"Combichem refers to a technique by which large chemical libraries can be generated by connecting together a diverse set of appropriate chemical building blocks in a systemic way. Combichem encompasses many strategies and processes for rapid synthesis of large, organised collection of compounds called as libraries. When planned intelligently combinatorial methods produce collections of molecularly diverse compounds that can be used for rapidly screening for biological activity.

Combichem has it’s conceptual roots in the immune system. In the body when a new antigen comes in contact with the pre-existing large collection of antibodies, the antibody that binds is selected and reproduced in large numbers to effect the immune response. In a similar way the combichem involves synthesis of a large no. of compounds. (This collection can be chemical mixture, physical mixture, physical mixture, or individual pure components.) This collection can be tested for the ‘biological activity’. Finally the active compound is identified and made in the quantity as the single compound.

Thus combichem has 2 phases :-

1. Making a library.
2. Finding the active compound

In 1984 1^st report on combichem was reported by Mario Geyson as use of peptide synthesis to probe viral Antigens." Geyson demonstrated that peptides can be synthesized in no. of several orders of magnitude greater than by the conventional one at a time methods. The peptides are synthesized on polyethylene rods arranged in microtiter plate format following ninety-six separate peptides to be simultaneously synthesized at the tips of the rods. This is called as ‘Pin-technology i.e. generating the libraries of single compounds.

Another report was from ‘Furka’ who described ‘Split-Pool’ method.

portions of the coupling to each of individual initial reactants. This gives uniform coupling since competition between reactants is eliminated. The individual polymers are combined in a single vessel for washing and deprotection and then divided again into individual portions for the next coupling. The resulting synthetic products exhibit a statistical distribution of sequences. Using this approach, a completed set of possible molecular combinations is rapidly prepared in approximately equimolar amounts.

are enclosed in porous polypropylene containers. The bags are immersed in individual solutions of the appropriate activated amino acids while deprotecting and washings are carried out by mixing all the bags together. The bags are then reseparated for subsequent coupling steps.(Split-pool method). Removal of peptides from the resins affords peptides in soluble form.

This method has the advantage that it affords fully characterizable non-modified, solution. Phase peptides better results than solid-support bound peptides. The method also affords variable quantities of soluble peptides for testing against any target and can be used with unnatural amino acids.

work on the ‘Solid-phase’ synthesis.

Now the whole combichem process can be summoned up in short as;

possible by the method of combichem, to couple or mixture of all the 20 aminoacids to another mixture of 20 aa’s to produce 400 dipeptides. If these newly formed 400are reacted with 20 additional aminoacids ; then 8000 tripeptides are obtained. This process describes the original formulation of combichem. The mixture of 8000 tripeptides would be called as the ‘Combinatorial library ’.

1. First the synthesis of mixture must be fast and efficient because you

need to prepare many variations of the libraries.

2. And the testing must be fast and easy because you need to rapidly test many compounds or mixtures to find active sequences.

Merrifield approach can be given as ;

And this is continued till the desired sequence is obtained.

Byproducts and excess reagents are not bound by the resin and can be removed from resin beads during washing cycles.

reagents are now easily available.

Till 1992 the solid-phase peptide methods rapidly matured but the solid phase methods for organic synthesis faded away.

libraries of the organic molecules. They gave the solid-phase synthesis of 1,4 benzodiazepines.

1. 2 amino benzophenones.
2. N-protected amino acids
3. Alkylating agents.

Solid phase synthesis requires –

1. Selection of building blocks with R₁, R₂, R₃ & R₄.
2. Suitable interconversion method like peptide synthesis.
3. A method for linking one of building blocks to solid support and conditions for cleaving the compound of the support at the end of cycle.

much more rigid with definite positioning by C or N functionalities to give bioactivity.

thus the application of combi. methods to produce small, drug related molecules.

The current list of successes includes benzodiozepines, peptoids, oligocarbamates and other synthetic compounds recently termed diversomers.

1. Solid support : - That should be stable to wide range of organic

solvents and reagents.

2. Linker : - Which connects support to the scaffold or target molecule.

Linker should be cleaved under mild conditions but stable to

proposed reaction conditions needed to build desired product.

1. Scaffold or target molecule : -

1. Kieselguhr – polyacrylamide composite :

It consists of polyacrylamide get trapped in porous structure of kieselguhar.

2. Polystyrene – polyacrylamide composite.

Composite of crosslinked polystyrene.

3. Polystyrene – polyethylene glycol composite : -

rely heavily on the formation of an ester or amide bond.

It is seen that many of the methods require harsh acid which makes the automation of simple synthesis difficult. So the mild conditions need to be developed.

In the early 1960’s the photactive grs. were used which upon the irradiation in the solution release the active groups.

Eg :

Photocleavable linkers.

Once the support and linker have been choosen it is possible to begin to plan the synthesis of library.

The use of solid support for the synthesis of libraries of organic compounds has the primary advantage of giving one the ability to quickly separate the product from soluble components of reaction mixture. Supports stability under planned reaction conditions must be examined and standardised. Once a support has been choosen, one must then look at the type of support . One must make certain that conditions necessary to cleave a linker is compatible with those of target compound. Once the synthesis of target compound begins one must be able to determine the extent to which reaction have gone to completion and then modify such conditions accordingly.

At present current challenge to organic chemist is to quickly and efficiently create libraries that are more sophisticated in the types of bonds created without dramatically sacrificing yeild or purity.

1. Poly functionalised core molecule :-

pharmaceutically valuable chemicals would produce large libraries of small organic molecules with a drug related structure.

organic molecules is given in figure below,

Core molecule Building blocks molecular library

Combine a rigid core molecule having multiple reactive sites with a mixture of building blocks to produce random mixture as polyfunctionalised structures.

Eg. Core molecule : -

Equimolar mixtures of amines A-Z to produce tetra substituted cubane compounds A , A , A , A , through Z , Z , Z .

This method has several advantages : -

1. In a single step cubane core 1 and 26 Amines A-Z would produce theoretically 38 , 701 different cubane tetramides.
2. These compounds are not on a solid support but in solution. This gives assaying of the compounds without worry of the solid support giving artifactual results.
3. The molecules have rigid carbon backbone as given by core molecule. This scaffold determines basic shape of the compounds.

down. It required chemists to think not in terms of synthesizing single,

well-characterized compounds, but in terms of simultaneously synthesizing large populations of compounds.

" Technology such as combinatorial chemistry and high-throughput screening generate masses of relatively unrefined data-data that are certainly less refined than what chemists produced in the past."

From the drug discovery perspective the design of large combinatorial libraries is driven by the requirement that individual reaction be highly reliable and versatile, while producing libraries with the highest possible degree of chemical diversity. Individuals steps must be optimized, the compatibility of building blocks must be examined thoroughly and the synthesis must be automated. As a consequence, a significant investment in time and resources must be made before a library can actually be produced.

Combinatorial libraries are created in the laboratory by one of two methods. – split synthesis or parallel synthesis. In split synthesis, compounds are assembled on the surfaces of microparticles or beads. In each step, beads from previous steps are partitioned into several groups and a new building block is added. The different groups of beads are then recombined and separated once again to form new groups. The next building block is added, and the process continues until the desired combinatorial library has been assembled.

" You could create diversity using separate reactions, so the components would have an equal chance to add in to a site , and then by mixing compounds together again you got the diversity you needed."

Libraries resulting from split synthesis are characterized by the phrase "One bead, one compound." Each bead in the library holds multiple copies of a single library member. Split synthesis greatly simplifies the isolation and identification of active agents because beads (and implicitly individual library members) are large enough to be observed visually and separated mechanically.

Parallel synthesis, in which different compounds are synthesized in separate vessels (without remixing). Often in an automated fashion. Unlike split synthesis, which requires a solid support, parallel synthesis can be done either on a solid support or in solution.

A commonly used format for parallel synthesis is the 96 –well microtiter plate.

Split synthesis is used to produce small quantities of a relatively large number of compounds, whereas parallel synthesis yields larger quantities of a relatively small number of compounds. And split synthesis requires that assays be performed on pools of compounds, whereas assays on individual compounds can be run on libraries created by parallel synthesis.

A special case of parallel synthesis is spatially addressable synthesis, pioneered by researchers at Affymax research Institute, Palo Alto, California , In this technique, libraries are synthesized in arrays on microchips, and all the compounds on a chip are assayed simultaneously for binding or activity.

1. Solid-phase synthesis permits use of excesses of reagents to drive reactions to completion, since excess reagents can be washed away from beads very easily afterwards.
2. However, solution-phase synthesis is more versatile because many organic solution-based reactions have not been adapted for solid-phase work.

Janta and coworkers at Scripps recently developed a liquid-phase synthesis procedure that combines some of the advantages of solution-phase synthesis [ Proc. Natl. Acad. Sci USA, 192 , 6419 (1995)] . The procedure involves use of polyethylene glycol monomethyl ether in place of solid-phase beads as a foundation for combinatorial assembly. The polymer is soluble in a variety of aqueous and organic solvents, making it possible to use solution-phase combinatorial synthesis. But the polymer can be precipitated out of solution by crystallization at each stage of the combinatorial process to facilitate purification.

Combinatorial chemistry began with the synthesis of large libraries of biopolymers such as peptides and olignucleotides.

However, peptides and olignucleotides are problematic for drug development because their oral bioavailability is poor and they are degraded rapidly by enzymes. Hence, the focus of combinatorial research has shifted in recent years to libraries of nonpolymeric small molecules having molecular weights of about 500 daltons or less.

Johathaqn A. Ellman and coworkers at the University of California, Berkeley, synthesized the first such library by creating variants of benzodiazepines, a class of compounds that has been a fertile source of successful drugs.

A major challenge of small-molecule combinatorial chemistry has been to adapt conventional solution-phase organic reactions to reactions on solid-phase particles.

Paralleling the increasing use of small-molecule libraries is a trend toward assaying libraries having smaller numbers of components "People seem to be much more comfortable working with smaller mixtures-probably a hundred components or less in a mixture, rather than the mixtures of 105 and 106 compounds per pool that we saw in the early experiments," says Ronald N. Zuckermann, associate director of bioorganic chemistry at Chiron corp. Emeryville, Calif, " The lower the number of compounds, the more confidence you can have in the biological data" because artifacts arise more readily in the screening of large pools of compounds.

In spatially addressable combinatorial synthesis active compounds can be identified by location. But in other forms of combinatorial chemistry identifying hits is not so easy because there’s often too little of each compound present for characterization with traditional analytical chemistry techniques.

1. Hence, many researchers now use some form of tagging or encoding to label compounds in large combinatorial libraries. The first such encoding schemes was proposed in 1992 by Scripps President Richard A. Lerner and molecular biologist Sydney Brenner at the institute. They suggested that a combinatorial library could be encoded with oligonucleotides synthesized in parallel with library compounds and linked to each one. Amplification or decoding of the attached oligonucleotide would serve to identify the small molecule bound to each bead.
2. In 1993, chemistry professor W. Clark Still and coworkers at Columbia University developed a second major type of encoding

scheme, in which chromatographically resolvable organic tags were

used as encoding elements for bead-based combinatorial libraries.

In Still’s technique, inert halogenated aromatic compounds are

used to encode the chemical reaction history experienced by each

bead. These tags are identified by capillary gas chromatography to

reveal the identify of active compounds in the library. Kahne, says

the method "is as good as it gets for identifying hits-a very simple

solution to a very important problem."

3. The most recent development in encoding technology involves the use of radiofrequency tags. Chemistry professor K. C. Nicolaou at Scripps and the University of California, San Diego, together with senior chemist Xiao-Yi Xiao, developed a technique in which memory devices are associated or coated directly with derivatized polymer during combinatorial synthesis. The chips encode relevant information about the synthetic pathway-including not only reagents used, but also reaction conditions such as temperature and pH. The device can then "report" this information to a receiver via radiofrequency transmission. The system will include radiofrequency memory devices in MicroKans, tiny spherical capsules with porous walls that also enclose polymer beads for combinatorial synthesis.
4. A related technique was developed independently by synthetic chemist Edmund J. Moran and coworkers at Ontogen Corp. Carlsbad,. Calif and the University of California,. Los Angeles. This approach differs form the Scripps technique in that reactions data from each stage of combinatorial synthesis are stored in a computer database, rather than being retained in the chip itself. An

identification number stored in the memory of each is a pointer to reaction information in the database.

Planning and performing combinatorial experiments in the laboratory is a complex and potentially tedious process. Hence,"A future trend is going to be greater availability of automation devices, "A lot of solutions are being developed for automating combinatorial split synthesis or multiple parallel synthesis."

1. Ontogen has developed OntoBLOCK an in-house combinatorial chemistry automation system that can produce 1,000 to 2,000 small organic molecules per day by parallel array synthesis. The system includes reaction blocks containing 96 reaction vessels, from which compounds can be transferred directly to standard 96-well microtiter plates for high-throughput screening.
2. Bohdan Automation Inc. , Mundelein. III markets a combinatorial chemistry reaction block that accommodates a wide variety of organic solvents and handles both solid-phase and solution-phase chemistry.
3. A combinatorial chemistry system still in the prototype stage is the Nautilus, a synthetic chemistry workstation being developed by Argonaut Technologies Inc., San Carlos, Calif. The instrument handles a wide range of reagents, with capabilities for temperature control and use of inert atmospheres.

Procedures that have been demonstrated on the Nautilus include a Suzuki coupling (a carbon-carbon bond-forming reaction at elevated temperature using an air-sensitive palladium catalyst), a butyllithium reaction, enolate reactions of the type developed by Ellman and coworkers, and synthesis of a solid-phase druglike molecule.

HTS involves the use of computer controlled robotic workstations and isolated molecules or cells as the drug targets. This enormously enhances the throughput and enables to rapidly identify the likely active molecules which upon further modifications using the combichem may lead to a drug candidate., this not only reduces the time taken in lead identification but also in minimizing the number of animals required for in vivo efficacy evaluation because the candidate drug molecules are subjected to in vivo screening.

To develop the HTS, the biological assay must be robust, cost-effective, user-friendly and safe and also amenability to automation. It should have as few steps as possible.

Biological systems for HTS range from simple, automated dilution devices to complex workstations in which multiple functions are performed by one or more mechanical arms.

An ideal assay is one that can be performed in a single well with no other manipulation except simple injection of samples to be tested. ELISA’S , Fluorescence based assays, high density screening formats on beads, nucleic acid probing and screening assays, as well as the cell based assays have been developed for HTS formats.

HTS simply helps in limiting the number of biologically active samples which need to be further evaluated by using suitable animal models, so to establish their efficacy , thus cutting down both the time required to identify potential active compound and number of animals.

The role of screening in the drug discovery process has undergone phenomenal change in the recent past. Since advent of the combinatorial chemistry has been able to bring in a very large number of comps for biological screening, the conventional methods has not been able to cope up with large number of compounds to be screened. Because of it’s ability to screen a large no. of comps quickly against defined specific targets, HTS has come in as a handy tool to face this challenge. i.e. until 1980’s only 10,000/- comps. could be screened per year. In 1990’s 25,000 comps. per week and now it is possible to screen as many as 1,00,000 comps per day.

This new technology has been able to achieve such speed and efficiency through combination of knowledge generated by molecular biology, structural biology, biochemistry, and immunology on one hand and robotics and computer engineering on the other hand. Since HTS helps in limiting the no. of biological active molecules, which can be further evaluated in suitable animal models, it has helped a great deal in cutting down both time and range of the potential lead comps as well as the number of animals required.

However there are some challenges regarding this system because there are also chances of missing a real active comp. if it fails to bind selected target. So it is important that more than one target could be used for testing the same comp. So that chances of missing an active lead are minimized.

Ellman also believes "there are some interesting opportunities in there of combining combinatorial strategies with computational strategies and structure-based design. The idea is to use information about three-dimensional structures of receptors and enzymes in combination with libraries to rapidly identify high-affinity ligands.

He says, "the real key to making it work is twofold First, you really need to have a good idea – a good basic structure that has a real chance of doing something really interesting and you want to manufacture that idea in as many variants as you can afford to screen .

A second and even greater challenge, says Still is devising novel and effective assays , " You need assays for the property you want that can be run in parallel.

" The amount of molecular diversity and the number of molecules that are going to become available are going to dwarf the present screening capacities." He says " what’s required is more individualized assays and assay miniaturization."

Different groups added to a core structure will affect the reactive of library members to a differential extent, leading to possible failures of key synthetic steps " So one needs to get a handle on how much of each component is being produced and whether you’re actually making all the components in your library."

People are trying to create a small library that would be very diverse that would get you leads to almost anything in the drug area. Some people call this combinatorial chemistry’s Holy grail."

In combinatorial chemistry, he says, " medicinal chemists design the first library of 1,000 to 10,000 compounds that has a good chance of acting on the target they are going after. Then they screen and make a new sublibrary based on the structure-activity relationships they find. I don’t think any medicinal chemist would believe any single library of 10,000 compounds, no matter how carefully chosen, will contain leads for every medical target."

Small substituted heterocylces are an exceptional diverse class of compounds, showing a wide spectrum of biological activity. These include antibacterial, antifungals(thiazolidinones, b-lactams), antihistamines and anti-inflammatory compounds (thiazolidinones), antihypertensives (dihydropyridines, pyrolidines), antiepileptics (hydantoins), anxiolytics and reverse transcriptase inhibitors

(benzodiazepines). The usefulness of heterocycles as scaffolds for library generation has not gone unnoticed. A wide variety of heterocyclic compound libraries have been synthesized by solid phase methods. These include benzodiazepins, pyrrolidines, hydantoins,

1,4-dihydropyridines, isoquinolinones, diketopiperazine, benzylypiperazines, quinolones, dihydro-and tetrhydroisoquinolines. Some solution phase heterocylic libraries have also been generated. Many biologically active compounds have been discovered from these libraries, including a potent tyrosine kinase inhibitor from a benzodiazepin library, a neurokinin-2 receptor antagonist from a diketopiprazine library, and an angiotensin-converting enzyme inhibitor from a pyrrolidine library, to name a few.

Example : - 2

Schreiber suggests that combinatorial molecular recognition could become a fundamental tool for understanding protein function. One of the ultimate goals of the Human Genome Project is to discover the functions of human proteins.

" Virtually all studies of the functions of proteins day involve making mutations in the genes that encode proteins and studying the effects." This genetic approach to studying protein functions is very powerful , but it is very slow and very inefficient. It is going to take centuries to study the function of all the proteins encoded by the human genome this way and that’s simply unacceptable."

In principle this problem could be solved, he says, by using a "chemical genetics approach-where instead of making mutations in the gene encoding the protein, you attack the protein itself by using organic ligands that bind to it." And such ligands can best be identified with combinatorial methods.

Hence, says Schreiber, "Chemical genetics be the way in the future to solve the problem of protein function. There’s big advantage if you do it that way- because the very act of understanding protein function gives you a molecule that actually alters function.

The ultimate usefulness of combinatorial chemistry for drug discovery and other applications remains to be proved. But Lilly’s Kaldor whose group developed by combinatorial means the CNS agent that has advanced to the clinic-is one. "These techniques are more broadly applicable than crystal-structure-guided design methods because you don’t have to have any knowledge of your receptor in order apply them. You can develop a pharmacophore hypothesis much more quickly than you might have otherwise been able to do so.

Lilly’s development of the CNS compound took less than two years from target identification the beginning of clinical trials. This is "very fast." Says Kaldor.

Example - 2

Merck represents the current state of the art of combinatorial chemistry-how you can use it to go from hits to potential clinical candidates."

At Merck, research fellow Scoott C. Berck and Coworkers used a combinatorial approach to identify nonpeptide agonists (activators) for each of five different somatostatin receptor subtypes. Their study "never could have been done so quickly without combinatorial chemistry, " says chemistry professor Ole Hindsgaul of the University of Alberta, Edmonton. Using conventional techniques, "it would have taken 10 years." He surmises.

Somatostatin is an endogenous peptide that plays important physiological roles as an inhibitor of hormones secretion in the pituitary, pancreas, and gastrointestinal tract; as a neurotransmitter in the brain; and as an inhibitor of tumor cell growth. Researchers therefore consider the five distinct human somatostatin receptor subtypes(sst1 through sst5) to b e promising targets for drug discovery.

`"Somatostatin doesn’t distinguish among the five different receptors subtypes it binds affinity to all five." Rohrer says. "So when you observe, a physiological response that’s due to somatostatin, you really don’t know which receptor subtype has mediate that effect" hence the functions of the different receptor subtypes are understood incompletely at best.

The Merck group was interesting in finding somatostatin analogs that could binds selectively to the different receptor subtypes—not only to identify potential drug leads, but also to help them better understand the specific physiological functions of each receptor subtype. Although analogs selective for sst2 had been found, compounds that bound to the other subtypes had not.

`By combinatorial means, Berk, Rohrer ande coworkers were able to identity nonpeptide against for each of the five subtypes. The researcher first searched the compound that turned out to bind with highest affinity to somatostatin was then used as a template for library construction. The Merck team synthesized the libraries on solid support beads by split-and-pool synthesis. They then cleaved the compounds from the beads and screened them as mixtures in solution.

After the subtypes selective compounds had been identified the researchers used them to define physiological roles for some of the receptor subtypes. For example they found out that sst2 & sst5 receptors subtypes regulate growth hormone release in rat pituitary glands.

The Merck combinatorial approach is "one of the better examples to date where mixture screening has produced viable candidates with the desired selectivity profile going in," says research scientist Davoid Mendel of Eli Lilly, Indianapolis. Mendel believes mixture-screening technology has improved recently and that the technique is currently "making a comeback."

Example - 3

The integrated application of structure-based design and combinatorial chemical technologies can produce synergistic improvements in the efficiency of drug discovery. The process begins with the knowledge based design of a library template or scaffold and involves synthesis of small subsets of library members.

The first step is the generation of chemical template or scaffold that can be derivatized at multiple sites using reliable chemical reactions to produce large combinatorial library. This chemical template is designed based on the structure of the target using the same heuristic set of rules for traditional structure based design. The combinatorial library designed so that few thousand to millions of discrete molecules can be produced by reaction of the designed template with appropriate proprietary and commercially available chemical building blocks.

The next step involves the implementation of the automated synthesis and generation of the library. Significant lead time is anticipated before a library can be produced because even the most reliable chemical reactions require optimization if they are to be carried out by a robot, particularly if the reaction are to be implemented with the template attached to a solid support. when the synthesis is optimized and fully automated , thousands to millions of compounds are accessible.

The preceding outlines a new drug-discovery paradigm that integrates structure based design, directed strategies for combinatorial chemical synthesis. Three dimensional structures provide the information required to most efficiently direct the design and optimization of new lead compounds. High throughput automated methods of chemical synthesis produce new classes of lead components and provide for the rapid generation of structure activity data.