Drug Discovery Process The identification of a chemical structure that has both the desired potency against a nominated biological target, and also a suitable bioavailability and efficacy in an appropriate animal model of the targeted disease. 1 st phase: Finding a lead compound or structure that has some degree of affinity for the biological target.

2 nd phase: Identifying the drug development candidate by improving the lead’s structure. When receptors and enzymes are therapeutic target, a synthesis process is needed for supplying thousands and millions of compounds rapidly. In these consequences combinatorial chemistry develops.

Combinatorial chemistry Combinatorial chemistry is a technology through which large numbers of structurally distinct molecules may be synthesized in a time and resource effective manner and then be efficiently used for a verity of application. Combinatorial chemistry Combinatorial chemistry is a synthetic strategy which utilizing different technique aims at the rapid synthesis of large collections of compounds.

Combinatorial chemistry offers a way by which many compounds can be synthesized very quickly in parallel to optimize a lead compounds activity in the absence of any binding model. It provides a way of rapidly exploring SAR. A+B  AB  ABC e.g. Compound A and Compound B react to produce AB, further react with C to produce ABC, which is isolated after reaction and purification through crystallization, distillation or chromatography in Orthodox synthesis. C

Where as in combinatorial chemistry every possible of compound A 1 to An with B 1 to Bn can be produced parallelly by solid phase or solution phase synthesis. e.g. a two stage synthesis with 10 starting compounds and 10 different building blocks at each stage would yield 1000 compounds.

Difference between orthodox and parallel synthesis

Design of Combinatorial Synthesis Two general strategies are usually followed: Two general strategies are usually followed: a. a. The sequential attachment of building blocks: Grow in one direction b. b. Non-sequential attachment of building blocks: Grow in different directions from an initial building block (Template)

The reactions used in designing a combinatorial sequence should satisfy certain criteria: The reactions used in designing a combinatorial sequence should satisfy certain criteria: From a bond between the building blocks Specific and high yielding Suitable for use in automated equipment Allow the formation of wide range of structure and all possible stereoisomers Easy availability of building blocks High diversity of building blocks for utilizing all types of bonding Accurately determinable structures of the final products.

General techniques for Combinatorial Synthesis General techniques for Combinatorial Synthesis Solid phase synthesis Solution phase synthesis Difference Involved: Reagents Purification Automation Flexibility Scale-up

Solid Phase Synthesis Combinatorial Synthesis is based on solid phase chemistry. It uses filtration as a separation and purification technique. The easy removal of unwanted substance by a simple filtration is the core of library synthesis. In this synthesis substituted resin beads are used as a solid phase which is actually a gel like matrix of connected polymeric molecules distended by the access of solvent molecules. Fig: Synthesis of dimer XY on resin beads using excess monomer Y and reagent R, which can be easily removed by filtration.

Resins beads Resins beads The use of solid support for organic synthesis requires A cross-linked, insoluble, but solvent swellable polymeric material that is inert to the conditions of synthesis. Some means of linking the substrate to this solid phase that permits selective cleavage of the product from the solid support during synthesis. A synthetic procedure compatible with the linker and the solid.

Preparation of resin beads The aditional and dispersion of an organic phase of monomer and cross linker in an aqueous solution dissolving a free radical initiator in organic phase and rising of temperature starts polymerization and produces small solid spherical resin beads. Size: µm usually.

Cross-linked polystyrene: Cross-linked polystyrene: Gel type polymer due to polymerization of styrene. 1% divinylbenzene acts as cross-linker. Its use is limited for high electronic reagents and above 130 C Fig: The molecular structure of polystyrene, Where X could be any suitable functionally.

Polyamide resins: It is polyacrylamide polymers, usually formed by using N,N- dimethylacrylamide as backbone monomer, N,N- bisacryloylethylenediamine, as cross linker and is functionalized through N-acryloyl-N-Boc-- alaninylhexamethylenediamine.

Controlled pore glass: It is rigid, glass derived bead material, compatible with any type of solvent, stable to aggressive reagents and extrems of pressure and temp. Due to ability of continuous solvent flow through macro pores it is suitable for continuous flow synthesis. Tenta Gel Resin: Consists of polyethylene glycol attached to cross-linked polystyrene through ether link. It is obtained by polymerization of ethylene oxide on cross linked polystyrene. Due to polarity it facilitates the release of products or screening of the beads in an aqueous environment. Fig: Polyethylene glycol chain grafted onto a cross-linked polystyrene.

Magnetic beads: Nitration of polyvinylbenzene and reducing the nitro group with ferrous sulfate hexahydrate result the incorporation of Fe +2 and Fe +3 within the bead. Thus the bead contain 24-32% iron by weight and can be manipulated by bar magnet. Figure

Merrifield solid support peptide synthesis

Parallel synthesis This technique is normally used to prepare combinatorial libraries that consist of separate compounds. It is not suitable for the production of libraries containing thousands to millions of compounds. In parallel synthesis the compounds are prepared in separate reaction vessels but at the same time, that is, in parallel. The array of individual reaction vessels often takes the form of either a grid of wells in a plastic plate or a grid of plastic rods called pins attached to a plastic base plate (Fig. 5.7) that fits into a corresponding set of wells. In the former case the synthesis is carried out on beads placed in the wells whilst in the latter case it takes place on so-called plastic ‘crowns’ pushed on to the tops of the pins, the building blocks being attached to these crowns by linkers similar to those found on the resin beads. Both the well and pin arrays are used in the same general manner; the position of each synthetic pathway in the array and hence the structure of the product of that pathway is usually identified by a grid code.

Figure 5.8 The reaction of amino acids with isocyanates to form hydantoins

The technique of parallel synthesis is best illustrated by means of an example. Consider the general theoretical steps that would be necessary for the preparation of a combinatorial library of hydantoins by the reaction of isocyanates with amino acids (Fig. 5.8) using a 96-well array. At each stage in this synthesis the product would be purified by washing with suitable reagents.

Eight N-protected amino acids (X1, X2_ _ _X8) are placed in the well array so that only one type of amino acid occupies a row, that is, row Awill only contain amino acid X1, row B will only contain amino acid X2, and so on (Fig.5.9a). Beads are added to each well and the array placed in a reaction environment that will join the X compound to the linker of the bead. The amino acids are deprotected by hydrogenolysis and 12 isocyanates (Y1, Y2_ _ _Y8) added to the wells so that each numbered row at right angles to the lettered rows contains only one type of isocyanate. In otherwords, compound Y1 is only added to rowone, compound Y2 is only added to row two, and so on (Fig. 5.9b). The isocyanates are allowed to react to form substituted ureas. Each well is treated with 6M hydrochloric acid and the whole array heated to simultaneously form the hydantoins and release them from the resin. Although it is possible to simultaneously synthesise a total of 96 different hydantoins (Z1–Z26, Fig.5.9c) by this technique, in practice it is likely that some of the reactions will be unsuccessful and a somewhat smaller library of compounds is normally obtained.

Fodor’s method for parallel synthesis In theory almost any solid material can be used as the solid support for parallel combinatorial synthesis. Fodor et al. (1991) produced peptide libraries using a form of parallel synthesis that could be performed on a glass plate. The plate is treated so that its surface is coated with hydrocarbon chains containing a terminal amino group. These amino groups are protected by the UV-labile 6-nitroveratryloxycarbonyl (NVOC) group.

Figure 5.10 A schematic representation of the Fodor approach to parallel synthesis. X represents an NVOCprotected amino group attached to the glass plate. The other letters correspond to the normal code used for amino acids. Each of these amino acids is in its NVOC-protected form

A photolithography mask (M1) is placed over the plate so that only a specific area of the plate can be irradiated with UV light (Fig. 5.10). This results in removal of the NVOC protecting group from the amino groups in the irradiated area. The entire plate is exposed to the first activated NVOC-protected amino acid. However, it will only bond to the amino groups exposed in the irradiated area (Step A). The process is repeated using a new mask (M2) and a second activated NVOC-protected amino acid attached to the exposed amino groups (Step B). This process is repeated using different masks (M3, etc.) until the desired library is obtained, the structure of the peptide occupying a point on the plate depending on the masks used and the activated NVOC-protected amino acid used at each stage in the synthesis. The technique is so precise that it has been reported that each compound occupies an area of about 50 mm × 50 mm. A record of the way in which the masks are used will determine both the order in which the amino acids are added and, as a result, the structures of each of the peptides at specific coordinates on the plate.

Split and Mix Synthesis Also known as : Split synthesis One-bead-one compound synthesis Selectide synthesis Proton mixing synthesis Divide-couple recombine synthesis Originated in peptide synthesis Simple efficient chemistry Long linear sequence of reactions Solid Phase approaches Fig No of reagents=10 No of reactions=steps reagents; 5 10= 50 No of products=reagents 10 5 =10000

Furka’s Spilt and Mix technique

Encoding Method Building Block: Oligonucletide Glycine: CACATG Methionine: ACGGTA

Stille reaction: Here aryl-aryl bonds are formed by palladium catalyzed rxn between resin bound aryl iodides and alkenyl stannanes. Fig. The palladium acetate catalyzed rxn of alkynes with resin bound o-iodoamines is used to synthesize indole. Fig.

Mitsunobu couplings: The Mitsunobu reaction is an organic reaction that converts an alcohol into a variety of functional groups, such as an ester using triphenylphosphine and an azodicarboxylate such as diethyl azodicarboxylate(DEAD) or diisopropyl azodicarboxylate (DIAD). The alcohol undergoes an inversion of stereochemistry. It was discovered by Oyo Mitsunobu (1934–2003).

Heterocyclic synthesis: Benzodiazepines: A range of independently synthesized Fmoc-protected 2-amino benzophenones were linked to the HMP linker on polystyrene resin through a phenolic carboxylic acid residue which were acylated with a set of Fmoc protected α-amino acid fluorides. After deprotection acid catalyzed eyelization gives Benzodiazepines. Further functionalization is achived by N-alkylation of the anilide. Fig

Quinolones: Wang resin is derivavatized with 2,4,5-trifluorobenzoylacetic acid in the first step Activation and addition of cyclopropylamine and formation of the quinolone template was achived by cyclization under tetramethylguanidine catalysis. Nucleophilic substitution with piperazine gives resin bound Ciprofloxacin. Fig.

The Pictet-Spengler reaction: This involves intramolecular reaction between iminium ions and nucleophilic aromatic groups. In the synthesis of carboline structure N-Boc tryptophan act as precursor which is attached to resin and cyclized with aldehyde under acid catalysis. Acetylation and sulfonylation introduce diversity. In this process the yield is 71% grater than individual yield. Fig.

Cycloadditions: A heterocyclic azadiene attached to carboxylated crosslinked polystyrene undergoes cycloaddition with electron-rich acetylenes or alkenes. By losing of nitrogen and aromatization, product 1,2-diazines were generated 16 different derivatives of this structure were produced offering four sites of diversity. Fig.

Ugi reaction: It is a multi component reactions, and useful route combinatorial library products. Ugi rxn is a four component consideration rxn where a carboxylic acid, an amine an aldehyde and an isonitrile substituted α-amide. Fig: Any of four component can attached to the resign bead. 96 compound can be prepared from a set of 12 carboxylic acid, 8 aldehydes, one isonitrile and Rink resin as the amine component. Fig:

1. Carboxylic acid linkers: By neucleophilic displacement of chloride of Merrifield resin with the help of caesium carboxylate salt in DMF. Fig. Can also be done on Wang linker Fig. Benzodiazepines Hydantoins Diketopiperazines

2.Carboxamide linkers: The MBHA (methyl BenzHydryAmine) linker on polystyrene resin beads. Fig. For the synthesis of peptide amides. Rink linkers can be used for synthesis of 1 and 2 sulphonamides. Fig.

3. Alcohol linkers: THP (Tetrahydropyranyl) based alcohol linkers are developed on Merrifield resin by reaction of Merrifield resin with the Na-salt of hydroxymethyldihydropyran. Stirring the THP-linked resin with alcohols results alcohol linkers. Fig. Mercaptoketones.

4. Amine linkers The carbamate linkers are prepared by the reaction of amine with a Polystyrene resin bound chloroformate. The resultant linkers contain amine (may be 1 and 2) Fig.

5. Traceless linkers: 6. Light-cleavable linkers: Nitro-substituted benzydrylamine linker for generation of Carboxamide (e.g. Thiazolidinone) Synthesis of Hydantoin using carbamate linkers.