[B0002]
 

Mark Meisenbach, Thomas Allmendinger, Ching-Pong Mak

Novartis Pharma AG, Chemical & Analytical Development, CH-4002 Basel, Switzerland.
[email protected]

Received: 27 July 2001 / Uploaded 7 August 2001

Introduction

Research in pharmaceutical industry and academia concentrate on combinatorial chemistry for lead finding and lead optimization. One of the practical approaches is the solid phase supported parallel synthesis to generate libraries for high-throughput screening. The number of hits may increase dramatically and the compounds as potential drugs are entering development. Process chemists are faced with the problem of rapid delivering the first amounts of drug substance necessary for early preclinical and clinical studies. Since the synthesis originated by a solid phase approach, no solution phase procedures for these compounds are available.

To tackle with this problem we were interested in gaining experience to scale up the solid phase syntheses directly. Time, an important factor in drug development, is saved by omitting the search for alternatives and by running solid phase multi step syntheses without isolation and purification of intermediates. Starting from a, b-unsaturated ketones 1 a library of different heterocycles was prepared in research [1].

As an example for scale-up directly on the solid support the synthesis of 4-(2-amino-6-phenylpyrimidin-4-yl)benzamide 10 was chosen to study the following items:

(1) Scale-up phenomena of different solid phase reactions and on-bead analytics

(2) Effect of loading (an equivalent to concentration in solution phase chemistry)

(3) Selection of appropriate equipment

Results and Discussion

(1) Scale up phenomena and on-bead analytics

Due to the fact, that the Rink resin (attached via an ether bond to the Merrifield resin as depicted in 1) is not available on large scale and extremely expensive, we used the so called Fmoc Rink linker 4-{(R,S)-1-[1-(9H-fluoren-9-yl)methoxycarbonylamino]-(2’,4’-dimethoxybenzyl)}-phenoxyacetic acid (3), well established in peptide synthesis and therefore readily available and fairly cheap. 3 was coupled to aminomethyl polystyrene 1.54 mmol/g (2a, AMPS), in a low excess (1.5 equiv.) using standard amid forming conditions (see scheme). The degree of loading was determined by UV ^[2], after the Kaisertest was negative. The Fmoc group was cleaved with 20% diethylamine in DMF and subsequently carboxybenzaldehyde (5) was coupled (diisopropyl carbodiimide, HOBt). Using prolonged reaction times, the excess of building block 5 could be reduced by a factor of 10 compared to the original protocol. The structure of the resulting solid phase supported aldehyde 6 was confirmed by IR spectroscopy showing a strong C=O band at 1710 cm^-1 and the characteristic C-H Band at 2730 cm^-1.
 

For the following Claisen-Schmidt reaction to chalcone (8) the research synthesis used a 20 fold excess of both LiOH*H₂O and acetophenone (7) in dimethoxyethane on a 100 µmol scale (reaction time 16 h). Employing these conditions on 35 mmol scale, no conversion could be obtained after 22 h, as shown by IR spectroscopy. After sample cleavage with 20% TFA in dichloromethane only formylbenzamide (11) could be detected by HPLC.

Suggesting a low solubility of LiOH in DME under dry/aprotic conditions, we added some EtOH which initiated a fast reaction^[3]. Samples cleavage from the resin showed the desired chalconamide, but also 20% Michael adduct 12, confirmed by LC-MS.

A short reaction screening provided considerable improvements: the reduction of the excess of acetophenone (7) and LiOH to 1.5 and 0.5 equivalents respectively, added to aldehyde resin 6a in a solvent mixture of THF and methanol gave complete conversion within one hour, with only 5% of Michael aduct as byproduct. For this Claisen-Schmidt reaction a Raman spectroscopic method was established to monitor the conversion online^[4].

The final reaction steps could be scaled up without any problems: Chalcone 8a and guanidine (liberated from its hydrochloride with sodium ethanolate) are forming pyrimidine 9a by heating in dimethylacetamide (DMA) while bubbling air through the mixture for aromatization. After 16 h and complete conversion, the product was cleaved from the support (20% trifluoro acetic acid in DCM). The filtrate was evaporated and the residue upon recrystallization from ethanol/water gave the desired pure 4-(2-amino-6-phenyl-pyrimidin-4-yl)-benzamide 10 as the trifluoro acetate salt in 56% yield based on supported formylbenzoic acid.

(2) Effect of different loadings

As the solid support carried throughout the synthesis is finally ending up as waste, the ratio of solid support to product formed, can be diminished by increasing the number of attachment sites on the resin^[5]. For the pyrimidine synthesis described, we looked for the effect of this loading quantified as mmol of functionality per g of resin. This was also a reason to switch from the ether-bonded Rink-linker to the acetic acid derivative to vary the loading of the resin easily by using aminomethylated polystyrenes (AMPS, 2) with different functionalization degrees. AMPS is commercially available with loading up to 2.9 mmol/g 2b (Novabiochem, Switzerland). A higher loaded resin was prepared by adapting and optimising the procedure of Adams^[5]. Thus polystyrene cross-linked with 1% divinylbenzene is reacted with N-chloromethyl phthalimide in dichloromethane using iron (III) chloride as the Friedel-Crafts catalyst. By running the reaction at room temperature a colourless resin is obtained. The phthaloyl protection group was removed by means of aminolyses using methylamine in water/THF avoiding the common but toxic hydrazine.

The desired loading can be obtained by adjusting the stoichiometry ratio of PS/chloromethyl phthalimide/FeCl₃. We synthezised a AMPS functioalized up to 4.48 mmol/g (2c), which means that 2 out of 3 phenylrings are aminomethylated, which has been not described so far.

To study the effect of loading we used 3 different resins (1.54, 2.86 and 4.48 mmol/g) and repeated the synthesis of 10. The higher loaded resins (2.86 and 4.48 mmol/g) didn't need any adjustments of conditions found for the less loaded example already described. The quality of the product was comparable and - as expected – the ratio of product to support was increased, as shown in the table.
 

Solid phase synthesis of pyrimidine 10 starting with resins of different loading
 

Aminomethyl polystyrene loading	Scale of synthesis / amount of starting resin	Mass of the resin 9	Yield of 10, crude	Purity of 10 [area% RP-HPLC]	Yield of 10 after re-crystallization (purity RP-HPLC)
1.54 mmol/g 2a	40 mmol / 26 g	49 g	14.9 g 92%	84%	56% (>98%)
2.86 mmol/g 2b	70 mmol / 24.5 g	61.3 g	17.8 g 66%	80%	47% (>97%)
4.48 mmol/g 2c	100 mmol / 22.3 g	74.9 g	31.83 g 82%	86%	55%  (> 98%)

The results shown above indicate that solid phase supported synthesis already optimized in research lab for the production of libraries on small scale can be scaled up quite easily. In principle, reactors for scale-up of solid phase peptide synthesis can be used. As peptide coupling and deprotection reactions are highly optimized to run at room temperature the used suction filter equipment does not have heating or cooling jacket. We therefore designed reactors with temperature adjustments by incorporating sintered filter plates into multiple necked double wall reactors with volumes up to 4 l. The outer compartment can be connected to a heating/cooling circuit (thermostate).
 

Conclusion

• The results show, that a fast scale-up of the synthesis of the desired pyrimidine derivative was possible on the solid support. The research protocols could be used directly with only minor modifications. This saves time, which is one of the most important factors in the early stage of drug development.

• The multistep synthesis can be completed in only 3 days with a purity of the crude product exceeding 80%.
• The high loading of aminomethylated polystyrene shifts the ratio support/product in the desired direction and makes this support attractive for the fast scale up of small molecules.

References

1. E. Felder, A. Marzinzik, J. Org. Chem. 1998, 63, 723.

2. C. D. Chang, M. Waki, M. Ahmad, J. Meienhofer, E. O. Lundell, J. D. Haug, Int. J. Pep. Prot. Res.,1980, 15, 59.
3. C. Chiu, Z. Tang, J. W. Ellingboe, J. Comb. Chem. 1999, 1, 73.
4. R. M. Dyson, A. G. Helg, M. Meisenbach, T. Allmendinger, 11^th International Symposim on Pharmaceutical and Biomedical Analysis, Basle, May 14-18 2000, P 007.
5. S. P. Raillard, G. Ji, A .D. Mann, T. A. Baer, Org. Proc. Res. Dev. 1999, 3, 177.
6. J. H. Adams, R. M. Cook, D. Hudson, V. Jammalamadaka, M. H. Lyttle, M. F. Songster, J. Org. Chem. 1998, 63, 3706.