Term Paper: Various Techniques in Drug Synthesis

Name: Osama Saeed

Roll No: 1284

Batch: Fifteen

Semester: Nine

Subject: Medicinal Chemistry

Subject In charge: Mam Huma Ahmad

FACULTY OF PHARMACY HAJVERY UNIVERSITY,

LAHORE
Various Techniques in drug synthesis
Abstract:
Drug discovery is a process which aims at identifying a compound therapeutically useful in curing and
treating disease. This process involves the identification of candidates, synthesis, characterization,
validation, optimization, screening and assays for therapeutic efficacy. Once a compound has shown its
significance in these investigations, it will initiate the process of drug development earlier to clinical trials.
New drug development process must continue through several stages in order to make a medicine that is
safe, effective, and has approved all regulatory requirements. One overall theme of our article is that the
process is sufficiently long, complex, and expensive so that many biological targets must be considered for
every new medicine ultimately approved for clinical use and new research tools may be needed to
investigate each new target. From initial discovery to a marketable medicine is a long, challenging task. It
takes about 12 - 15 years from discovery to the approved medicine and requires an investment of about US
$1 billion. On an average, a million molecules screened but only a single is explored in late stage clinical
trials and is finally made obtainable for patients. This article provides a brief outline of the processes of new
drug discovery and development
Introduction
Drug discovery is the process through which potential new therapeutic entities are identified, using a
combination of computational, experimental, translational, and clinical models. Despite advances in
biotechnology and understanding of biological systems, drug discovery is still a lengthy, costly, difficult,
and inefficient process with a high attrition rate of new therapeutic discovery. Drug design is the inventive
process of finding new medications based on the knowledge of a biological target. In the most basic sense,
drug design involves the design of molecules that are complementary in shape and charge to the molecular
target with which they interact and bind. Drug design frequently but not necessarily relies on computer
modeling techniques and bioinformatics approaches in the big data era. In addition to small molecules,
biopharmaceuticals and especially therapeutic antibodies are an increasingly important class of drugs and
computational methods for improving the affinity, selectivity, and stability of these protein-based
therapeutics have also gained great advances . Drug development and discovery includes preclinical research
on cell-based and animal models and clinical trials on humans, and finally move forward to the step of
obtaining regulatory approval in order to market the drug. Modern drug discovery involves the identification
of screening hits, medicinal chemistry and optimization of those hits to increase the affinity, selectivity (to
reduce the potential of side effects), efficacy/potency, metabolic stability (to increase the half-life), and oral
bioavailability. Once a compound that fulfills all of these requirements has been identified, it will begin the
process of drug development prior to clinical trials.
Drug discovery process
Drug discovery process basically is a patient oriented science, where researchers strive to improve the
existing drugs or invent a totally new chemical entity, which should be ideally more potent than any existing
drug of a similar category. If not, then at least it should be safer than those existing. This process is a very
time consuming and expensive activity, calling for the expertise of many eminent researchers. It takes nearly
12-14 years of exhaustive research and a huge amount of financial investment for the discovery of a single
drug. Right from the chemical synthesis to its clinical development and finally formulating it to a suitable
form. Failure at any stage would mean a huge loss for the company. Hence, a lot of planning is required
even before the project is underway. Recently, with the use of technology the process is becoming a less
risky business, because of the ability of the computers to predict the possible outcomes. This will surely
reduce the efforts in fruitless directions.
Drug design
 Drug design, sometimes referred to as rational drug design or more simply rational design, is the
inventive process of finding new medications based on the knowledge of a biological target.
 There are two types of drug designing:
1.Ligand based designing.
2.Structure based Designing.
Ligand-based drug design
 Also called indirect drug design.
 “Which relies on knowledge of other molecules that bind to the biological target of interest”.
 These other molecules may be used to derive a pharmacophore*(“a molecular framework that carries the
essential features responsible for a drug’s biological activity”) model that defines the minimum
necessary structural characteristics a molecule must possess in order to bind to the target.
 In other words, a model of the biological target may be built based on the knowledge of what binds to it,
and this model in turn may be used to design new molecular entities that interact with the target.
Example:
 An example of a pharmacophore model of the benzodiazepine binding site on the GABAA receptor.
White sticks represent the carbon atoms of the benzodiazepine diazepam, while green represents carbon
atoms of the non-benzodiazepin. Red and blue sticks are oxygen and nitrogen atoms that are present in
both structures. The red spheres labeled H1 and H2/A3 are, respectively, hydrogen bond donating and
accepting sites in the receptor, while L1, L2, and L3 denote lipophilic binding sites.
Structure base drug designing
 Also called direct drug design.
 which relies on knowledge of the three dimensional structure of the biological target obtained through
methods such as x-ray crystallography or NMR spectroscopy.
 If an experimental structure of a target is not available, it may be possible to create a homology model
of the target based on the experimental structure of a related protein.
 Note: The information about the structural dynamics and electronic properties about ligands increased
with more information concerning 3D structures of biomolecular targets.
 Current methods for structure based drug design can be divided roughly into two categories.
(1)“finding” ligands
 for a given receptor using database search a large number of potential ligand molecules are screened to
find those fitting the binding pocket of the receptor. The key advantage of database searching is that it
saves synthetic effort to obtain new lead compounds.
(2) “building” ligands
 Ligand molecules are built up within the constraints of the binding pocket by assembling small pieces in
a stepwise manner. These pieces can be either individual atoms or molecular fragments. The key
advantage of such a method is that novel structures, not contained in any database, can be suggested.
Requirements of rational drug discovery:
 The traditional methods of drug discovery rely on trial-and-error testing of chemical substances on
cultured cells or animals, and matching the apparent effects to treatments. Due to the complexity of the
drug design process, two terms of interest are still serendipity and bounded rationality.
 Rational drug design begins with a hypothesis that modulation of a specific biological target may have
therapeutic value. In order for a biomolecule to be selected as a drug target, two essential pieces of
information are required.
 The first is evidence that modulation of the target will have therapeutic value.
For example, disease linkage studies that show an association between mutations in the biological target
and certain disease states.
 The second is that the target is "drugable". This means that it is capable of binding to a small molecule
and that its activity can be modulated by the small molecule.
Process of rational drug design
 Once a suitable biomolecule target has been identified, the target is normally cloned and expressed.
 The expressed target is then used to establish a screening assay.
 In addition, the three-dimensional structure of the target may be determined.
 The search for small molecules that bind to the target is begun by screening libraries of potential drug
compounds. This may be done by using the screening assay (a "wet screen").
 If the structure of the target is available, a virtual screen may be performed of candidate drugs.
There are the following steps of structure based drug design:
Choice of a Drug Target
 The choice of a drug target is primarily made on a biological and biochemical basis. The ideal target
macromolecule for structure-based drug design is one that is closely linked to human disease and binds a
small molecule in order to carry out a function. The target molecule usually has a well-defined binding
pocket.
Example:
1.Many good drug targets are proteins.
2.Drug design against RNA targets with well-defined secondary structure like the bacterial ribosome and
portions of the HIV genome, has also been effective.
Literature Review:
The following paragraphs shall discuss the various stages of drug discovery process.
Identification of biological targets
The human body functions normally by the virtue of the biochemical process which go on, producing all the
necessary chemicals required for numerous functions to undergo smoothly within the body. Many of these
processes are regulated by the enzymes and the endogenous effector molecules via their respective receptors.
A diseased state, may hence, be identified by, either the abnormal biochemical functioning or, over or
underproduction of some of the intermediates. Hence , the most important and most common biological
targets for drug discovery are either enzymes regulating the biochemistry or the receptors through which
many hormones and endogenous effectors show their response. For example, inhibition of human
dihydrofolate reductase, by methotrexate, brought under control the growth of tumour in humans (Borsa and
Whitemore, 1969). Similarly, blocking of the betaadrenoceptors in the cardiac muscles was found to reduce
the hypertensive state (Pearson, et al., 1989). Another type of biological targets are nucleic acids. Though
they are rarely targeted as compared to those mentioned above, yet they are important targets. (Overington,
et al., 2006).
Validation of biological targets
Once the target is identified, it becomes absolutely necessary to confirm, that the correct target has been
identified. The use of reliable and suitable animal models and the latest techniques in gene targeting and
expression are all essential to the validation process. (Abuin, et al., 2002). Validation also helps researchers
to identify any secondary target that the drug may bind to, which may lead to any sort of unwanted or
adverse reaction. Ideally the drug candidate should be such that it binds to a single target only, but this
seldom happens. Thus, binding to other targets, apart from the correct target leads to unwanted
pharmacological actions. These cannot be completely avoided. It can be minimized to negligible extent.
(Marton, et al., 1998). G-protein coupled receptors (GCPRs) are the most common and the major targets
where a drug binds. Hence, over 30% of drugs in market are modulators of GPCR. The quantitative
polymerase chain reaction (qPCR) analysis is one of the techniques used to measure the mRNA expression
on the receptor. (Wise, et al., 2002).
Lead structure search:
A lead compound is the one that has basic structural requirements for exhibiting the desired action. This
means that, a lead compound has many structural spaces for further development of the structure, to give a
compound with further enhanced action. Highthroughput screening is a technique, which helps to identify
the lead compound out of the many synthesized compounds or those compounds which are collected from
the natural source. Hence, it becomes utmost important to identify the lead compound, as this forms the
basis for further development of the molecule(s). (Bleicher, et al., 2003). Figure 2 illustrates the design cycle
for lead search. The various other techniques involved in lead identification are virtual screening,

informatics, pharmacaphore mapping, High throughput docking, NMR-based screening and chemical
genetics. (Xue and Bajorath, 2000).

Lead optimization
As soon as the lead structure is identified, the next step is to optimize the same. Here, the chemists in close
collaboration with the pharmacologists will carefully study the structureactivity relationship and will
synthesize such other derivatives, so as to get a compound with the best possible desired activity. The
various other approaches for lead optimization are Structure-Based Drug Design (SBDD), Quantitative
Structure-Activity Relationship (QSAR) and Computer-Assisted Drug Design
(CADD). All such approaches generate a huge amount of data, so as to assist the chemist in optimizing the
lead to the best possible structure, with best possible desired action. These aforementioned approaches shall
be dealt in detail in the later part of the chapter. (Joseph-McCarthy, 1999 & Ooms, 2000). Figure 3
represents the design cycle for lead optimization and drug development.
Preclinical studies:
The main objective of preclinical studies is to ascertain the safety of the newly developed molecule. A newly
developed molecule is never permitted to be tested on the human body, unless supported by a confirmed
data about the pharmacology and toxicology of the molecule which is, based on animal studies is obtained.
This phase, generally deals with elucidating the mode of action the molecule and getting an idea about the
pharmacokinetics (PK) and pharmacodynamics (PD) of the molecule. However, the most important is the
toxicological data obtained from the animal study, which gives the rough estimate about the possible adverse
reactions that may be likely to be seen during the course of the therapy. These are carried out in two stages,
in-vitro studies and in-vivo studies. The in-vitro studies make use of different cell-lines and tissue
preparations. The in-vivo studies are performed on the live animals and are observed the changes in the
animal’s behavior.
Clinical trials:
The next stage after preclinical studies is the clinical studies, actual testing of the molecule in the human
volunteers. This phase allows to assess the safety and efficacy of the new molecule. This phase also allows
to gather information about the toxicological effects in the human body, as seldom the toxicity shown by
animals, cannot be always directly correlated to the humans. Before the start of this stage, the innovator
should file an application, namely, “Investigational New Drug (IND)”, as the FDA approves based on the
preclinical data, the innovator can proceed for clinical studies. This stage consists of three phases, phase 1,
phase 2, phase 3 and the phase 4 studies are carried out after the drug has been launched in to the market.
Phase 1 studies are usually carried out on healthy human volunteers and on a small group of people. This
phase evaluates the safety, tolerability and PK and PD of the new molecule. Phase 2 studies are generally
carried out on a small population with the target disease. In this phase, the drug’s efficacy and safety,
metabolism and PK are evaluated in a diseased human body. Phase 3 studies are extensive and multiple site
studies. This phase, covers a large group of individuals with target disease. This phase basically is a
therapeutic confirmatory phase, as all the parameters studied in the phase 2 of the study are confirmed in this
phase. This phase may take somewhere about 3-6 years to complete. After this phase is successfully
completed, the company files the “NEW DRUG APPLICATION (NDA)”to the FDA. Once the FDA issues
an approval to the company, based on their data compiled from the clinical trials, the drug can be launched
in the market. Phase 4 (Post-Marketing survilience) studies are carried out, after the drug has been launched
into the market. The company continues its monitoring of the drug. The rationale behind this phase is to
check for any new adverse or serious reaction which was not detected in the earlier phases and may be
observed in this phase. If so happens that some serious adverse reaction is observed, the company may
withdraw the drug from the market.
Formulations for clinical studies:
The formulations for clinical studies are usually prepared as capsule dosage form, as it is easy for
formulation and also easy for administration. Apart from this advantage, there is another key factor to be
considered while formulating a trial batch, as the drug itself has not been tested in humans, any untoward
action can be directly ascertained to the drug in the absence of any excipients. Capsules, unlike the tablets
can be formulated without any or minimal excipients. Liquid dosage forms may also be formulated,
provided the drug is water-soluble, for the ease of preparation and water being the safest medium.
Formulations should be properly tested for its stability and must be stable at least for the period the trials are
underway. The other reason for choosing simple formulations is to avoid any time lag, as the process of
trials itself is lengthy. Any more delay, may further lead to the delay in marketing the drug.
Computer-aided drug design
Computers, have found their way in every field of science and technology today. The boon of computers is
that a large number of calculations and observations can be done in no time. Drug discovery and designing is
no exception to this generalization. Drug designing has received a many fold face-lift by the virtue of
computer software dedicated to the designing of ligands and identifying the biological targets. Computer
generated structures serve to be good predictive models for the evaluation of biological activity. A drug
exhibits its action when it binds to its biological target, usually receptors. Receptors are nothing but proteins
with active sites for the binding of ligands. Hence, in order to design a good ligand, it becomes necessary to
know the structure of such receptors and to identify their active sites accurately. The two important aspects
involved in predicting molecular-interactions in computer-aided drug design (CADD) are development of
pharmacophore-based and molecular docking and scoring techniques. Computerized structure of the known
proteins is based on the experimental data present in various literatures and protein data banks. With this, it
is possible to deduce the 3D structure of the all the known proteins with the help of sequence homology
approach. Hence, these hypothetical proteins behave more or less like the real proteins in their native
biological environment (Taft, et al., 2008). Recently, many computer-assisted models are being developed
and several thousand candidates are being screened for various activities using these models. The methods
of choice for this purpose are computer programs that superimpose molecules by a flexible alignment to
derive pharmacophoric patterns and/or quantitative structure-activity relationships, dock molecules to the
surface of a protein 3D structure or to a hypothetical pseudoreceptor, or construct new ligands within a
predefined binding site (Klebe, 1995 & Kubinyi, 1998a). Different molecular property fields, such as
electrostatic, steric, hydrophobic, hydrogen bond acceptor and donor fields, as well as their weighed
combinations, have been used to achieve a fully automated alignment of the molecules. (Mestres, et al.,
1997). The process of docking process involves the prediction of ligand conformation and orientation within
a targeted binding site. Docking is basically performed for accurate structural modelling and correct
prediction of the biological activity.

Molecular modelling and drug design;

Theoretical studies of biological molecules permit the study of the relationships between structure, function
and dynamics at the atomic level. The entire process is about simulation of the biological processes and
quantum mechanical calculation based on the principles of chemistry and physics.
Molecular mechanics- force field (Potential energy function)
Current generation force fields (or potential energy functions) provide a reasonably good compromise
between accuracy and computational efficiency. They are often calibrated to experimental results and
quantum mechanical calculations of small model compounds. Their ability to reproduce physical properties
measurable by experiment is tested; these www.intechopen.com Drug Designing, Discovery and
Development Techniques 27 properties include structural data obtained from x-ray crystallography and
NMR, dynamic data obtained from spectroscopy and inelastic neutron scattering and thermodynamic data.
(MacKerell, et al., 1995). The molecular structures, properties and energies of a molecule are better
understood through the use of the mechanical molecular model. This model involves the development of a
simple molecular mechanics energy equation representing the sum of various energy interaction terms
comprised of bonds, angles, torsions of both bonded and non-bonded atoms. Force fields the model serves as
a simple descriptor for vibrations in molecules. The concept of force fields is now widely employed as one
of the simplest tools in molecular modeling. Force fields are fundamentally important in de novo drug
design programs, in pharmacophore mapping, and represent the “scoring functions” in many docking
programs. As scoring functions, force fields are used to rank “ligand poses” obtained by a docking
algorithm, or in de novo ligand design programs to suggest placement of fragments in the sites in the
enzyme with the highest binding affinity. In all these applications, force fields are mainly used to compute
the interaction energy between the protein and the ligand as pair-wise interaction potentials consisting of van
der Waals and electrostatic interactions, in addition to H-bond energy between the ligand and the enzyme.
Energy minimization methods
The goal of energy minimization is to find a route from an initial conformation to the nearest minimum
energy conformation using the smallest number of calculations possible. NMR and X-ray crystal structures
tend to have high energy interactions like Pauli repulsions. That is because the methods to retrieve molecular
structures are not perfect and especially in x-ray-structures there are crystal contacts, which lead to a
compaction of the molecules. Moreover, hydrogen atoms are added to relatively arbitrary positions near
their neighbors. Thus, there are atoms lying too close together so that the Pauli repulsion outweighs the
dispersion attraction and the energy is raised high above natural energy levels. These high energy
interactions lead to local distortions which result in an unstable simulation. They can be released by
minimizing the energy of the structure before starting a run. The minimization results in a structure with
energy near the lowest possible energy the system can have.

Conformational analysis
Conformational analysis deals with the computation of minimal energy configurations of deformable
molecules and docking involves matching one molecular structure to the receptor site of another molecule
and computing the most energetically favorable 3-D conformation.
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