MP 5) Acetylation 6) cleavge S S CTC Ac-le- slide 13

14 Solution to selective Allyl / Aloc Cleavage Catalyst Pd(Ph3)4 Pd(Ac)2 / P(oTol)3 PdCl2(Ph3)4 Scavengers / proton donor C/DIEA Dimedone Morpholine Ac/MM Phenylsilane Me2.B3 (DMAB) Design of Experiments slide 14

15 DoE Approach - Screening Response Surface Robustness Test Factor Screening DoE Design Space ptimization DoE slide 15

16 Individual Coupling ptimization MW = MW = Purity 93A% + Prod. + 2 SM β Ala impurity Prod.-Boc Fmoc-Arg(Pbf)-Trp(Boc)-Lys(Aloc)- slide 16

17 Final Global Deprotection S 1) reaction: TFA solution + Scavenger mixture 2) Precipitation in ether F F F carbamate PI-001-P C: Molecular Weight = Exact Mass =1618 Molecular Formula =C S PI-001-roh C: Molecular Weight = Exact Mass =1138 Molecular Formula =C C2F32 slide 17

18 ptimization Reaction optimization (CAD): First set of experiments (screening) cocktail composition Second set of experiments (optimization) reaction conditions: Temp. / Time. residual solvent from SPPS Anisole TIS slide 18

19 Coefficient plot of the model Investigation: PI-001-roh(sreening corr yield) (MLR) Scaled & Centered Coefficients for Yield PL 2 DCM AI TIS PL*AI PL*TIS 2*TIS DCM*AI % =19 R2=0.946 R2 Adj.=0.893 DF=9 Q2=0.888 RSD= Conf. lev.=0.95 slide 19

20 Influence of TIS and phenol slide 20

21 Influence of TFA and Time Yield slide 21

22 Crude Material before DSP slide 22

23 Factors to Success - SPPS Approach Be innovative in the choice of protecting groups Strong sourcing department Back integration of key starting materials (Facility in China) State of the art cyclization is on resin Use DoE for screening & optimization Availability of tank farm for large volume & railway connection n site solvent recovery expert and assets slide 23

24 Summary 29 Yield Years of expertise slide 24

25 Contact Details Dr. Matthieu Giraud Director Research & Development Peptides Lonza Exclusive Synthesis - E52 Lonza Ltd Valais Work C-3930 Visp, Switzerland phone: fax: mobile: mailto: matthieu.giraud@lonza.com slide 25