EP1015502A1

EP1015502A1 - Formulation

Info

Publication number: EP1015502A1
Application number: EP98951330A
Authority: EP
Inventors: Nicholas William-SmithKline Beecham Pharmac HIRD; Ian-SmithKline Beecham Pharmaceuticals HUGHES; Vincenzo-SmithKline Beecham Pharmaceuticals RE; Nigel Charles-SmithKline Beecham Pharmaceu NORTH
Original assignee: SmithKline Beecham Ltd
Current assignee: SmithKline Beecham Ltd
Priority date: 1997-08-29
Filing date: 1998-08-26
Publication date: 2000-07-05
Also published as: JP2001514266A; WO1999011676A1; GB9718415D0

Abstract

Novel formulations, processes for their preparation and their use in solid phase synthesis.

Description

FORMULATION

The present invention relates to novel formulations, processes for their preparation and their use in solid phase synthesis.

The combinatorial synthetic approach involves the creation of 'compound libraries' consisting of collections of chemical compounds based upon a common template/core structure. The template/core structure will generally have a discrete number of independently variable substituents, each of which can have one of a defined range of values. Preferably, a library is designed so that, for the range of values selected for each of the independently variable substituents, compounds containing substantially all possible permutations of these substituents will be present in the library. A typical library will contain 5 to 10,000, preferably 10 to 10,000, or more compounds.

The use of solid phase methods to facilitate the assembly of such libraries is well known in the art, particularly in the fields of peptide and oligonucleotide synthesis. Solid phase methods are particularly suited to the preparation of libraries as a large excesses of reagents can be employed to drive reactions to completion and that excess can be readily removed. More recently, such methods have also been used for the synthesis of small molecule libraries wherein the template/core structure has a number of functional sites each of which can be reacted in a stepwise fashion with a number of different reagents.

For maximum synthetic efficiency the introduction of different substituents at each functional site should be accomplished as a single step, using a mixture of reagents. A diverse range of substituents can however translate into a diverse range of reactivities for the reagents. Therefore, it is often convenient to adopt the so-called 'split and mix' approach whereby the evolving library is split into a series of parallel aliquots, each containing the same mixture. Each aliquot is then reacted with a single but different reagent, to introduce a further variant, and the new sub-libraries recombined before splitting again, for a further synthetic cycle (Furka et al, 14th Intl. Congress of

Biochemistry, Prague, July 1988; Furka et al, Int. J. Peptide Protein Res., 1991, 37, 487). A complementary approach to creating a library of compounds is to use the parallel synthesis method, whereby the compounds comprising the library are prepared separately and in parallel. The use of solid phase supports is applicable to libraries prepared by the combinatorial 'split and mix' approach or by parallel synthesis. In either approach the template/core structure may be linked to the solid phase support through a linker group. Linker groups are chosen so that they are not only compatible with the conditions to be used for introducing the substituents on the template but will also allow release of the library from the solid phase support once synthesis is complete. Suitable linker groups include those present in Wang and Merrifield resins.

Solid phase supports such as resins are notoriously difficult to handle, e.g. to weigh out, primarily because of their electrostatic properties. Weighing aliquots of resin for solid phase synthesis is particularly time consuming and tedious when a large number of aliquots are involved. One approach taken to overcome this problem is to dispense aliquots of resin from a suspension of the solid support in a solvent mixture having the same density as the solid support (a so-called "isopycnic" suspension or slurry) as described in Int. J. Peptide Protein Res., 1992, 40, 497. This method does, however, suffer from certain shortcomings that limit its use and applicability. In particular, there is no single isopycnic solvent mixture that can be used for all resins, i.e. it is highly resin dependent. There is consequently still a requirement for a more generic, robust, reliable and reproducible means of delivering pre-determined quantities of resin to reaction vessels.

The present invention provides a solid formulation comprising a solid phase support dispersed within an inert matrix.

The solid formulations may be prepared by blending the matrix with a solid phase support and optionally forming the resulting mixture into discrete pieces having a defined shape. The formulation will typically contain 5 to 75% w/w, or more typically 20 to 50% w/w of the solid phase support. The formulation is preferably in the form of discrete pieces having a defined shape, for instance, tablets, discs or spheres. Discrete pieces of the formulation may be prepared, for example, by compression in a suitable die, either manually or by an automated press; by solidification of a melt; or by extrusion. Suitably, a discrete piece of the formulation will contain a unit amount of solid phase support, typically up to 1 g, e.g. 5 to 100 mg of solid phase support. Discrete pieces of the formulation containing different unit amounts may be differentiated by having different shapes and/or sizes. Different shapes may also be used to differentiate between formulations comprising different solid supports. Additionally, colour or impressed markings may be used to differentiate between materials.

The inert matrix, which may comprise organic or inorganic materials, facilitates the delivery of the solid phase support to the reaction vessel and is dissolved and optionally removed prior to synthesis. Consequently the matrix should be soluble either in common organic solvents, such as dichloromethane, methanol, toluene or tetrahydrofuran; or in aqueous media. In addition, the matrix should be chemically inert, in the sense of not being capable of modifying the solid phase support in anyway. It will also be appreciated that the choice of matrix will be influenced by its physical properties, e.g. the ease of formulation and its ability to maintain its shape if it is formed into discrete pieces. The matrix material may comprise a single material, or of a mixture of materials, to confer suitable blending and/or compression characteristics.

Suitably the matrix comprises materials commonly employed as binders or diluents in the field of pharmaceutical formulations. Suitable matrix materials include alginic acid, polyethylene glycols such as polyethylene glycol 6000, hydrogenated vegetable oil, glycerylpalmitostearate, ethyl cellulose, hydroxypropyl cellulose, polymethacrylates and povidone. Preferred matrix materials include polyethylene glycols such as polyethylene glycol 6000. It will also be appreciated that the choice of matrix is not limited to substances that are suitable for human dosing. Suitably, the matrix could be selected from inorganic compounds such as potassium bromide and sodium chloride, or organic compounds such as anthracene and naphthalene.

Suitable solid phase supports are well known in the art and include resin beads, pellets, disks, capillaries, hollow fibers, needles, solid fibers, cellulose beads, pore-glass beads, silica gels, glass particles coated with a hydrophobic polymer such as POLY- HIPE™, TENTAGEL™, etc. Suitable resin beads include polystyrene beads optionally cross-linked with divinylbenzene, cross-linked polystyrene and polyethylene glycol beads, grafted co-poly beads, poly-acrylamide beads, latex beads, and dimethylacrylamide beads optionally cross-linked with N,N-bis-acryloyl ethylene diamine. A range of bead sizes are in common use and are available from commercial suppliers (for instance, ranges of about 38 to 75 μm, 75 to 150 μm, and 150 to 300 μm). The solid phase support may be derivitised e.g. with linker groups and/or template/core structures prior to or after formulation with the inert matrix. The solid phase support may also comprise a scavenger resin, e.g. N-(2-aminoethyl)aminomethyl polystyrene or N-(2-mercaptoethyl)aminomethyl polystyrene which are used for scavenging electrophilic reagents; or an immobilised reagent, e.g. bis-(6-carboxy-HOBt)-N-(2- aminoethyl)aminomethyl polystyrene which is a solid phase catalyst for acylation reactions.

The formulations of the invention are particularly useful for dispensing solid phase supports into vessels, particularly reaction vessels for use in chemical synthesis. Thus according to a further aspect the invention provides a method for dispensing a solid phase support into a vessel, comprising dispensing a solid formulation comprising a solid phase support dispersed within an inert matrix into the vessel. The method according to this aspect of the invention is preferably used to dispense a known quantity of solid phase support into a vessel. The method finds particular application in automated techniques for dispensing solid phase supports into multiple vessels.

The solid formulations of the invention have numerous advantages, in particular the handling and dispensing of solid phase supports into reaction vessels is greatly simplified. The solid formulations have flexibility in terms of their shape and size and hence formulations can easily be adjusted to suit the particular reaction vessel in which they are to be used; a specific example is the dispensing of resin into mesh cans used in library synthesis, e.g. IRORI MICROKANS™, which are low volume and have a limited aperture for the introduction of the solid phase support The solid formulations may be produced and dispensed volumetric ally rather than by weighing, the mixing of the solid phase support with an inert matrix prior to dispensing improves the flow of the solid phase support into a volumetric measure since it reduces or eliminates static build up on the solid phase support.

The invention is illustrated by the following examples:

Example 1: Preparation of discs from polyethylene glycol

FMOC phenylalanine on Wang resin (75 to 150 μm bead size, 0.76 mmol/g loading) (500 mg) was mixed thoroughly with finely milled polyethylene glycol 6000 (1.0 g). Aliquots (0.25 ml) were compressed at 2 tonnes for 15 sec to give opaque white discs. The compression was carried out in a conventional press which is typically used for the formation of KBr discs utilised in infra-red spectroscopy. Example 2: Use of discs in automated synthesis of 4-tert-butylbenzoylphenylalanine using ACT 396 Multiple Peptide Synthesiser (MPS)

Eight discs from Example 1 were individually placed into the ACT 396 reaction block. The wells were then washed according to ACT programme 1 as shown below. The beads all appeared free from polyethylene glycol excipient. The automated synthesis procedure was then carried out using ACT programme 2 as shown below. To each well, trifluoroacetic acid in dichloromethane (1 ml, 1:1 v/v) was added and the reaction block was allowed to stand for 90 minutes. The contents of the wells were drained under vacuum to separate vials, and the residue was washed with dichloromethane (1 ml). The combined filtrate was evaporated using a ZYMARK TURBO VAP™ to give the following product yields and purities.

Example 3: Preparation of discs from naphthalene

In a similar manner to Example 1, approximately equal volumes of FMOC phenylalanine on Wang resin (75 to 150 μm bead size, 0.87 mmol/g loading) (500 mg) mixed thoroughly with finely powdered naphthalene (2 g) were formed into discs at 10 tonnes pressure for 1 minute.

Example 4: Recovery of beads from discs

Four of the discs from Example 3 were separately washed with dichloromethane (3x5 ml) and methanol (3x5 ml) and dried to give bead recoveries of 32.1, 31.6, 33.5 and 37.3 mg (average weight of resin per disc = 33.6 mg). Example 5: Use of discs in automated synthesis of 4-tert-butyIbenzoylphenylalanine using ACT 396 MPS.

In a similar manner to Example 2, five discs from Example 3 each containing approximately 33.6 mg of resin were individually placed into the ACT 396 reaction block, and FMOC phenylalanine on Wang resin (0.87 mmol/g loading) (50 mg) was placed in the sixth well. Washing, automated synthesis and cleavage were performed as described in Example 2 to give slightly yellow oils with the following analytical data. Average yield from discs 1 to 5 = 10.3 mg. Theoretical yield from 33.6 mg resin based on Well 6 is 9.7 mg.

Example 6: Preparation of tablets from polyethylene glycol

Equal weights of Merrifield resin (150 to 300 μm bead size) and finely milled polyethylene glycol 6000 were mixed thoroughly. The mixture was formed into 6 mm diameter tablets using a Manesty F single punch eccentric press. The weights of twelve randomly selected tablets were 104.9, 104.8, 104.5, 104.0, 104.1, 104.6, 104.8, 104.2, 104.8, 104.0, 104.1 and 103.9 mg (average weight of tablets = 104.4 mg). Example 7: Recovery of resin from tablets

Four of the tablets from Example 6 were separately dissolved in dichloromethane (1 ml), filtered, then washed with dichloromethane (5x1 ml). Following drying, the resin recoveries were 52, 51, 51, and 51 mg (average recovery = 51.2 mg, theoretical recovery = 52.2 mg).

ACT Programme 1

1 Dispense System Fluid dcml 1.00ml to Reactionblock[l-8]

2 Mix for 2.00 minutes at 300 rpm(s) 3 Empty Reactionblock for 2.000 minute(s) 4 Repeat from step 1, 2 times

ACT Programme 2

1 Dispense System Fluid dmf2 1.00ml to Reactionblock[l-8]

2 Mix for 30 seconds at 300 rpm(s)

3 Empty Chamberl for 2.000 minute(s) 4 Repeat from step 1, 2 times

5

6 REM Fmoc CLEAVAGE

7 Transfer 1.00ml from monomeri to32[l] (PIPERIDINE) to Reactionblock[l-8] using dmf2 8 Mix for 5.00 minutes at 300 rρm(s)

9 Empty Chamberl for 2.000 minute(s)

10 Transfer 1.00ml from monomeri to32[l] (PIPERIDINE) to Reactionblock[l-8] using dmf2

11 Mix for 5.00 minutes at 300 rpm(s) 12 Wait for 5.000 minute(s)

13 Mix for 5.00 minutes at 300 rpm(s)

14 Empty Chamberl for 2.000 minute(s) 15

16 Dispense System Fluid dmf2 1.00ml to Reactionblock [1-8] 17 Mix for 30 seconds at 300 rpm(s)

18 Empty Chamberl for 2.000 minute(s)

19 Repeat from step 16, 2 times 20

21 Dispense System Fluid dcml 1.00ml to Reactionblock[l-8] 22 Mix for 30 seconds at 300 rpm(s) 23 Empty Chamberl for 2.000 minute(s)

24 Repeat from step 21, 2 times 25

26 REM ADD tBUTYLBENZOYL CHLORIDE 27 Transfer 500ul from monomeri to32[2] (tBuPhCOCl) to Reactionblock[l-8] using dcml

28 Mix for 30 seconds at 300 rρm(s)

29 Transfer 500ul from monomeri to32[3] (TEA) to Reactionblock[l-8] using dcml

30 Mix for 5.00 minutes at 300 rρm(s) 31 Wait for 5.000 minute(s)

32 Repeat from step 30, 12 times 33

34 REM WORK UP

35 Dispense System Fluid dcml 1.00ml to Reactionblock[l-8] 36 Mix for 30 seconds at 300 rpm(s)

37 Empty Chamberl for 2.000 minute(s)

38 Repeat from step 35, 2 times 39

40 Dispense System Fluid dmf2 1.00ml to Reactionblock[l-8] 41 Mix for 30 seconds at 300 rpm(s)

42 Empty Chamberl for 2.000 minute(s)

43 Repeat from step 40, 2 times 44

45 Transfer 1.00ml from monomeri to32[4] (MeOH) to Reactionblock[l-8] using dcml 46 Mix for 30 seconds at 300 rpm(s)

47 Empty Chamberl for 2.000 minute(s)

48 Repeat from step 45, 2 times 49

50 Dispense System Fluid dcml 1.00ml to Reactionblock[l-8] 51 Mix for 30 seconds at 300 rpm(s)

52 Empty Chamberl for 2.000 minute(s)

53 Repeat from step 50, 2 times 54

Claims

CLAMS

1. A solid formulation comprising a solid phase support dispersed within an inert matrix.

2. A formulation according to claim 1 in which the matrix is readily soluble in an organic solvent or aqueous media.

3. A formulation according to claim 1 or 2 in which the matrix is a material used as a matrix for pharmaceutical formulations.

4. A formulation according to claim 1 or 2 in which the matrix is polyethylene glycol, anthracene or naphthalene.

5. A formulation according to claims 1 or 2 in which the matrix is potassium bromide or sodium chloride.

6. A formulation according to any one of the preceding claims in which the solid phase support is a resin.

7. A formulation according to any one of the preceding claims in which the solid phase support is polystyrene beads.

8. A formulation according to any one of the preceding claims in which the formulations are in the form of discrete pieces having a defined shape.

9. A process for the preparation of a formulation according to any one of the preceding claims which comprises blending the matrix and the solid support and optionally forming the resulting mixture into discrete pieces having a defined shape.

10. A method for dispensing a solid phase support into a vessel, comprising dispensing a solid formulation according to any one of claims 1 to 8 into the vessel.