Practise School - Chemistry

Fourth Year B.
Pharm Semester –VII

Practice School – Chemistry & Analysis
Expt. No. Introduction to drug discovery and development Date: Roll No.
1 process. Structure drawing (2D and 3D), protein A038
structure selection and protein preparation.
Aim: Introduction to drug discovery and development process. Structure drawing (2D and 3D),
protein structure selection and protein preparation.
References:
PhD thesis of Dr. Ashish M. Kanhed
ChemAxon (Marvin Sketch) [https://chemaxon.com/products/marvin]
Protein data bank [https://www.rcsb.org/]
UCSF Chimera Software [https://www.cgl.ucsf.edu/chimera/]
Avogadro software [https://avogadro.cc/]
AutoDock
Software: Marvin Sketch, UCSF Chimera, Avogadro, MGL Tools and AutoDock.
Hardware requirement: Hardware with minimum 2 GB ram, Windows / Linux / Mac OS
(AutoDock will not work on Mac OS).
Introduction to drug discovery and development process:

There are so many uneasy and cruel health conditions affecting human beings that are
collectively termed as diseases or disorders. To achieve wellness and comfort over such
conditions one of the methods is to use specific drug molecules which act on the human body
relieving such uneasy conditions with very minimum of other responses. The designing of such
new chemical molecules and making them available to the patients is a very complex, time
consuming and costly process. It may take from 12 to 15 years of time and cost around 350
million dollars on an average for a new drug to be introduced into the market. To minimize the
cost and time factors, rational drug design approach is required.
It is well know that the traditional method of getting a drug with good safety is time
consuming and costly. To minimize the money and time so many evolutionary techniques are
introduced. Computer Aided Drug Design (CADD), also known as rational drug design/
computer aided molecular modeling/in silico drug design, is one such evolutionary technique.
SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Fourth Year B. Pharm Semester –VII
Computer based designing of molecules is robust, reliable and a rapid method that boosts the
process of drug development and also reduces the uncertainty of process, huge random use of
chemicals and of animals for testing the new chemical entities.
Computational chemistry is a branch of chemistry that uses computer simulations in
solving chemical problems and designing newer chemical molecules for the desired biological
targets. CADD involves diverse disciplinary aspects of physics and chemistry. Different
computational chemistry tools fall within the domain of rational drug designing. Recent
technological advances like structure based drug design, ligand based drug design and
availability of large number of chemical and biological databases have made this technique more
useful and interesting. So, the drug design and discovery process is a multidisciplinary team
effort where computational chemists play a central role.
Ligand based drug design (LBDD) (or indirect drug design) involves the concept of
similarity searching, pharmacophore searching and quantitative structure activity relationship
(QSAR). Ligand-based drug design relies on the knowledge of other molecules that bind to the
biological target of interest. These other molecules may be used to derive a
pharmacophore 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. Alternatively, in QSAR a correlation between
calculated properties of the molecules and their experimentally determined biological activities
are derived. These QSAR relationships in turn may be used to predict the activity of new
analogs.
Structure based drug design (SBDD) (or direct drug design) involves the concept of
protein-ligand docking i.e. molecular docking. Structure-based drug design relies on the
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 is possible to create a homology model of the target on the basis of the
experimental structure of a related protein(s). Using the structure of the biological target,
candidate drugs that are predicted to bind with high affinity and selectivity to the target may be
designed using interactive graphics and the intuition of a medicinal chemist.

Molecular Docking
In the field of molecular modeling, docking is a method which predicts the preferred
orientation of one molecule to a second one when bound to each other to form a
stable complex. Knowledge of the preferred orientation in turn may be used to predict the
strength of association or binding affinity between the two molecules using, for example, scoring
functions. Docking is frequently used to predict the binding orientation of small
molecule drug candidates to their protein targets in order to predict the affinity and activity of the
small molecule drugs. Hence docking plays an important role in the rational design of drugs.
To perform a docking screen, the first requirement is the structure of a protein of interest.
Usually the structure has been determined using biophysical techniques such as X-ray
crystallography or NMR spectroscopy. This protein structure and a database of potential ligands
serve as the inputs to a docking program. The success of a docking program depends on two
components- the search algorithm and the scoring function.
Search algorithm is nothing but searching the conformational space for docking.
The search space in theory consists of all possible orientations and conformations of the protein
paired with the ligand. However in practice with current computational resources, it is
impossible to exhaustively explore the search space—this would involve enumerating all
possible distortions of each molecule (molecules are dynamic and exist in an ensemble of
conformational states) and all possible rotational and translational orientations of the ligand
relative to the protein at a given level of granularity. Most docking programs utilize flexible
ligands, and attempt to model a partially flexible protein receptor. Each "snapshot" of the pair is
referred to as a pose. A variety of conformational search strategies have been applied to the
ligand and to the receptor. These include: Systematic or stochastic torsion which searches
conformations about rotatable bonds; Molecular dynamics simulations and Genetic algorithms.
The scoring function (force field) takes a pose as input and returns a number indicating the
likelihood that the pose represents a favorable binding interaction. Scoring functions are physics-
based molecular mechanics force fields that estimate the energy of the pose; a low (negative)
energy indicates a stable system and thus a likely binding interaction.

Pharmacophore modeling
As per the IUPAC (1998) “A pharmacophore is the ensemble of steric and electronic
features that is necessary to ensure the optimal supramolecular interactions with a specific
biological target structure and to trigger (or to block) its biological response.”
In medicinal chemistry most of the time this term is used incorrectly. A pharmacophore
never represent a real molecule/skeleton such as flavones, steroids etc or never present a real
association of functional groups like hydroxyl, amine, guanidines etc, but it is only a summarized
concept that accounts for the common molecular interaction capacities of a group of compounds
towards their target structure. Pharmacophore modeling helps to identify a set of common
features that interacts with a set of complementary sites on the biological target. Generally these
features are, hydrogen bond donors, hydrogen bond acceptors, negatively and positively charged
groups and hydrophobic regions. Pharmacophore concept is closely related with the concept of
bioisosterism. In addition, in the 3D pharmacophore methods the spatial relationship between the
pharmacophoric features is also precise. These pharmacophoric features may be positioned on
the ligand itself or may be presumed as counter features to be located in the receptor.
In general the process of pharmacophore model development involves five steps.

Selection of data set as training set should include the structurally diverse set of molecules. Also
the data set should include both active and inactive compounds in order to train the model to
discriminate between active and inactive compounds. The second step involves the development
of low energy conformations that are likely to contain the biologically active conformations of
individual ligands. The alignment of different conformations generated for ligands either
considering atom to atom pairing or feature based pairing is the next step for the pharmacophore
model development. Here a set of conformations (one for each ligand) which show good fitting
or alignment is presumed to be the most active conformation. Next step is abstraction or
representation of the molecules with the common identified features such as aromatic ring,
hydrophobic feature, hydrogen bond donor/acceptor etc. And the final step includes the
validation of the developed model. One of the basic validation steps is that the model should
differentiate the molecules with high and low biological activity. One can also validate the

generated pharmacophore model by comparing the mapped features with the active site of the
receptor under study, if the receptor 3D structure is available. It is also useful to compare the
features of the pharmacophore with the features obtained from contours of nD-QSAR.
Apart from this traditional concept of pharmacophore, one can generate receptor
dependent pharmacophore i.e. mapping of pharmacophore on the receptor active site.
Quantitative Structure Activity Relationship
Quantitative structure activity relationship (QSAR) or quantitative structure property

relationship (QSPR), is the process in which chemical structure is quantitatively correlated with
some well defined process, such as biological activity or chemical reactivity. When
physicochemical properties or structures are expressed with numbers, one can form a
mathematical relation between the structure and the activity. The mathematical expression can
then be used to predict the biological response of other chemical structures. QSAR is based on
the assumption that there is an underlying relationship between the molecular structure and
biological activity. On this assumption QSAR attempts to establish a correlation between various
molecular properties of a set of molecules with their experimentally determined biological
activity. There are two main objectives for the development of QSAR:
Development of predictive and robust QSAR, with a specified chemical domain, for the
prediction of activity of untested molecules.
It acts as an informative tool by extracting significant patterns in descriptors related to the

measured biological activity leading to understanding the mechanism of the biological
activity. This could help in the designing of novel molecules with improved activity profile.
A general mathematical equation for QSAR is:
Activity = f (physicochemical and/or structural properties)

Structure Drawing (2D & 3D):

Chemical structure drawing is the essential step in in-silico drug design. Drawing any
structure in its correct form (skeleton, stereochemistry etc) helps to generate correct physic-
chemical properties and hence help to generate accurate drug discovery models. These developed
models will help in designing of new chemical entities.
ChemAxon, free for academic, tool is very useful for drawing the structures and
calculating the properties of chemical molecules. To download the Marvin sketch product of
ChemAxon, go to https://chemaxon.com/academic-license and create a account for free
academic license. To register and download the product, go to above link and follow the steps.
Install the software and activate using license received in your ChemAxon account.
Image: Working area of Marvin Sketch

Assignment:
1. Using the tools given in Marvin sketch, draw the following structures and calculate the
said properties. Save the structure in *.mol2 format. Copy the drawn structure using
snipping tool and paste here along with the said properties in table format.
a. Ibuprofen
b. Levocetirizine
c. Ciprofloxacin
Calculate i) elemental properties; ii) molecular formula; iii) pKa; iv) logP; v) determine
IUPAC name.
2. Draw chemical reaction (synthesis of paracetamol from link:
https://en.wikipedia.org/wiki/Paracetamol#Green_synthesis) using Marvin sketch and
copy using “copy as” option and paste here.
(Mention all the answers below in Observations and Results section).
Observations and Results:

a) Ibuprofen

1. Elemental Analysis- C (75.698%), H(8.80%), O(15.51%), Atom count= 13, Mass

spectrum- 206: 1.00 207: 0.14 208: 0.01
2. Molecular Weight- 206.285
3. Molecular weight- C13 H18 O2
4. pKa- 15.76
5. log P- 3.84
6. IUPAC Formula- 2-[4-(2-methylpropyl)phenyl] propanoic acid
b) Levocetrizine
1. Elemental Analysis- C (64.868%), H(6.488%), O(9.128%) N(7.208) O(12.348), Atom

count= 52, Mass spectrum- 388: 1.00 389: 0.24 390: 0.35 391: 0.08 392: 0.01
3. Molecular weight- C21 H25 Cl N2 O3
4. pKa- N- 1.55, 7.42, OH- 3.59
5. log P- 3.58
6. IUPAC Formula- 2-(2-{4-[(R)-(4- chlorophenyl) (phenyl)methyl] piperazin-1-yl}
ethoxy} acetic acid
c) Ciprofloxacin

1. Elemental Analysis- C (61.62%), H(5.488%), O(14.498%) N(12.68) F(5.73), Atom

count= 42, Mass spectrum- 331: 1.00 332: 0.20 333: 0.02
3. Molecular weight- C17 H18 F N3 O3
4. pKa- N- 0.22, NH- 8.77, OH- 5.56
5. log P- 1.55
6. IUPAC Formula- 1- cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)- 1,4-dihydoquinoline-
3-carboxylic acid
Synthesis of Paracetamol

Protein structure selection and protein preparation:
Selection of a 3D protein structure from rcsb protein data bank site is very crucial step in
structure / target based drug design.
In search option, type name of specific target. Individual structure will be displayed with
multiple information like title, resolution, PDB accession code (four digit code), method of
development of structure (either X-ray or NMR), DOI etc. Here lower the resolution value, better
is the protein structure in terms of correctness of atoms, sequence and structure.
Structures developed using X-ray methodology is more accurate as compared to
structures developed using NMR technique. To download the protein structures go to specific
PDB structure in RCSB (for e.g. type 1ACJ in search area and press enter). Click on download
files and download PDB format.

The downloaded protein structure further needs some corrections and fixations along with
addition of charges. This process is called as protein preparation. To carry out protein
preparation in AutoDock Tools, follow the following steps:
1. Go to select- Select from string- click chain list- Click 3v99b to delete it
2. Edit- delete water, select- select from string- choose ligand from residue sets, edit- delete
non polar hydrogens
3. Edit- Charges- calculate gasteogar values
Take screenshot of protein structure before protein preparation (paste here). After protein
preparation, save it as PDB, then open in Chimera and prepare good image and paste that image
here in Observations and Results.
Observations and Results:

Before Treatment

After Treatment

Practical marks:
Attendance Performance Viva Total Signature of

(Out of 02 Marks) (Out of 05 Marks) (Out of 03 Marks) ( Out of 10 Marks) Faculty

Expt. No. Physicochemical property analysis and ADMET Date: Roll No.
2 study A038
Aim: Physicochemical property analysis and ADMET study.
References:
ChemAxon (Marvin Sketch) [https://chemaxon.com/products/marvin]
Chemicalize online server [https://chemicalize.com/welcome]
Swiss ADME online server [http://www.swissadme.ch/index.php;
https://www.nature.com/articles/srep42717]
Software: Chemicalize online server, Swiss ADME online server

Hardware requirement: Hardware with minimum 2 GB ram, Windows / Linux / Mac OS with
active internet connection.
Introduction:
To be effective as a drug, a potent molecule must reach its target in the body in sufficient
concentration, and stay there in a bioactive form long enough for the expected biologic events to
occur. Drug development involves assessment of absorption, distribution, metabolism and
excretion (ADME) increasingly earlier in the discovery process, at a stage when considered
compounds are numerous but access to the physical samples is limited. In that context, computer
models constitute valid alternatives to experiments.
Many online as well as paid software are available for this purpose. Chemicalize is one of
such tool from ChemAxon. This tool is free for calculation of various physicochemical
properties of molecules with heavy atom count 12 or less. The tool is not free for the evaluation
of molecules with heavy atom count more than 12. One can draw a structure using Marvin plugin
provided in Chemicalize tool and submit it for prediction of various physicochemical properties.
The SwissADME web tool is freely accessible at http://www.swissadme.ch and meant for
user-friendly submission and easy analysis of the results, also for nonexpert in CADD.
Compared to the state-of-the art of free web-based tools for ADME and pharmacokinetics (e.g.
pk-CSM and admetSAR) and apart from unique access to proficient methods (e.g. iLOGP or the

BOILED-Egg), SwissADME strong points are, non-exhaustively: different input methods,

computation for multiple molecules, and the possibility to display, save and share results per
individual molecule or through global intuitive and interactive graphs. Finally, SwissADME is
integrated in the SwissDrugDesign workspace. One-click interoperability gives access to various
CADD tools developed by the Molecular Modeling Group of the SIB (Swiss Institute of
Bioinformatics), e.g. ligand-based virtual screening (SwissSimilarity), biotarget prediction
(SwissTargetPrediction), molecular docking (SwissDock), bioisosteric design
(SwissBioisostere), or molecular mechanics (SwissParam).
Following steps are the pictorial explanation of SwissADME web tool.
SwissADME submission page. The actual input is a list of SMILES, which contains one
molecule per line with an optional name separated by a space. Molecules can be directly pasted
or typed in SMILES format, or inserted through the molecular sketcher.
Computed parameter values are grouped in the different sections of the one-panel-par-molecule
output (Physicochemical Properties, Lipophilicity, Pharmacokinetics, Drug-likeness and
Medicinal Chemistry). The panel is headed by the molecule name and an up-arrow button to
scroll to the top of the page. The molecule is first described by its chemical structure and

canonical SMILES together with the Bioavailability Radar (see Fig. below). Contextual help can
be displayed by leaving the mouse over the radar or different question mark icons next to some
parameters.
The Bioavailability Radar enables a first glance at the drug-likeness of a molecule. The pink
area represents the optimal range for each properties (lipophilicity: XLOGP3 between − 0.7 and
+ 5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å2, solubility:
log S not higher than 6, saturation: fraction of carbons in the sp3 hybridization not less than 0.25,
and flexibility: no more than 9 rotatable bonds.

Assignment:
1. Using the Chemicalize tool determine physicochemical properties of following
compounds. Convert the generated data in pdf format and attach with the module.
a. Ibuprofen
b. Metformin
c. Acetyl salicylic acid
2. Using the Chemicalize tool determine physicochemical properties of following
compounds. Carry out similarity screening using SwissSimilarity option and submit the
report in pdf format with module.
a. Ciprofloxacin
b. Diclofenac
Observations and Results: Combine file with generated report as single pdf for submission.
1. a.
Chemicalize - Ibuprofen.pdf
b.
Chemicalize -Metformin.pdf
c.
Chemicalize -Acetyl Salicylic Acid.pdf

2.a.
Chemicalize -Ciprofloxacin.pdf
b.
Chemicalize - Diclofenac.pdf
Ciprofloxacin

Diclofenac
Practical marks:

Expt. No. Date: Roll No.

3 Molecular docking study and result analysis A038
Aim: Molecular docking study and result analysis.
References:
▪ PhD thesis of Dr. Ashish M. Kanhed
▪ ChemAxon (Marvin Sketch) [https://chemaxon.com/products/marvin]
▪ Protein data bank [https://www.rcsb.org/]
▪ UCSF Chimera Software [https://www.cgl.ucsf.edu/chimera/]
▪ Avogadro software [https://avogadro.cc/]
▪ AutoDock
Introduction:
In the field of molecular modeling, docking is a method which predicts the preferred orientation
of one molecule to a second one when bound to each other to form a stable complex. Knowledge
of the preferred orientation in turn may be used to predict the strength of association or binding
affinity between the two molecules using, for example, scoring functions. Docking is frequently
used to predict the binding orientation of small molecule drug candidates to their protein targets in
order to predict the affinity and activity of the small molecule drugs. Hence docking plays an
important role in the rational design of drugs.
To perform a docking screen, the first requirement is the structure of a protein of interest. Usually
the structure has been determined using biophysical techniques such as X-ray crystallography
or NMR spectroscopy. This protein structure and a database of potential ligands serve as the inputs
to a docking program. The success of a docking program depends on two components- the search
algorithm and the scoring function.

Search algorithm is nothing but searching the conformational space for docking. The search
space in theory consists of all possible orientations and conformations of the protein paired with
the ligand. However in practice with current computational resources, it is impossible to
exhaustively explore the search space—this would involve enumerating all possible distortions of
each molecule (molecules are dynamic and exist in an ensemble of conformational states) and all
possible rotational and translational orientations of the ligand relative to the protein at a given level
of granularity. Most docking programs utilize flexible ligands, and attempt to model a partially
flexible protein receptor. Each "snapshot" of the pair is referred to as a pose. A variety of
conformational search strategies have been applied to the ligand and to the receptor. These include:
Systematic or stochastic torsion which searches conformations about rotatable bonds; Molecular
dynamics simulations and Genetic algorithms. The scoring function (force field) takes a pose as
input and returns a number indicating the likelihood that the pose represents a favorable binding
interaction. Scoring functions are physics-based molecular mechanics force fields that estimate the
energy of the pose; a low (negative) energy indicates a stable system and thus a likely binding
interaction.
Procedure:
1. Import the protein 1acj from the web and then delete water molecules (edit delete water),
add hydrogens (edit add hydrogens) and then check for missing residues and repair them.
Following this, we have to add charges (edit charges compute gasteiger). With this, our
protein is ready for further docking process.
2. Now, go to the grid and choose the macromolecule and save it in pdbqt format. Once done,
insert a grid box to cover the ligand. Change the coordinates to cover the ligand and save the
coordinates. Now go to grid output save as gpf give .gpf extension while saving. Further
we have to run autogrid. Go to Run run autogrid and choose the gpf file that we saved to earlier
and click on launch.
3. To insert the ligand tacrine in the working space, go to ligand input select mol2 format
of the ligand and the ligand comes to the working space.
4. To continue with docking, go to docking macromolecule set rigid filename select
the pdbqt file saved before. Now again go to docking ligand choose select the tacrine
molecule in the dialogue box that has appeared. Now under docking, go to search parameters and

then genetic algorithm, a dialogue box appears and we have to click on accept. Now, docking
output Lamarckian GA save as dpf. Further, we have to run autodock. Go to run run
autodock and choose the dpf save earlier and click on launch.
5. Edit delete delete all molecules
6. We have to now analyze the docking parameters. Go to analyze and open the dlg file and
also bring the macromolecule in the working space. Now, analyze conformations play. Then,
check the conformations with highest binding energy. In the dialogue box, click write complex
and save the best conformation as pdbqt.
7. For converting the pdbqt to pdb, use autodock or open babel.
8. Bring the pdb format of the best conformation to chimera and work the ligand. Give labels
to the interacting residues and hide the ribbons. Save the ligand along with interacting residues in
the best orientation possible.
Assignment:
a. From RCSB take PDB: 1ACJ. Prepare the protein for docking. Prepare tacrine molecules
using Marvin Sketch, save in Mol2 format. Perform the molecular docking between 1ACJ and
Tacrine. Report the binding energy, docking interaction diagram showing hydrogen bonds (use
UCSF Chimera tool for image generation).
Observations and Results: (i) Binding energy; (ii) 3D Interaction diagram / image using UCSF
Chimera.
i) Binding energy: 2.71
ii) Image from UCSF Chimera

Practical marks:



Molecular docking in presence of metal ions and
4 A038
docking with DNA
Aim: Molecular docking in presence of metal ions and docking with DNA.
References:
▪ PhD thesis of Dr. Ashish M. Kanhed
▪ ChemAxon (Marvin Sketch) [https://chemaxon.com/products/marvin]
▪ Protein data bank [https://www.rcsb.org/]
▪ UCSF Chimera Software [https://www.cgl.ucsf.edu/chimera/]
▪ Avogadro software [https://avogadro.cc/]
▪ AutoDock
Introduction:
In the field of molecular modeling, docking is a method which predicts the preferred orientation
of one molecule to a second one when bound to each other to form a stable complex. Knowledge
of the preferred orientation in turn may be used to predict the strength of association or binding
affinity between the two molecules using, for example, scoring functions. Docking is frequently
used to predict the binding orientation of small molecule drug candidates to their protein targets in
order to predict the affinity and activity of the small molecule drugs. Hence docking plays an
important role in the rational design of drugs.
To perform a docking screen, the first requirement is the structure of a protein of interest. Usually
the structure has been determined using biophysical techniques such as X-ray crystallography
or NMR spectroscopy. This protein structure and a database of potential ligands serve as the inputs

to a docking program. The success of a docking program depends on two components- the search
algorithm and the scoring function.
Search algorithm is nothing but searching the conformational space for docking. The search
space in theory consists of all possible orientations and conformations of the protein paired with
the ligand. However in practice with current computational resources, it is impossible to
exhaustively explore the search space—this would involve enumerating all possible distortions of
each molecule (molecules are dynamic and exist in an ensemble of conformational states) and all
possible rotational and translational orientations of the ligand relative to the protein at a given level
of granularity. Most docking programs utilize flexible ligands, and attempt to model a partially
flexible protein receptor. Each "snapshot" of the pair is referred to as a pose. A variety of
conformational search strategies have been applied to the ligand and to the receptor. These include:
Systematic or stochastic torsion which searches conformations about rotatable bonds; Molecular
dynamics simulations and Genetic algorithms. The scoring function (force field) takes a pose as
input and returns a number indicating the likelihood that the pose represents a favorable binding
interaction. Scoring functions are physics-based molecular mechanics force fields that estimate the
energy of the pose; a low (negative) energy indicates a stable system and thus a likely binding
interaction.
Metal ions are present in some metalloenzymes where, the metal ion perform important
physiological role in the active site. In AutoDock tools instead of Kollman charges, Gasteiger
charges needs to be given if metal ions are present in the system in the active site region.
Similarly, there are many drugs which intercalate with DNA and exhibit their action. Such drugs
are mainly involved in Cancer, bacterial infection etc treatments. Understanding the interaction
pattern between DNA-drug (drug like molecule) helps in designing better and more potent drug
candidate. Here also in AutoDock Gasteiger charges are implemented.
Procedure:
1. Import the macromolecule 1BNA from the web and then delete water molecules (edit
delete water), add hydrogens (edit add hydrogens) and then check for missing residues and
repair them. Following this, we have to add charges (edit charges compute gasteiger).
With this, our macromolecule is ready for further docking process.

2. Now, go to the grid and choose the macromolecule and save it in pdbqt format. Once done,
insert a grid box to cover the ligand. Change the coordinates to cover the ligand and save the
coordinates. Now go to grid output save as gpf give .gpf extension while saving. Further
we have to run autogrid. Go to Run run Auto Grid and choose the .gpf file that we saved to
earlier and click on launch.
3. To insert the ligand tacrine in the working space, go to ligand input select .mol2
format of the ligand and the ligand comes to the working space.
4. To continue with docking, go to docking macromolecule set rigid filename select
the pdbqt file saved before. Now again go to docking ligand choose select the tacrine
molecule in the dialogue box that has appeared. Now under docking, go to search parameters
and then genetic algorithm, a dialogue box appears and we have to click on accept. Now,
docking output Lamarckian GA save as dpf. Further, we have to run autodock. Go to
run run autodock and choose the dpf save earlier and click on launch.
5. Edit delete delete all molecules
6. We have to now analyze the docking parameters. Go to analyze and open the dlg file and
also bring the macromolecule in the working space. Now, analyze conformations play. Then,
check the conformations with highest binding energy. In the dialogue box, click write complex
and save the best conformation as pdbqt.
7. For converting the pdbqt to pdb, use autodock or open babel.
8. Bring the pdb format of the best conformation to chimera and work the ligand. Give labels
to the interacting residues and hide the ribbons. Save the ligand along with interacting residues in
the best orientation possible.
Assignment:
1. Download 1bna from RCSB.org. Prepare this DNA ladder in AutoDock tools. Perform the
blind docking with Cyclophosphamide. Report the binding affinity and interaction diagram using
UCSF Chimera tool.
Observations and Results: (i) Binding energy; (ii) 3D Interaction diagram / image using UCSF
Chimera.

(i) Binding Energy
Binding Energy= -7.35
(ii) 3D Interaction diagram / image using UCSF Chimera.

Practical marks:


Expt. No. Pharmacophore: Receptor based and ligand- Date: Roll No.
5 based pharmacophore model development A038
Aim: Pharmacophore: Receptor based and ligand-based pharmacophore model development.
References:
https://link.springer.com/article/10.1007/s11030-020-10130-1
Introduction:
As per the IUPAC (1998) “A pharmacophore is the ensemble of steric and electronic
features that is necessary to ensure the optimal supramolecular interactions with a specific
biological target structure and to trigger (or to block) its biological response.”
In medicinal chemistry most of the time this term is used incorrectly. A pharmacophore
never represent a real molecule/skeleton such as flavones, steroids etc or never present a real
association of functional groups like hydroxyl, amine, guanidines etc, but it is only a summarized
concept that accounts for the common molecular interaction capacities of a group of compounds
towards their target structure. Pharmacophore modeling helps to identify a set of common
features that interacts with a set of complementary sites on the biological target. Generally these
features are, hydrogen bond donors, hydrogen bond acceptors, negatively and positively charged
groups and hydrophobic regions. Pharmacophore concept is closely related with the concept of
bioisosterism. In addition, in the 3D pharmacophore methods the spatial relationship between the
pharmacophoric features is also precise. These pharmacophoric features may be positioned on
the ligand itself or may be presumed as counter features to be located in the receptor.
In general, the process of pharmacophore model development involves five steps.

Selection of data set as training set should include the structurally diverse set of molecules. Also
the data set should include both active and inactive compounds in order to train the model to
discriminate between active and inactive compounds. The second step involves the development
of low energy conformations that are likely to contain the biologically active conformations of
individual ligands. The alignment of different conformations generated for ligands either

considering atom to atom pairing or feature based pairing is the next step for the pharmacophore
model development. Here a set of conformations (one for each ligand) which show good fitting
or alignment is presumed to be the most active conformation. Next step is abstraction or
representation of the molecules with the common identified features such as aromatic ring,
hydrophobic feature, hydrogen bond donor/acceptor etc. And the final step includes the
validation of the developed model. One of the basic validation steps is that the model should
differentiate the molecules with high and low biological activity. One can also validate the
generated pharmacophore model by comparing the mapped features with the active site of the
receptor under study, if the receptor 3D structure is available. It is also useful to compare the
features of the pharmacophore with the features obtained from contours of nD-QSAR.
Apart from this traditional concept of pharmacophore, one can generate receptor
dependent pharmacophore i.e. mapping of pharmacophore on the receptor active site.
The generation of ligand-receptor/protein complex-based pharmacophore specifies only

the important steric and electrostatic features present in that ligand, which is co crystallized with
the protein structure. Thus, the development of a ligand-receptor/protein complex-based
pharmacophore model carries the highest chances of missing out other possible sites for
interactions present in the protein active site. Also, to generate a ligand-based pharmacophore
model, enough ligands, with some scaffold similarity, acting against the same target is required.
Thus, in absence of sufficient number of ligand library with wide range of activity or absence of
protein in complex with highly active ligand in active site, then in such cases receptor active site-
based pharmacophore model is suitable option. In receptor active site-based pharmacophore, a
fragment library of different feature fragments (like hydrophobic, aromatic, positive, negative
polar functionalities etc) are allowed to interact with the receptor active site and interaction
scores are recorded. On the basis of interaction scores, features are assigned to particular sub-
cavity of receptor active site. Further these features together can be used for the virtual screening
of various data bases or libraries.
Virtual Screening
Virtual screening (VS) is a computational technique used in the drug design process to
screen a library of small molecules in order to identify the probable active structures which are

most likely to bind with the active site of the target receptor or enzyme. VS can search
enormous chemical space of over 1060 plausible compounds to a handy number that can be
synthesized and evaluated against a particular condition. Although searching in of such a large
data may be interesting theoretically, but in practical terms VS focuses more on selective or
targeted combinatorial libraries.
The VS technique can be either structure based, or ligand based. Structure-based VS

involves docking of candidate ligands into the active site of the target protein or receptor and
then applying a scoring function. The ligands that show high affinity for the active site of the
target receptor are identified.
The ligand based or pharmacophore-based VS or logic-based rules describe features of

substructures and chemical properties related to the activity using support vector inductive logic
programming. Another approach is molecular similarity searching which is based upon similar
property principle and attempts to predict properties of molecules on the basis of knowledge
derived from properties of another set of molecules. The chemical similarity study is useful to
compare the biological similarity between a set of molecules.
Assignment:
1. Read the publication on pharmacophore and docking based virtual screening
“Identification of potential Mpro inhibitors for the treatment of COVID-19 by using
systematic virtual screening approach” (free download link is available at:
https://link.springer.com/article/10.1007/s11030-020-10130-1). Write summary of
workflow in 5-10 lines as per your understanding from the research paper.

Summary:
The article is about the development of a potential treatment option for COIVD-19. The
identification of the polyproteins and its cleavage sites which help in replication of the virus are
mediated by the main proteases (Mpro). Therefore, Mpro site is further discussed in the article as
a target site of action for inhibiting the life cycle of the novel coronavirus. Pharmacophore model
development was preferred from the studies conducted as the drugs were a perfect match to these
sites derived from the Asinex bio design library. The drugs that were chosen are after the
modelling and docking studies: ritonavir, nelfinavir, saquinavir. Thus, the repurposing of these
drugs may be a faster and a promising way out to get a drug that can treat the novel coronavirus.
Diagnostic agents like pralmorelin, iodixanol, iotrolan carry certain limitations and the
pharmacophoric changes in these can lead to the development of potential drug candidate for
SARS-COV-2 viral attack treatment. The Asinex bio design library helped identify 20 molecules
as the Mpro inhibitors (with 3 mentioned earlier having the most potential) that can possibly be
one of the mechanisms of actions for inhibiting the replication coronavirus.
Practical marks:


Expt. No. Prediction of unknown molecule activity by using Date: Roll No.
6 Quantitative structure activity relationship A038
(QSAR) and Prediction of unknown molecule
property by using Quantitative structure
property relationship (QSPR)
Aim: Prediction of unknown molecule activity by using Quantitative structure activity

relationship (QSAR) and Prediction of unknown molecule property by using Quantitative
structure property relationship (QSPR).
References:
https://drive.google.com/file/d/1NpI0zl2N8SL3CtGCrIoxS-
nZNmsu3OY0/view?usp=sharing
Introduction:
Quantitative structure activity relationship (QSAR)11 or quantitative structure property
relationship (QSPR), is the process in which chemical structure is quantitatively correlated with
some well-defined process, such as biological activity or chemical reactivity. When
physicochemical properties or structures are expressed with numbers, one can form a
mathematical relation between the structure and the activity. The mathematical expression can
then be used to predict the biological response of other chemical structures. QSAR assumes that
there is an underlying relationship between the molecular structure and biological activity. On
this assumption QSAR attempts to establish a correlation between various molecular properties
of a set of molecules with their experimentally determined biological activity. There are two
main objectives for the development of QSAR:
Development of predictive and robust QSAR, with a specified chemical domain, for the
prediction of activity of untested molecules.
It acts as an informative tool by extracting significant patterns in descriptors related to the

measured biological activity leading to understanding the mechanism of the biological
activity. This could help in the designing of novel molecules with improved activity profile.

A general mathematical equation for QSAR is:

Activity = f (physicochemical and/or structural properties)
For the generation of QSAR or to perform QSAR study the general method involves the
following steps:
For QSAR analysis, a set of series of synthesized molecules tested for their desired
activity is required. Its quality totally depends on the quality of the experimental data used for
building the model. The biological activity used is in terms of mathematical data obtained by
applying some statistics. This can be of two type; continuous response type: such as Minimum
effective concentration (MEC), Inhibitory concentration (IC50), Effective dose (ED50), %
inhibition, and categorical response such as active or inactive etc.
Molecular descriptors can be defined as numerical representation of chemical information
encoded with molecular structure via some mathematical procedure. There are so many
descriptors available for building a QSAR model, but all are not important for a specific model
generation, and hence to select an optimal set of descriptors, various selection methods are used
which include systematic variable selection and stochastic variable selection. In systematic
variable selection leave one out rule is used and it is applied by stepwise forward, stepwise
forward-backward or by stepwise backward methods. Stochastic variable selection methods are
based on simulation of various physical or biological processes. These methods create models
starting from randomly generated models and later modifying these models by using different
process operators, such as perturbation, crossover etc to get better model(s). The descriptors used
in QSAR are based on the dimensionality of the molecule. For example from zero dimensional
molecule, molecular weight, number and type of atoms etc are calculated; from one dimensional
molecular representation functional groups, ring, bonds etc are calculated; from two dimensional,
types of bonding and interaction of particular atoms is possible; from three dimensional
molecular representation various descriptors, such as molecular surface, molecular volume,
electronic, steric and geometric descriptors are derived. Apart from these dimensions one can
consider the fourth dimension, generally in terms of conformational flexibility or the fifth
dimension in terms of additional different induced fit features.
In the QSAR study the model need to be validated and for this purpose the dataset is to be
divided into training set (for building the QSAR model) and test set (for examining its predictive

ability). For any QSAR model, it is of importance that the training set selected to create a model
exhibits a well-balanced distribution of biological activity and contains representative molecules.
Various methods such as manual selection method, random selection method, sphere exclusion
method, factorial design, cluster analysis method etc are used for the division of dataset into
training and test set.
The selected variable descriptors when coupled with a suitable statistical method allow analyses
of data subsets in order to establish a QSAR model for the selected descriptors. Such model
should be statistically significant in determining the biological activity. The statistical methods
used in QSAR can be broadly divided into linear and non-linear methods. In statistics a
correlation is established between dependent variable(s) (biological activity) and independent
variable(s) (molecular descriptors). The linear method fits a line between the selected descriptors
and activity as compared to non-linear method which fits a curve between the selected
descriptors and activity. The statistical method to build the QSAR model is decided based on
type of biological activity data. Various statistical methods include discriminant analysis, k-
nearest neighbor classification, multiple regression, principle component regression, continuum
regression, partial least square analysis etc. which can be used for specific set of data.
Assignment:
1. Read the publication on QSAR “Structural requirements for imidazo[1,2-a]pyrazine
derivatives as Aurora A kinase inhibitors and validation of the model” (pdf is available
at: https://drive.google.com/file/d/1NpI0zl2N8SL3CtGCrIoxS-
nZNmsu3OY0/view?usp=sharing). Write summary of workflow in 5-10 lines as per your
understanding from the research paper.

Summary:
The article aptly explains the potential of aurora A kinase inhibitors as a treatment for cancer.
QSAR of Imidazo[1,2-a]pyrazine derivatives are studied in order to find out a new lead for
curing cancer. The highlight of the article is technological software’s that are used in order to
carry out the study. Autodock4 and 3D-QSAR techniques were used to find out important
parameters about all the 51 derivatives. Comparative molecular field analysis (CoMFA) and
Comparative molecular similarity indices analysis (CoM-SIA) models were developed with the
aid of Sybyl7.0. A model was developed after sufficient information was collected from all the
parameters and this was model was stable and showed good predictive ability for the compounds
as Aurora A kinase inhibitors. The model also explained the appropriate role of steric,
electrostatic, hydrophobic, and hydrogen bonding parameters in the development of active
inhibitors. Therefore, the observations from this study can now be used for the development of
more potent Aurora kinase inhibitors. The newly developed model was found to be robust and
could be reliable.
Practical marks:



7 IR spectroscopy – demonstration A038
Aim: IR spectroscopy – demonstration.
References:
Organic Spectroscopy by Y. R. Sharma
Spectroscopy by Pavia
https://drive.google.com/drive/folders/10Hv8RUAEn37s12u6iJcQsVz2BY0augvQ?usp=
sharing
Principle:
- The absorption of infra-red radiation causes an excitation of molecule from lower to the
higher vibrational level. As each vibrational level is closely related with the number of
rotational levels, thus this spectrum is considered as vibrational-rotational spectra.
- All the bonds in the molecule are not capable to absorb IR energy, but only those bonds
having dipole moment / charge absorbs in the IR region. Such transitions accompanied by
change in dipole moment are considered as infrared active transitions like C=O, N-H.
The transitions which are not accompanied by change in dipole moment are IR inactive
like C-C, C=C.
- In IR spectroscopy the absorbed energy brings about the predominant changes in the
vibrational energy which depends upon; Masses of the atoms present in the molecule,
Strength of the bond, The arrangement of the atoms within the molecule. It is found that
no two compounds except enantiomers can have similar IR spectrum
- When IR radiation can interact with the sample, vibrational and rotational energy changes
are observed in the molecule. These are stretching vibrations and bending vibrations
- Therefore, all in all, IR spectroscopy detects the absorption of light by a compound, in the
IR region of the electromagnetic spectrum. To absorb light a molecule must have a bond
within its structure that can exhibit what is referred to as a ‘dipole moment’ which means
electrons within a bond are not shared equally.

- Hooke’s law: The value of stretching vibrational frequency can be calculated fairly
accurately by the application of Hooke’s law – which states that the force (F) needed to
extend or compress a spring (bond) by some distance (x) scales linearly with respect to
that distance. The vibrational frequency is proportional to the strength of the spring
(bond); the stronger the spring (bond), the higher the frequency. The vibrational
frequency is inversely proportional to the masses at the ends of the spring (bond); the
lighter the weights, the higher the frequency
Instrumentation: [include images of different parts]
INSTRUMENTATION OF IR SPECTROSCOPY
1) Radiation source: Nernst glower, globar source, carbon dioxide laser
A Nernst glowers

2) Mirror system: Are used to adjust the radiation direction towards the sample, gratings and
detector system.
3) Sample techniques: Depending on the nature of the sample (i.e. solid, liquid or gas)
sample handling changes. Example: solution sample holder, liquid samples, gas sample
holder.
4) Chopper: used to block the radiation path
5) Monochromator: diffraction gratings are used.
6) Detectors: various types of detectors are used like thermocouple, bolometer, golay cell,
photoconducting detector.
A bolometer
A golay cell
Applications:

- Infrared spectroscopy is a simple and reliable technique widely used in both organic and
inorganic chemistry, in research and industry. It is used in quality control, dynamic
measurement, and monitoring applications such as the long-term unattended
measurement of CO2 concentrations in greenhouses and growth chambers by infrared gas
analyzers.
- Infrared spectroscopy is also useful in measuring the degree of polymerization

in polymer manufacture.
- Infrared spectroscopy has also been successfully utilized in the field of semiconductor
microelectronics
- Another important application of Infrared Spectroscopy is in the food industry to measure

the concentration of various compounds in different food products
- IR-spectroscopy has been successfully used in analysis and identification

of pigments in paintings
- In the study of geometrical isomerism, conformational analysis, chemical reactions,

rotational isomerism.
- Detection of impurities in the compounds, distinction between 2 groups of hydrogen

bonds
- Quantitative analysis and qualitative analysis of functional groups.

Assignment:
1. What is Fourier Transform in FT-IR?
2. Suggest the IR peak region for C=O, aliphatic C-H, aromatic C-H, hydroxyl –OH, amine
–NH, C#C, C-O-C.
Answers:
A1) Fourier Transform-Infrared Spectroscopy (FTIR) is an analytical technique used to identify
organic (and in some cases inorganic) materials. This technique measures the absorption of
infrared radiation by the sample material versus wavelength. The infrared absorption bands
identify molecular components and structures. The FTIR spectrometer uses an interferometer to
modulate the wavelength from a broadband infrared source. A detector measures the intensity of
transmitted or reflected light as a function of its wavelength. The signal obtained from the
detector is an interferogram, which must be analyzed with a computer using Fourier transforms
to obtain a single-beam infrared spectrum. The FTIR spectra are usually presented as plots of
intensity versus wavenumber (in cm-1). Wavenumber is the reciprocal of the wavelength. The
intensity can be plotted as the percentage of light transmittance or absorbance at each
wavenumber.
A2)
C=O: 1732 cm-1
aliphatic C-H: 2945-2879 cm-1
aromatic C-H: 3058 cm-1
hydroxyl –OH: 3500-2500 cm-1
amine –NH: 3490-3180 cm-1
C#C: inactive
C-O-C : 1294 cm-1
Practical marks:



8 LC-MS/MS – demonstration A038
Aim: LC-MS/MS – demonstration.
References:
Organic Spectroscopy by Y. R. Sharma
Spectroscopy by Pavia
sharing
Theory:
LC-MS/MS is a tandem technique that combines the physical separation capabilities of

chromatography and mass analysis capabilities of mass spectrometry. MS provides fingerprint
mass spectrum which contains molecular weight and structure specific fragment ions.
Advantages:
- It has major advantage in handling the mixture of compounds.
- It is More Specific than only LC-MS technique.
- Require less time and quicker results for faster decision making.
Liquid chromatography:
Partitioning of analyte /sample between mobile phase and stationary phase. Columns used are
much smaller having internal diameter of 0.1 to 4.6mm. LC systems operate at flow rates of 0.1
– 1.5ml per min.
Mass spectroscopy:
Mass spectrometer produces a beam of ions and separates them according to the mass/charge
ratio. Heart of mass spectrometer is an analyzer. It uses electrical or magnetic field or
combination of to deflect ions from the region where they are produced to a detector, where they
produce a signal which is amplified.
Tandem mass spectroscopy:
In this technique sequential mass analyzers are used to both separate and identify ions in a single
instrument. The first analyzer generates parent ions from the sample that are based on their m/z
ratio. Some of these ions are then selected for further fragmentation to form daughter ions that
can be mass analyzed.

Instrumentation: [include images of different parts along with description]
Interfaces:
1) Moving belt interface:

The moving-belt interface separates the condensed
liquid-phase side of the LC from the high vacuum of
the MS and uses a belt to transport the analytes from
one to the other. The mobile phase of the LC is
deposited on a band and evaporated. The analytes
remain on the continuously cycling belt and are
transported from atmospheric pressure into the
vacuum of the ion source through two differentially
pumped vacuum locks. A heater in the ion source evaporates the sample from the belt allowing
MS analysis. Most moving-belt analyses deal with volatile analytes using CI/EI; however, less
volatile molecules such as nucleosides and nucleotides are analyzed using this system.
2) Particle beam interface:
This system is now known as particle beam. The
LC eluent is forced through a small nebulizer using
a He gas flow to form a stream of uniform droplets.
These droplets move through a desolvation
chamber and evaporate to a solid particle. These
particles are separated from the gas and transported
into the MS source using a differentially pumped
momentum separator. The PB interface allows
flowrates from 0.1–0.5 mL/min. Most analytes that are amenable to PB LC–MS can be analysed
using GC–MS as well.
3) Thermo spray:
Thermospray is a soft ionization source by which a
solvent flow of liquid sample passes through a very
thin heated column to become a spray of fine liquid
droplets. As a form of atmospheric pressure
ionization in mass spectrometry these droplets are
then ionized via a low-current discharge electrode to
create a solvent ion plasma. A repeller then directs
these charged particles through the skimmer and
acceleration region to introduce the aerosolized sample to a mass spectrometer. It is particularly
useful in liquid chromatography-mass spectrometry (LC-MS).

4) Electrospray ionization (esi):

Electrospray ionization (ESI) is a technique used in
mass spectrometry to produce ions using
an electrospray in which a high voltage is applied to a
liquid to create an aerosol. It is especially useful in
producing ions from macromolecules because it overcomes
the propensity of these molecules to fragment when
ionized. Electrospray ionization is the ion source of choice
to couple liquid
chromatography with mass spectrometry (LC-MS).
5) Atmospheric pressure chemical ionization (apci): Using APCI the liquid flow from the
LC is sprayed and rapidly evaporated by a coaxial nitrogen stream and heating the
nebulizer to high temperature (350–500 °C). APCI finds most of its applications in
molecular weights below 1000 Da for medium- to low-polarity molecules.
Applications:
1) In bioanalytical studies: Quantitative measurement and structural characterization of

drug, drug metabolites or chemicals in biological fluids like:
- Determination of granisteron in plasma.
- Determination of orlistat in plasma.
- Diuretics screening in urine sample.
- Inborn error of metabolism.
- Bioanalysis of Vitamin D in Serum.
2) In proteomics: Identification of biomarkers, analysis of product of gene expression
including identification, quantification and characterization of proteins.
3) In impurity profiling: Quantification of impurities is important to establish
biological safety of individual impurity like identification of polar impurity in
Mosapride, a gastroprokinetic drug.
4) Forensic and toxicological applications: Recently LC-MS/MS is used as
analytical tool in forensic toxicological study. It can be used for non-volatile, heat
labile and high molecular weight compounds. Example: Screening for drug of abuse
in hair with LC-MS/MS.

Practical marks:


Expt. No. Structure elucidation of organic compounds by Date: Roll No.

9 using various spectral data viz. UV, IR, NMR, A038
Mass and elemental analysis
Aim: Structure elucidation of organic compounds by using various spectral data viz. UV, IR,
NMR, Mass and elemental analysis.
References:
▪ Organic Spectroscopy by Y. R. Sharma
▪ Spectroscopy by Pavia
▪ https://drive.google.com/drive/folders/10Hv8RUAEn37s12u6iJcQsVz2BY0augvQ?usp=
sharing
Theory: Structure elucidation of organic compounds is very essential in the field of

phytochemistry, new drug design and development, biochemistry, organic synthesis and reaction
monitoring, process development & validation etc.
Mainly the technique of structure elucidation includes techniques such as elemental analysis, UV-
Visible spectroscopy, IR spectroscopy, different NMR spectroscopies and Mass spectrometry.
Apart from these techniques, X-ray diffraction, fluorescence etc techniques can be used in the
identification of structure of unknown chemical molecules.
Correlation tables: [include IR and proton NMR tables here from provided ppt].
Assignment:
1. Determine the structure of compound from following data (elaborate solution steps): Mass-
120. IR- 3067-2907, 1608, 1477 cm-1. NMR: 6.79 singlet, 2.26 singlet.
2. Mass: 130. Elements: C = 73.85% & H = 13.85%. IR = 2960-2851 (m), 1342 (w), 1075
(s). NMR: 1.05 singlet.

3. Mass: 108. IR= 3342 (s,b), 3065 (w), 2288 (m), 1499 (w), 1455 (m). NMR = 7.26 multiplet
(24.5 square), 4.6 singlet (9.5 sq), 3.9 singlet (4.8 sq).
Solutions:
1. Mass-120. IR- 3067-2907, 1608, 1477 cm-1. NMR: 6.79 singlet, 2.26 singlet
● The broad band at 3067 - 2907 suggests aliphatic or aromatic C-H stretching
● 1608 - suggests C-C stretching
● NMR = 6.79, aromatic C-H protons and 2.26 suggests that there is some aliphatic structure
on the aromatic ring. Singlet- no proton on neighbouring atoms.
● If the total mass is 120 consisting of C and H then let's consider there are 9 atoms of C; 9
* 12 = 108. Therefore H = 12. C9H12.
● Now we know that there is a possibility of an H atom. Therefore, double bond equivalence:(
9 + 1 - 12/2) = 4; which means that the sum of unsaturation and aromatic ring equals 4.
● Molecular formula : C9H12
2. Mass: 130. Elements: C = 73.85% & H = 13.85%. IR = 2960-2851 (m), 1342 (w), 1075
(s). NMR: 1.05 singlet.
● C= 73.85%, H= 13.85%, therefore 12.3% other element.
● The band at 2960-2851 is medium. This suggests aliphatic stretching.
● Weak band at 1342 and strong band at 1075, according to correlation table suggests
presence of ether linkage [C-O-C or C-O stretching ]
● Presence of aliphatic proton in the same environment because of single peak according to
NMR data.

● Now that we know the third element is O, therefore mass of C(73.85%) = 96, H(13.85%)
= 18 and similarly O(12.3%) = 16.
● To have a singlet all the protons should be in the same environment and the neighbouring
carbon of a proton should not have any proton.
● Thus, the structure which satisfies this condition is Di-tertbutly-ether.
Molecular formula: C8H18O
Molecular Wt: 130
3. Mass: 108. IR= 3342 (s,b), 3065 (w), 2288 (m), 1499 (w), 1455 (m). NMR = 7.26
multiplet (24.5 square), 4.6 singlet (9.5 sq), 3.9 singlet (4.8 sq).
● The band at 3342 is strong and broad. This signifies the presence of hydroxyl group [O-H
stretching]
● Band at 3065 signifies the presence of aromatic ring [C-H stretching]
● Bands at 1499 and 1455 could signify the presence of carboxylic compounds [C-O-H in
plane bending] and methylene group [C-H in plane bending]
● Let 4.8 sq be rounded to 5 sq and let that signify 1 proton [presence of hydroxyl group]
● Thus, 9.5 sq will be rounded to 10 sq which would represent 10 / 5 = 2 protons [presence

of methylene bridge]
● Thus, 24.5 sq will be rounded to 25 sq which would represent 25/ 5 = 5 protons [Presence
of aromatic ring]
● So, the protons are present in the ratio of 5:2:1.

● Double bond equivalence: [(2 x 7) + 2 – 8] / 2 = 4
Thus, the structure which satisfies this condition is Benzyl Alcohol
Molecular formula: C7H8O
Molecular weight: 108 g/mol
Practical marks:



Purification of organic compounds by;
10 A038
(a) Salt preparation; (b) Acid base treatment
Aim: Purification of organic compounds by;

a. Salt preparation
b. Acid base treatment.
References:
Practical Organic Chemistry by Vogel
Theory:
Preparation of salt of organic compound is one of the purification techniques which can
help to remove the inorganic as well as organic impurities trapped in the final API during
synthetic process. Salt preparation includes reacting of acidic or basic functionality of chemical
molecules with some strong or weak base or acid respectively.
The salt preparation technique also improves the stability of product by reducing the
chances of degradation due to environmental effects.
Acid base preparation technique involve reacting acidic or basic drugs with base or acid
to make salt which can easily dissolve in the aqueous phase leaving behind all the impurities in
the organic phase. One can wash the aqueous layer with small volume of immiscible organic
solvent to remove the trapped impurities. The separately collected aqueous layer with the drug
sample in salt form can be made free using some counter ions like acid or base to precipitate out
the drug in free pure form.

Assignment:
1. Draw the structures of two drugs available in market in hydrochloride form and two
drugs in sulphate form.
Practical marks:


Expt. No. Purification of organic compounds by: Date: Roll No.

11 (a) Prep TLC A038
(b) Column chromatography
Aim: Purification of organic compounds by:

a. Prep TLC
b. Column chromatography
References:
Practical Organic Chemistry by Vogel
Theory: [[[Mention Principle of Chromatography]]]

PRINCIPLE: Chromatography is an important biophysical technique that enables
the separation, identification, and purification of the components of a mixture for
qualitative and quantitative analysis. Chromatography is based on the principle
where molecules in mixture applied onto the surface or into the solid, and fluid
stationary phase (stable phase) is separating from each other while moving with the
aid of a mobile phase. When a solute in a solvent (or a mobile phase) is passed
through or around the outside of a matrix (or a stationary phase), interactions occur
between the solute and the stationary phase. Solutes with different properties are
separated based on differences in these interactions and their polarities.
Procedure: [Mention procedure of Prep TLC and Column chromatography]]]
Prep TLC:
1) Cut the plate in half by scoring with a pencil, so there is no contact of silica
across the middle of the plate.
2) Gently mark a pencil line roughly 1-1.5 inches from one side of the plate
(careful not to scrape the silica gel). This is the "origin" line.
3) Prepare a relatively concentrated solution of your crude sample (~1-2 mL) in

a fairly low-boiling solvent (i.e. DCM or Et2O).

4) Using a short pipet, carefully deposit a thin line of sample across pencil line,
avoiding the edges
5) Apply remaining solution repeatedly on the original line of sample, drying in

between applications.
6) Obtain a prep TLC chamber. Then in a dry chamber prepare about 100 mL
of eluent.
7) Place the plate in the chamber and seal the top with the lid or aluminum foil.
A typical run takes 40 mins to 1 hour.
8) Remove the plate and visualize under UV.
9) If your desired band(s) are not separated enough, repeat run another elution.
Column chromatography:
1) Mobile phase – This phase is made up of solvents and it performs the

functions like acting as a solvent, eluting agent, and a developing agent.
2) Stationary phase – It is a solid material which should have good adsorption

property.
3) The stationary phase is made wet with the help of solvent as the upper level
of the mobile phase and the stationary phase should match. The mobile
phase or eluent is either solvent or mixture of solvents. In the first step the
compound mixture that needs to be separated, is added from the top of the
column without disturbing the top level. The tap is turned on and the
adsorption process on the surface of silica begins.
4) Without disturbing the stationary phase solvent mixture is added slowly by
touching the sides of the glass column. The solvent is added throughout the
experiment as per the requirement.
5) The tap is turned on to initiate the movement of compounds in the mixture.
The movement is based on the polarity of molecules in the sample. The non-
polar components move at a greater speed when compared to the polar
components.

6) For example, a compound mixture consists of three different compounds viz

red, blue, green then their order based on polarity will be as follows
blue>red>green
7) As the polarity of the green compound is less, it will move first. When it
arrives at the end of the column it is collected in a clean test tube. After this,
the red compound is collected and at last blue compound is collected. All
these are collected in separate test tubes.
Practical marks:



Purification of organic compounds by Flash
12 A038
chromatography - demonstration
Aim: Purification of organic compounds by:

a. Flash chromatography
References:
sharing
Theory: [[[Mention Instrumentation of Flash Chromatography]]]
Main components of flash chromatography are:
1) Pump systems: this includes the pump controller and the vacuum or the peristaltic
pumps: A pressure range up to either 10 bar or 50 bar gives optimum separation results
for a broad range of applications. The pump modules can be controlled by three different
units. The Pump Controller C610 (for isocratic separation up to 10 bar), the Pump
Manager C615 (for isocratic and gradient separation up to 50 bar) and the Control Unit
C620. Vacuum Pump/peristaltic Pump: Transfer Solvent From Mobile phase
Reservoir to Flash Pump.
2) Sample injection systems: Injection systems are designed to facilitate column loading
with liquids and low solubility oils and solids. Regardless of the nature or quantity of the
material. Sample Chamber 100 ml: Sample Chamber 100 ml for use
in combination with the Injection Unit for loading sample volumes of 10 to 100 ml
including N2 gas valve (on/off).
3) Columns: glass column: A wide range of columns offer maximum flexibility for every
situation. Depending on the nature and the quantity of the sample offers a series of
column types which vary in form, size and performance.
Plastic + Glass Column: Plastic + Glass-coated Glass Columns are available for
larger sample amounts and higher-pressure applications on a high safety level. The
columns are designed for sample amounts from1 – 100 g and pressures up to 50 bar
during preparative separations. Easy fixation on a support rod by using the corresponding
pivoting clamp.

4) Pre columns: Precolumn are minimizing dead volumes and enhance the life time of the
main column by trapping contaminants. The small Precolumn, fits to Glass Columns of
inner diameter of ID 15, 26, 36 and 49 mm. The large Precolumn, fits to Glass Columns
of ID 70- and 100-mm inner diameter.
5) Fraction collector: For simple separations a column, pump and pump controller may be
enough. For a greater level of automation with precision, performance, and ease of use
the Fraction Collector can be incorporated into most setups. Example: fraction collector
C-660
6) Detectors: For most applications one of the robust UV/Vis detectors would be sufficient
for the systems detection needs. Both detectors are delivered in combination with a
preparative flow cell. In the absence of adequate UV/Vis absorption, likely for sugars or
polymers, a Differential Refractometer (RI Detector) in combination with a UV/Vis
detector is the preferred setup.
7) LCD display
INSTRUMENTATION OF FLASH CHROMATOGRAPHY

Applications: [Mention 5-7 applications]]]
1) Natural Products/Nutraceuticals Application:
Isolation and Purification of Catechins from Green Tea Extract.
Separation and Isolation of α-Santalol and β-Santalol from Sandalwood Extraction
Isolation and Purification of Flavonoids from Ginkgo Biloba Leaves Extract.
2) Carbohydrate Application:
Isolation of Aminosugar and Acarbose.

Flavanone Glycoside Purification.
Isolation of Aminoglycoside Antibiotics.
3) Lipids Applications:
Purification of Fatty Acid Methyl Esters (FAMEs), Purification of a Mixture of

Glycerides, Mono-, Di-, and Tristearin.
Purification of Sterols.
4) Pharmaceutical/Small Molecules Application:
Bile Acid Purification During Lead Generation in Drug Discovery. In Impurity Isolation
During Drug Purification.
Mestranol Purification During Chemical Synthesis.
In Anti-malarial Drug Purification in Drug Discovery.
Practical marks:


Expt. No. Date: Roll No

Application of microwave reactor in organic
13 A038
synthesis
Aim: Application of microwave reactor in organic synthesis
References:
▪ https://drive.google.com/drive/folders/10Hv8RUAEn37s12u6iJcQsVz2BY0augvQ?usp=
sharing
Principle:
Microwave radiation is the form of energy that falls within 300-30000 MHz. Microwaves are low
frequency forms of energy that only cause the molecules to rotate. Microwaves move at a speed
of light and comprise of oscillating electric field and magnetic fields which swing at right angles
to each other and to the source of energy. It is only the electric field of microwave which interacts
with molecules, causes the transfer of energy and generation of heat. Microwave heat substance
by – dipolar polarization and ionic conduction.
Dipolar polarization – If a molecule possess a dipole moment. It tries to align with the electric
field. Since, the field is oscillating, the dipolar constantly tries to realign. At 2.45 MHz, are able to
align with the electric field but are not able to follow oscillating field. This continuous reorientation
of molecule leads to friction and heat.
Ionic conduction – If a molecule is charged then the electric field of microwave moves the ion
back and forth the sample. This movement generates heat and is known as ionic conduction.

Heating Mechanism Diagrams:
Advantages over conventional heating:
Compared to using a hot plate to heat reaction mixture, microwave radiation is more efficient and
it reduces reaction time. Hot plat relies on thermal conductivity and convention current to heat the
mixture. That energy must first heat the vessel and then pass heat to reactant. Whereas, microwave
energy directly interacts with molecules in reactants, heating it faster. In ionic conduction, since
energy is interacting with molecules at a fast rate, the molecules don’t have time to release and
heat generated can be much greater than the overall recorded temperature of such reaction mixture.

Practical marks:


Instrumental Methods of Analysis
Expt. No. Effect of temperature, concentration and Date: Roll No.

14 peroxide on acid / base hydrolysis of molecules A038
Aim: To investigate the effect of temperature, concentration and peroxide on acid / base
hydrolysis of molecules
Reference:
Bajaj, Sanjay, and Saranjit Singh, eds. Methods for Stability Testing of Pharmaceuticals.
Humana Press, 2018.
Baertschi, Steven W., Karen M. Alsante, and Robert A. Reed, eds. Pharmaceutical stress
testing: predicting drug degradation. CRC Press, 2016.
Apparatus: Volumetric Flask (100 ml), Volumetric Pipette, beaker, glass funnel
Chemicals: Water, HCl, NaOH, Hydrogen peroxide, Sample Molecule
Instrument: HPLC, LC-MS

Procedure:
1 mg/ml solutions of a given molecule will be prepared.
Following conditions are to be created within the above sample
Experimental Condition Other conditions Sampling time in Min
0.1 M HCl 20 o C 5, 10, 20, 30
0.1 M HCl 30 o C 5, 10, 20, 30
o
0.1 M HCl 40 C 5, 10, 20, 30
0.1 M HCl 3% H2O2 5, 10, 20, 30
0.1 M NaOH 20 o C 5, 10, 20, 30
0.1 M NaOH 30 o C 5, 10, 20, 30
o
0.1 M NaOH 40 C 5, 10, 20, 30
0.1 M NaOH 3% H2O2 5, 10, 20, 30
Also perform the above experiments with 2 mg/ml and 4 mg/ml at room temperature with
0.1 M HCl and 0.1M NaOH only
Quantitative analysis of the initial and final samples to be done by LC-MS, and/or HPLC
analysis
SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS

Principle:
Hydrolysis is a chemical reaction in which the water molecule is responsible for breakdown one
or more chemical bods. These reactions are responsible for the degradation of the many type of
pharmaceutical API products and therefore, its understanding is important for the proper storage
of the pharmaceutical products and formulations. These reactions are generally facilitated by the
presence of acid or base and therefore broadly categorized as acid or base catalyzed hydrolysis
reactions. Meanwhile during and hydrolysis reaction, increase or decrease in reaction
temperature, starting material concentration as well as presence of concentration and presence of
peroxide entities have critical role in the kinetics of the hydrolysis process. Considering these
facts, the current experiment is to demonstrate the actual effect of the above three factors on the
hydrolysis process of the chemical entities.
Practical marks:


Expt. No. Force degradation study and stability testing of Date: Roll No.
15 API / synthetic drug molecule A038
Aim: Force degradation study and stability testing of a given sample of API and/or synthetic
drug molecule
Reference:
Bajaj, Sanjay, and Saranjit Singh, eds. Methods for Stability Testing of Pharmaceuticals.
Humana Press, 2018.
Baertschi, Steven W., Karen M. Alsante, and Robert A. Reed, eds. Pharmaceutical stress
testing: predicting drug degradation. CRC Press, 2016.
Apparatus: Volumetric Flask (100 ml), Volumetric Pipette, beaker, glass funnel
Chemicals: Water, DMSO, Acetic acid, Propanoic acid, HCl, NaOH, Hydrogen peroxide, Drug Sample
Instrument: Stability Chamber, HPLC, LC-MS

Procedure:
1 mg/ml solutions of a given drug molecule will be used and if drug is poorly soluble is
water inert organic co-solvents (e.g., DMSO, acetic acid or propionic acid) are used
For force degradation study, following stress conditions are to be created within the
above 1 mg/ml solutions sample
Degradation Type Experimental Condition Storage condition Sampling time in Days

o o
Hydrolysis 0.1 M HCl 40 , 60 C 1,3,5
0.1 M NaOH
Oxidation 3% H2O2 40o, 60o C 1,3,5
Thermal 60o C 40o, 60o C 1,3,5

o
60 C/75%RH
Quantitative analysis of the final samples to be done by LC-MS, and HPLC based
technique

Principle:
Force degradation studies are that are used to identify the undesirable reactions that may occur
while storage, and processing of the chemicals and pharmaceutical formulations. In force
degradation studies, an external stress is applied so that we can rapidly judge the probability of
the degradation of the pharmaceutical products under normal storage conditions.
Long term storage tests are the time-consuming processes and also require very high expenses,
however, forced degradation studies require very less time and money to estimate the
degradation processes within the chemical entities. The common stresses that can be easily
examined using forced degradation studies are pH, Temperature, Oxidation and effect of
concentration etc
Practical marks:

Practise School - Chemistry

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Practise School - Chemistry

Uploaded by

Copyright:

Available Formats

Fourth Year B.

Pharm Semester –VII

Introduction to drug discovery and development process:

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

In general the process of pharmacophore model development involves five steps.

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Quantitative Structure Activity Relationship

Quantitative structure activity relationship (QSAR) or quantitative structure property

It acts as an informative tool by extracting significant patterns in descriptors related to the

A general mathematical equation for QSAR is:

Activity = f (physicochemical and/or structural properties)

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Structure Drawing (2D & 3D):

Image: Working area of Marvin Sketch

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Observations and Results:

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

1. Elemental Analysis- C (75.698%), H(8.80%), O(15.51%), Atom count= 13, Mass

1. Elemental Analysis- C (64.868%), H(6.488%), O(9.128%) N(7.208) O(12.348), Atom

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

1. Elemental Analysis- C (61.62%), H(5.488%), O(14.498%) N(12.68) F(5.73), Atom

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Protein structure selection and protein preparation:

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Observations and Results:

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Attendance Performance Viva Total Signature of

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Aim: Physicochemical property analysis and ADMET study.

Software: Chemicalize online server, Swiss ADME online server

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

BOILED-Egg), SwissADME strong points are, non-exhaustively: different input methods,

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Chemicalize -Acetyl Salicylic Acid.pdf

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Expt. No. Date: Roll No.

Aim: Molecular docking study and result analysis.

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

i) Binding energy: 2.71

ii) Image from UCSF Chimera

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Attendance Performance Viva Total Signature of

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Expt. No. Date: Roll No.

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

(i) Binding Energy

Binding Energy= -7.35

(ii) 3D Interaction diagram / image using UCSF Chimera.

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Attendance Performance Viva Total Signature of

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai

Aim: Pharmacophore: Receptor based and ligand-based pharmacophore model development.

In general, the process of pharmacophore model development involves five steps.

SPP-School of Pharmacy and Technology Management, SVKM’s NMIMS, Mumbai