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3. EXPERIMENTAL

3.1 MOLECULAR MODELING TECHNIQUES

In recent years, as computational modeling of binding and biological activity has matured into a

well accepted field of drug discovery, there has been a concomitant increase in the application of

computational modeling to Absorption, Distribution, Metabolism, Excretion and Toxicity

(ADMET) properties.

3.1.1 COMPUTATIONAL METHODOLOGY

The currently available molecular modeling techniques can be broadly classified into four major

heads: a) Quantum chemical methods, b) Correlation methods, c) Docking methods and d)

Mapping methods.

Analysis based on all these techniques start with the identification of the bioactive conformation

of the molecule concerned. The bioactive conformation may be obtained from X-ray crystal

structure analysis. If such information is not available, global minimum conformation is

considered as the bioactive conformation. Global minimum structure of any given drug molecule

can be obtained by carrying out a series of molecular modeling exercises. The general procedure

for these exercises involves estimation of energy using either quantum chemical or molecular

mechanical methods (details given below), followed by performing energy minimization. Thus a

possible conformation of a drug molecule with reasonably reliable chemical representation of

the structure can be obtained. This conformation may be local minimum on the potential energy

surface of the drug molecule. To obtain the global minimum we have to perform conformational

search by manual or automated methods. Energy estimation using quantum chemical methods,

molecular mechanics methods, energy minimization, conformational analysis are presented

below.

Energy Minimization

Molecular structures generated in the computer have to be optimized to find the individual

energy minimum state. This is normally carried out with molecular mechanics or quantum

mechanical methods. In the course of minimization procedure, the molecular structure is

relaxed. The internal strain in the structures due to small deviation in the bond lengths and

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angles are rectified. Before starting the geometry optimization, van der Waals contacts should be

removed. Several advantages like speed, sufficient accuracy and the broad applicability on the

small molecules as well as on large systems have established the force field geometry

optimization as the most important standard method. The common energy minimization

procedures used by molecular mechanics include steepest descent minimizer, conjugate gradient

method and Newton-Raphson method.421

Hence the choice of minimizer depends upon the size of the system and the current state of the

optimization. The convergence criterion in non-gradient methods like the steepest descent is the

increments in the energy or the coordinates while for the gradient systems atomic gradients are

used like root mean square gradient of the forces on each atom of the molecule.

Conformational Analysis

Each molecule containing freely rotatable bonds exists at each moment in many different

conformations. The transformation from one conformer to the other is related to the change in

torsion angles. The changes in molecular conformations can be regarded as movements on a

multi-dimensional surface that describes the relationship between the potential energy and the

geometry of a molecule. Each point on the potential energy surface represents the potential

energy of a single conformation. Stable conformations of a molecule correspond to local minima

on this surface. The relative population of a conformation depends on its statistical weight which

is influenced by potential energy and entropy. Therefore global minimum having the lowest

potential energy is not necessarily the structure with the highest statistical weight. The biological

activity of a drug molecule is supposed to depend on a single unique conformation on the

potential energy surface which is called as the bioactive conformation that can bind to the active

site of the molecule. It is widely accepted that the bioactive conformation is not necessarily the

lowest-energy conformation. Thus identification of various conformations is an important task

in medicinal chemistry (scheme 1.4). Various automated methods of conformational analysis are

known which include, the systematic search procedures, Monte Carlo methods, random search

methods, genetic algorithms, expert systems, molecular dynamics etc.422

The systematic search is the most natural of all the different conformational analysis methods. It is

performed by varying systematically each of the torsion angles of a molecule to generate all possible

conformations. The step size for torsion angle change is normally 30°. The first step in the data

reduction is a van der Waals screening or "bump check". The screening procedure excludes all the

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conformations where a van der Waals volume overlap of atoms, which are not directly bound to

each other, is detected. The validity of the conformation is detected by the sum of the van der Waals

radii of non-bonded atoms. The next step is the calculation of the potential energy of the

conformers. It is in general calculated by neglecting the electrostatic interactions. After the

calculation of the conformational energies, another possibility to reduce the number of

conformations is the use of energy window. It is based on the fact that the conformations containing

much more energy than those close to the minimum are neglected. This method is of advantage to

relatively rigid molecules. Monte Carlo methods or random search procedures follow a different

route and are of statistical nature. Molecular dynamics is another method of carrying out systematic

conformational search for a flexible molecule.

3.1.1.1 Quantum chemical methods

Quantum mechanics is a fundamental physical theory which extends and corrects Newtonian

mechanics, especially at the atomic and subatomic levels. It gives a set of laws that describe the

behavior of small particles such as electrons and nuclei of atoms and molecules. It is the

underlying framework of many fields of physics and chemistry, including condensed matter

physics, quantum chemistry, and particle physics. The term quantum (Latin, “how much”) refers

to the discrete units that the theory assigns to certain physical quantities, such as the energy of

an atom at rest. Quantum chemistry is a branch of theoretical chemistry. It is the application of

quantum mechanics to problems in chemistry. The description of the electronic behavior of

atoms and molecules pertains to their reactivity is an application of quantum chemistry.

Quantum chemistry lies on the border between chemistry and physics, and significant

contributions have been made by scientists from both branches of science. Organic chemists use

quantum chemistry to estimate the relative stabilities of molecules, to calculate properties of

reaction intermediates, to investigate the mechanisms of chemical reactions, to predict the

aromaticity of compounds and to analyze the NMR spectra. It is also applied by analytical

chemists for using the spectroscopic methods extensively and by inorganic chemists to study the

properties of determines the natural atomic orbitals, natural hybrid orbitals, natural bond

orbitals, natural localized molecular orbitals and uses them to perform natural population

analysis (NPA) and other tasks pertaining to localized analysis of wave function properties. This

program can be used extensively to study various kinds of second order interactions present in

the molecules hence is a tool to understand the different electronic interactions in the molecules

under investigation. 423,424

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3.1.1.2 Quantitative Structure Activity Relationship (QSAR)

It is almost 40 years since QSAR is in practice in the fields of agrochemistry, pharmaceutical

chemistry, toxicology and other facets of chemistry. It constitutes an important ligand based

technique as a drug design approach. This method employs the construction of QSAR models to

design new molecules. More than a century ago indications came from various groups that

physiological action of a substance is dependent on its chemical composition. QSARs attempt to

relate physical and chemical properties of molecules to their biological activities. This can be

achieved by using easily calculable descriptors (for example, molecular weight, number of

rotatable bonds, Log P). Further developments over the years and contributions of Hammett and

Taft laid the mechanistic basis for the development of QSAR paradigm by Hansch and Fujita.

Initially 2D QSAR or the Hansch approach took different kinds of descriptors, which did not

involve any 3D properties. It was known that the structural changes that affect biological

properties are electronic, steric and hydrophobic in nature. These properties were described by

Hammett as the electronic constants, Verloop Sterimol parameters and hydrophobic constants.

These type of descriptors are simple to calculate and allow for a relatively fast analysis but often

fail to take into account the 3D nature of chemical structures (which obviously play a part in

ligand-receptor binding, and hence activity).

The development in the field of molecular modeling and X-ray crystallography spawned 3D

QSAR methodologies that were based on molecular shape approaches developed independently

by Simon et at. and Hopfinger, distance geometry method by 'Crippen'. 3D QSAR uses probe-

based sampling within a molecular lattice to determine three- dimensional properties of

molecules (particularly steric and electrostatic values) and can then correlate these 3D

descriptors with biological activity. These are routinely used in CADD.4 Then evolved the most

successful module CoMFA (Comparative Molecular Field Analysis) developed by Crammer in

1988425

Similar approach was adopted in developing modules like CoMSIA (Comparative

Molecular Similarity Index Analysis),426

SOMFA (Self Organizing Molecular Field Analysis)

and COMMA (Comparative Molecular Moment Analysis). The broad history of the QSAR

is described in table 1.4. Utilization and predictivity of CoMFA itself has improved sufficiently

in accordance to the objectives to be achieved by it.427

Despite the formal differences between

the various methodologies, any QSAR method must include some identifiers of chemical

structures, reliably measured biological activities and molecular descriptors. In 3D QSAR,

structural descriptors are of immense importance in every QSAR model. Superimposition of the

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molecules is necessary to construct good models. The main problems encountered are related to

improper superposition of molecules, greater flexibility of the molecules, uncertainties about the

bioactive conformation and about different binding modes of ligands. The major assumptions in

3D QSAR include, the effect is produced by modeled compound and not it's metabolites, the

proposed conformation is the bioactive one, the binding site is the same for all modeled

compounds, the biological activity is largely explained by enthalpic processes, entropic terms

are similar for all the compounds, the system is considered to be at equilibrium, and kinetics

aspects are usually not considered, pharmacokinetics parameters like solvent effects, diffusion,

transport are not included. While considering the template, conformation from the crystal can be

extracted or conformation at the binding site can be taken. Otherwise conformational search can

be performed using anyone of the options like grid search, systematic search, random search or

distance, geometry (NMR-NOE). Alignment of Molecules is carried out using RMS atoms

alignment, moments alignment or field alignment. The relationship between the biological

activity and the structural parameters is obtained by linear or multiple linear regression analysis.

CoMFA (Comparative Molecular Field Analysis)

The CoMFA methodology is a 3D QSAR technique which allows one to design and predict

activities of molecules. The database of molecules with known properties is suitably aligned in

3D space according to various methodologies. After consistently aligning the molecules within a

molecular lattice, a probe atom (typically carbon) samples the steric and electrostatic

interactions between the probe atom and the rest of the molecule. Charges are then calculated for

each molecule at a level of theory deemed appropriate. These values are stored in a large

spreadsheet within SYBYL, from where they are accessed during the partial least squares (PLS)

routines. One then attempts to correlate these field energy terms with a property of interest by

the use of PLS with cross-validation, giving a measure of the predictive power of the model.

Electrostatic maps are generated indicating red contours around regions where high electron

density (negative charge) is expected to increase activity, and blue contours represent areas

where low electron density (partial positive charge) is expected to increase activity. Steric maps

indicate areas where steric bulk is predicted to increase (green) or decrease (yellow) activity.425,

427

CoMSIA (Comparative Molecular Similarity Index Analysis)

A technique similar to CoMFA called CoMSIA is also in use. Due to the problems associated

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with Lennard-Jones potential used in CoMFA methods, Klebe et al. have developed a similarity

indices-based method named as CoMSIA. It uses Gaussian-type functions to, model the probe

interactions which allows for much smoother sampling of the fields around the molecules, as

well as incorporating new field information such as hydrophobic and hydrogen bonding fields

instead of the traditional CoMFA potentials. The clear advantage of CoMSIA lies in the

functions used to describe the molecules as well as the resulting contour maps. It also avoids the

cutoff values used in CoMFA to prevent the potential functions from assuming unacceptably

large values.426

Apex-3D

Apex-3D is used to build 3D QSAR models which can be used for activity classification and

prediction. The importance of Apex-3D methodology lies in the automated identification of

pharmacophores. These biophores can be used for constructing 3D QSAR models when good

quantitative data is available, and for database searching. Combination of a 3D pharmacophore

with a quantitative regression equation is unique to the Apex-3D approach.428,429

4D QSAR

4D QSAR analysis developed by Vedani et al. incorporates the conformational, alignment, and

pharmacophore degrees of freedom in the development of 3D QSAR models. It is used to create

and screen against 3D-pharmacophore QSAR models and can be used in receptor-independent

or receptor-dependent modes. 4D QSAR can be used as a CoMFA pre-processor to provide

conformations and alignments in combination with CoMFA to combine the field descriptors of

CoMFA with the grid cell occupancy descriptors (GCOD) of 4D QSAR to build a "best" model,

or in addition to CoMFA because it treats multiple alignments, conformations and embedded

pharmacophores which are CoMFA limitations.430

5D QSAR

4D QSAR concept has been extended by an additional degree of freedom, the fifth dimension,

allowing for a multiple representation of the topology of the quasi- atomistic receptor surrogate.

While this entity may be generated using up to six different induced-fit protocols, it has been

demonstrated that the simulated evolution converges to a single model and that 5D QSAR, due

to the fact that model selection may vary throughout the entire simulation, yields less biased

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results than 4D QSAR where only a single induced-fit model can be evaluated at a time

(software Quasar).431

6D QSAR

The program Quasar generates a family of quasi-atomistic receptor surrogates that are optimized

by means of a genetic algorithm. The hypothetical receptor site is characterized, the surface of

which mimics the 3D shape of the binding site. It maps properties of interest, such as

hydrophobicity, electrostatic potential, and hydrogen-bonding propensity. The fourth dimension

(4D QSAR) takes in account an ensemble of conformations, orientations, and protonation states,

of each ligand molecule thereby reducing the bias in identifying the bioactive conformation and

orientation. Quasar evaluates the manifestation and magnitude of the induced fit of ligands to the

target protein, up to six different induced-fit protocols (5D QSAR). The most recent extension of

the Quasar concept to six dimensions (6D QSAR) allows for the simultaneous consideration of

different solvation models.422

This can be achieved explicitly by mapping parts of the surface

area with solvent properties (position and size are optimized by the genetic algorithm) or

implicitly. In Quasar, the binding energy is calculated as:

Ebinding = Eligand-receptor - Edesolvation, ligand - TΔS - Eintemal strain - Einduced fit

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The 3D QSAR methodology has been adopted in chapter 3.1

3.1.1.3 Molecular Docking

There are several possible conformations in which the ligand may bind to active site called as

binding modes. Molecular docking involves a computational process of searching for a

conformation of the ligand that is able to fit both geometrically and energetically into the

binding site of a protein. Docking calculations are required to predict the binding mode of new

hypothetical compounds. Docking procedure consists of three interrelated components, i.e.

identification of binding site, a search algorithm to effectively sample the search space (the set

of possible ligand positions and conformations on the protein surface) and scoring function. In

most docking algorithms, the binding site be predefined, so that the search space is limited to a

comparatively small region of protein. The search algorithm effectively samples the search

space of the ligand-protein complex. The scoring function used by the docking algorithm gives a

ranking to the set of final solutions generated by the search. Different energy calculations are

needed to identify the best candidate. The stable structures of a small molecule correspond to

minima on the multidimensional energy surface. Different forces that are involved in binding are

considered - electrostatic origin, electrodynamic origin, steric forces and solvent related forces.

The free energy of a particular conformation is equal to the solvated free energy at the minimum

with the small entropy correction. All energy calculations are based on the assumption that the

small molecule adopts a binding mode of lowest free energy within the binding site. The free

energy of binding is the change in free energy that occurs on binding.

ΔGbinding = Gcomplex - (Gprotein + Gligand)

Where Gcomplex is the energy of the complexed protein and ligand, Gprotein is the free energy of

noninteracting separated protein and Gligand is the free energy of noninteracting separated ligand.

The common search algorithms used for the conformational search, that provide a balance

between the computational expense and the conformational search include, molecular dynamics,

Monte Carlo methods, genetic algorithms, fragment-based methods, point complementary

methods, distance geometry methods, tabu searches, systematic searches.433

Scoring functions are used to estimate the binding affinity of a molecule or an individual

molecular fragment in a given position inside the receptor pocket. Three main classes of scoring

functions are known which includes force field based methods, empirical scoring functions and

knowledge based scoring functions.

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The force field scoring function uses nonbonded energies of existing, well established molecular

mechanics force fields for estimating binding affinity. The AMBER and CHARMM nonbonded

terms are used as scoring functions in several docking programs. The van der Waals term of

force fields is mainly responsible for penalising docking solutions with respect to overlap

between receptor and ligand atoms. It is often omitted when only the binding of experimentally

determined complex structure is analysed. A new contribution to the list of force field based

scoring methods is OWFEG (One Window Free Energy Grid) by Charifson and Pearlman.

In case of empirical scoring functions the binding free energy of the noncovalent receptor-ligand

complex can be factorised into some of localised, chemically intuitive interactions. Such

decompositions are useful to gain insight into binding phenomenon even without analysing 3D

structures of receptor-ligand complexes. Empirical scoring functions usually contain individual

terms for hydrogen bonds, ionic interactions, hydrophobic interactions and binding entropy, as

in the case of SCORE employed in DOCK4 and Bohm scoring functions used in FlexX.

Empirical scoring functions regard only explicit interactions. Less frequent interactions are

usually neglected.

Knowledge based scoring functions overcome this drawback and try to capture the knowledge

about protein-ligand binding that is implicitly stored in the protein data bank by means of

statistical analysis of structural data, for example PMF and DrugScore functions, Wallqvist

scoring function, the Verkhivker scoring function.422,434,435,436,437

Various molecular docking softwares are available of which widely used ones are FlexX, 438

Flexidock, 439

DOCK, 440

AUTODOCK, 441

etc.

FlexX

FlexX is fragment-based method. It handles the flexibility of the ligand by decomposing the

ligand into fragments and performs the incremental construction procedure directly inside the

protein active site. It allows conformational flexibility of the ligand while keeping the protein

rigid.

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MCDOCK

MCDOCK is a non conventional Monte Carlo simulation technique in which Monte Carlo

simulation method is used to search the global minimum. The scoring function used is based

upon molecular mechanics as used in CHARMM and AMBER programs. It allows full

flexibility of ligands by explicitly sampling all the rotatable bonds of the ligand and the ligand

conformational energy is assessed by the atomic interactions of the ligands. A number of

techniques have been integrated to optimize its docking efficiency.435, 442

FlexE

It addresses the problem of protein structure variations during docking calculations. It can dock

flexible ligands into an ensemble of protein structures which represent the flexibility, point

mutations or alternate models of a protein. It is based on the united protein description generated

from the superimposed structures of the ensemble. For varying parts of the protein discrete

alternative conformations are explicitly taken into account which can be combinatorially joined

to create new valid protein structures. FlexE is based on FlexX.443

DREAM++

DREAM++ (Docking and Reaction programs using Efficient search Methods) is a set of

programs to design combinatorial libraries that uses techniques of a hybrid algorithm between

backtrack and incremental construction algorithms and inheritance of conformations through

reactions. The correlation between the binding affinity and the number of conformations can be

examined to find the best way to screen virtually generated molecules.444

3.1.1.4 Pharmacophore mapping

The pharmacophore is an important and unifying concept in rational drug design which

embodies the notion that molecules are active at a particular receptor because they possess a

number of key features that interact favorably with this receptor and which possess geometric

complementarity to it. It is the spatial arrangement of key chemical features that are recognized

by a receptor and are thus responsible for ligand-receptor binding. Pharmacophore models are

typically used when some active compounds have been identified but the three dimensional (3D)

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structure of the target protein or receptor in unknown. It is possible to derive pharmacophores in

several ways, by analogy to a natural substrate, by inference from a series of dissimilar

biologically active molecules (active analog approach) or by direct analysis of the structure of

known ligand, and target protein. The active compounds are superimposed to determine their

common features to provide a pharmacophore map that explains ligand-receptor binding. Given

a set of active molecules, the mapping of a pharmacophore involves two steps: (i) analyzing the

molecules to identify pharmacophore features, that is to identify atoms that interact with a

receptor and (ii) align the active conformations of the molecules to find the best overlay of the

corresponding features. The main difficulty in pharmacophore mapping is in handling

conformational flexibility. The major differences between the programs lie in the algorithms

used for the alignment and in the way in which conformational flexibility is handled.445

Besides ligand-based approach, pharmacophores can also be generated manually. Common

structural features are identified from a set of experimentally known active compounds.

Conformational analysis is carried out to generate different conformations of the molecules and

interfeature distances are inferred to develop the final models.

The receptor mapping technique is also currently in practice to develop pharmacophores. The

pharmacophore models can be derived using the receptor active site. The important residues

required for binding the pharmacophores are identified, which are employed for generating the

pharmacophores. The structure of protein can be used to generate interaction sites or grids to

characterize favorable positions for ligands. Four types of interactions sites, which are hydrogen-

bond donors, hydrogen-bond acceptors, aliphatic and aromatic groups, are characterized.

Aliphatic and aromatic side chains are important as they also pack closely to form hydrophobic

core or proteins. Molecular dynamics simulation for generating diverse protein conformations

have been proposed to introduce protein flexibility in the pharmacophore development.

After a pharmacophore map has been derived there are two ways to identify molecules which

share its features and thus elicit the desired response. (a) First is the de novo drug design which

seeks to link the disjoint parts of the pharmacophore together with fragments in order to generate

hypothetical structures that are chemically novel. (b) The second is the 3D database searching,

where large databases comprising 3D structures are searched for those that match to a

pharmacophoric pattern. One advantage of this method is that it allows the ready identification

of existing molecules which are either easily available or have a known synthesis.446

The two

distinct approaches being used for 3D database searches include shape based methods in which a

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protein structure is used to formulate the database query, to search for compounds whose

structure complements the receptor's steric characteristics and pharmacophore based methods

which search for compounds whose structure satisfies a certain pharmacophoric pattern.

Generally pharmacophores are taken from the literature and are used as 3D search queries or are

taken from protein crystal structure.447

To propose the 3D requirements for a molecule to exhibit a particular bioactivity, bioactive

conformation and a superposition rule for every active compound needs to be supplied. For this

pharmacophore mapping, generation and optimization of the molecules, the location of ligand

points and site points (projections from ligand atoms to atoms in the macromolecule) is carried

out. Typical ligand and site points are hydrogen bond donors, hydrogen bond acceptors, and

hydrophobic regions such as centers of mass of aromatic rings. A pharmacophore map identifies

both the bioactive conformation of each active molecule and how to superimpose, compare in

3D, the various active compounds. The map identifies what types of points match in what

conformations of the compounds.

A complication for pharmacophore mapping is that different ligands might approach a polar site

point from different directions, the result is that in a pharmacophore map, the positions of the

ligand atoms may not overlap even though their projections to the macromolecule do, in the case

of hydrogen bonding interactions. Hence to match these features in the ligands superimposition

of only the ligand based points is done. For the pharmacophore mapping of potent small

molecules, it is preferred that the proposed bioactive conformation be a low energy one. It is

assumed that the interacting atoms of the biomolecule can move slightly and with little energy

cost to make optimal interaction with a ligand. Thus a larger tolerance is accepted between

overlapping points if the resulting pharmacophore map includes significantly lower energy

ligand structures.445

Experience suggests that it is computationally expensive to search

conformational space of a molecule. Especially if a molecule is flexible, even 100 structures

may not be enough. Using molecular graphics to compare many conformations of several

molecules can take much human time. Moreover these comparisons can be biased by the order

in which one studies them. The pharmacophore mapping softwares therefore sought an objective

and fast method that would compare all low energy conformations of all molecules

simultaneously. Of the various methods employed to superimpose two defined 3D structures

when the knowledge of the corresponding atoms is unknown, the most attractive solution is a

method given by Brint and Willett based on clique detection technique. DISCO softwares

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became popular for performing pharmacophore mapping.

Distance Comparison method (DISCO)

The various steps involved in DISCO are conformational analysis, calculation of the location of

the ligand and site points, to find potential pharmacophore maps and graphics analysis of the

results. In the process of conformational search, 3D structures can be generated using any

building program like CONCORD, from crystal structures or from conformational searching and

energy minimization with any molecular or quantum mechanical technique. Comparisons of all

the duplicate conformations are excluded while comparing all the conformations. If each

corresponding interatomic distance between these atoms in the two conformations is less than a

threshold (0.4A), then the higher energy conformation is rejected. DISCO calculates the location

of site points to be considered for the pharmacophore. These points can be the location of ligand

atoms, or other atom-based points, like centers of rings or a halogen atom are points of potential

hydrophobic groups. The other point is the location of the hydrogen bond acceptors or donors.

The default locations of site hydrogen bond donor and acceptor points are based on literature

compilations of observed intermolecular crystallographic contacts in proteins and between the

small molecules. Hydrogen bond donors and acceptors such as OH and NH2 groups can rotate to

change the locations of the hydrogen atom.

During the process of performing pharmacophore mapping in DISCO, its set up iterates the

tolerance used to set inter-point distances the same. The starting and the final values and an

iteration increment are given. Alternatively, the user may input the tolerance for each type of

inter-point distance. The user may direct DISCO algorithm to consider all the potential points

and to stop when a pharmacophore map with a certain total number of points is found.

Alternatively they may specify the types of fewer features an inhibitor maps to, the poorer its fit

to them and lower its predicted affinity.448, 449

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Theoretical generation of pharmacophoric features using theoretical structure activity

relationship (SAR) are highlighted in chapter1.

3.1.2 VIRTUAL SCREENING

Virtual screening also called as in silico screening is a new branch of medicinal chemistry that

represents a fast and cost effective tool computationally screening compound databases in search

for novel drug leads. Various changes have occurred in the ways of drug discovery, the major

ones taking place in the field of high throughput synthesis and screening techniques. The basic

goal of virtual screening is the reduction of the huge chemical space of synthesized molecules

and to screen them against a specific target protein. Thus the field of virtual screening has

become an important component of drug discovery programs. Substantial efforts in this area

have been put in by large pharmaceutical companies. However, the area of virtual screening is

highly diverse and is evolving rapidly. Various reports describing the development of new

methods are coming up. Many chemists and biologists perceive this field as an `algorithmic

jungle` that lacks well defined standards .435

Types of virtual libraries

Two types of virtual libraries can be generated. One is produced by computational design. The

basic idea behind this is of course not to synthesize all compounds (as in combinatorial library

design, for example) but to select only preferred molecules. Other libraries focus on compounds

that already exist from commercial sources or company inventories. It is possible to extract

millions of compounds from these sources and create virtual library. These libraries are useful in

the absence of knowledge about the specific drug targets for virtual screening. More focused

libraries are important sometimes to save resources as it may be more prudent not to run the

entire HTS file against the target protein instead of focused library with higher chances of

containing hits may be scrutinized .450

Applications

Virtual screening can be applied to target-based subset selection from the data bases. Statistical

approaches like binary QSAR or recursive partitioning can be applied to process HTS results

and develop predictive models for biological activity. The developed models are employed to

select candidate molecules from databases. Similarly, hits from HTS are used in fingerprint

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searches or compound classification analysis to identify set of similar molecules. Based on these

results, a few compounds are selected for additional testing. Many assumptions are made in

virtual screening and a positive outcome can not be guaranteed every time. However, the overall

process is extremely cost effective and fast. Virtual screening has the ability to produce leads

that otherwise may not have been identified. In silico mining of compounds no longer requires

an expensive infrastructure. Clustering of low-cost computers make it possible to process,

analyze, and search a large amount of data that is otherwise difficult to handle. With the merging

of bioinformatics and cheminformatics, virtual screening is expected to become increasingly

better established. Improvements in current techniques and the development of new methods

will continue at a fast pace, keeping the areas stimulating and interesting.435,450.

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3.1.3 COMPUTATIONAL ANALYSIS

3D-QSAR tools are applied to derive indirect binding information from the correlation between

the biological activity of a training set of molecules and their 3D structures which could

facilitate in designing New Chemical Entities (NCEs) for therapeutic purpose. The importance

of steric and electrophilic characteristics is revealed by aligning structurally similar analogues

using pharmacophoric features as structural superimposition guides.23

The results of

Comparative Molecular Field Analysis (CoMFA) performed on a series of Eosinophil Inhibitors

as Antiasthmatic Agents have been reported. CoMFA, the most popular 3D-QSAR method has

been chosen because of the renowned robustness of the model it produces. The aim was to

analyze structural requirements of the eosinophil inhibitors to understand the structural basis for

their affinity of the theoretically designed new molecules and to guide the design and synthesis

of more potent inhibitors with predetermined affinities.

MATERIALS AND METHODS

Dataset for analysis

The Dataset used for QSAR studies, was collected from two reference papers* available in

literature reporting % eosinophil inhibition. Sixty four compounds were chosen from the quoted

literature to build the data set (Table CI). The two molecules; 25 and 56 were found to be the

most active molecules with highest %inhibition activities with respect to lowest molar dose

(Table CI). The dataset molecules were classified into two sub-datasets of 43 (Table CII) and 22

(Table CIII) compounds according to their eosinophil inhibition activity on different dose i.e.

30mg/kg and 3mg/kg respectively. Data set with activities reported for a fixed molar dose was

not reported. Therefore, the % inhibition activities of the data set molecules at a particular dose

were converted to molar dose for both types of compounds.

First of all the molar dose for the 43 compounds of the first sub-data set were calculated

followed by the calculation of the average. None of the molecules were found to have a molar

dose within the range of 10%, 15%, even 20% of that of the most active molecules. So this set

(Table CII) was discarded. Then the molar dose of 22 compounds of the second sub-data set and

average were calculated in a similar fashion (Table CIII). Eighteen molecules from this set were

found to have a molar dose within the range of 15% of that of the most active molecules. So, 18

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compounds thus selected constituted the data set molecules for generating the CoMFA model.

Leave One Out (LOO) analysis to build a good predictive model was carried out with this

dataset of 18 molecules (Table C1a). It was found during the analysis that deleting the molecule

(18) improved the cross validated regression coefficient (r2

cv) that measures the predictive ability

of the CoMFA model. Consequently, the rest 17 compounds were kept in the data set for further

analysis. However, the (r2

cv) obtained from this dataset was not good enough for a good

predictive model. Two more molecules (9, 15) were left out from the 17 compounds dataset

(Table C1b) during the process of further LOO analysis to finally remain with a dataset

consisting of 15 compounds. The statistical results of the models generated have been shown in

Tables C2a, C2b1 and C2b2 respectively. This set was divided into a training set of 12 molecules

(Table C3) and a test set of 3 molecules. The model generated by this training set was found to

be with appreciable statistical results of r2

cv = 0.445 (near to 0.5), r2

ncv= 0.999 (~1) and lower

value of standard error of estimation (SEE) (Table 2b2). The number of test set molecules was

increased by adding two more molecules 8 and 13, previously well predicted, to give a test set of

5 molecules (Table C4).

MOLECULAR MODELING AND MINIMIZATIONS

Molecular structures generated in the computer have to be optimized to find the individual

energy minimum state. This is normally carried out with molecular mechanics or quantum

mechanical methods. In the course of minimization procedure, the molecular structure is

relaxed. The internal strain in the structures due to small deviation in the bond lengths and

angles are rectified. Before starting the geometry optimization, Van der Waals contacts should

be removed. Several advantages like speed, sufficient accuracy and the broad applicability on

the small molecules as well as on large systems have established the force field geometry

optimization as the most important standard method.

The 3D-QSAR was performed using SYBYL 7.3 installed on a RedHat Enterprise Linux

workstation available at our in silico Drug Design Lab. The structure of 1,2,4 triazole based

template (25, Table CI, Figure CA)* was constructed using the sketch module available in the

molecular modeling software package SYBL 7.3.*

Gasteiger-Huckel partial atomic charges were

assigned using Tripos force field. A constrained minimization followed by full minimization

was carried out on this molecule in order to prevent the conformations moving to false region.

121

Powell’s conjugate gradient method was used for minimization*. The minimum energy

difference of 0.001 kcal/mol was set as convergence criterion. The energy minimized structure

was taken as the template (Sr. No. 1, Table C4) and the molecules of the training and test set

thus selected were modeled onto it and minimized in a similar way and subsequently subjected

to database alignment(Figures C4 and C5).

MOLECULAR ALIGNMENT

The most active molecule (25, Table C1,) was built by the sketch module of SYBYL 7.1*

installed on a Silicon graphics IRIX workstation (NIPER, Mohali) for FlexS alignment and also

on SYBYL 7.3 installed on a RedHat Enterprise Linux workstation available at our in silico

Drug Design Lab for generating CoMFA model. This was taken as the template (Figure CA)

after minimization. All the 84 theoretically designed molecules (Table C5-C10) were modeled

onto the template and minimized. All the energy optimized molecules were subjected to

Database Alignment (Figure C1) and Fit Atom Alignment (Figure C2). All these molecules

were also specifically subjected to FlexS Alignment studies (Figure C3) to generate the Total

and Normalized Score to aid in selecting the molecules for further synthesis. All the designed

molecules were also subjected to fit atom alignment using the set of atoms from the template as

shown in Figure CA.

The most active molecule (25, Table C1,)was built by the sketch module of Sybyl 7.3* installed

on RedHaT enterprise a Silicon graphics IRIX workstation (NIPER, Mohali) for FlexS

alignment.

CoMFA (COMPARATIVE MOLECULAR FIELD ANALYSIS)

The CoMFA methodology could be utilized to design and predict activities of molecules. The

database of molecules with known properties is suitably aligned in 3D space according to

various methodologies. This generates a model by the relationship between molecular field

differences of a set of molecules and differences in their biological activity. Molecular fields are

defined in terms of the interaction energies of some probe atom placed at the nodes of a grid

surrounding the molecules. After consistently aligning the molecules within a molecular lattice,

122

a probe atom (typically carbon) samples the steric and electrostatic interactions between the

probe atom and the rest of the molecule. Charges are then calculated for each molecule at a level

of theory deemed appropriate. These values are stored in a large spreadsheet within SYBYL,

from where they are accessed during the partial least squares (PLS) routines. One then attempts

to correlate these field energy terms with a property of interest by the use of Partial Least Square

Analysis (PLS) with cross-validation, giving a measure of the predictive power of the model.

The QSAR produced by the CoMFA model, are usually represented as 3D ‘coefficient contour

maps’. The steric contour maps are represented in green and yellow while the electrostatic

contour maps are represented in red and blue. Contour maps were generated as scalar product of

coefficients and the standard deviation associated with each CoMFA column. Electrostatic maps

are generated indicating red contours around regions where high electron density (negative

charge) is expected to increase activity, and blue contours represent areas where low electron

density (partial positive charge) is expected to increase activity. In other words, the red contours

represent regions that lead to the enhancement of activity with electron rich groups, and contrary

to that the blue regions represent electron deficient regions and can lead to an increase in the

activity of molecules by similar substitutions. On the other hand green contours are indicative

of favorable regions for sterically bulkier groups and the yellow contours are indicative of

regions that are sterically less favorable. The 3D-QSAR contour maps revealing the contribution

of CoMFA fields are shown in Figures C8a, C9 with the template 1 and 2 inside (Figure CA).

After generating the best predictive model with the training set available, most of the

theoretically designed molecules were placed inside the contours obtained from the model to

judge how better the designed molecules fit into the contours (Figures C11- C23). Thus

analyzing and justifying the desired alignment (Fit Atom and FlexS), steric and electrostatic

requirements (CoMFA) for better activity as a criterion has been developed to select molecules

for further synthesis, also taking into account the predicted alignment scores and activities

(Tables C5-C10, Figure C1 and C3)

123

CoMFA Interaction Energy Calculation

The steric and electrostatic CoMFA fields were calculated at each lattice intersection of a

regularly spaced grid of 2.0 Å in all three dimensions within defined region. The Van der Waals

potential and columbic terms representing the steric and electrostatic fields respectively were

calculated using standard Tripos force fields. A distance dependent dielectric constant of 1.00

was used. A sp3

carbon atom with +1.00 charges was used as a probe atom. The steric and

electrostatic fields were ignored at the lattice points with maximal steric interactions.

Partial Least Square (PLS) analysis

PLS method was used to linearly correlate the CoMFA fields to the inhibitory activity values.

The cross-validation analysis was performed using leave one out method (LOO) in which one

compound is removed from the dataset and its activity is predicted using the model derived from

the rest of the dataset. The cross validated r2

that resulted in optimum number of components

and lowest standard error of prediction were considered for further analysis. Equal weights were

assigned to steric and electrostatic fields using CoMFA-standard scaling option. To speed up the

analysis and reduce noise, a minimum filter value σ of 2.00 kcal/mol was used. Final analysis

was performed to calculate conventional r2

using the optimum number of components.

124

RESULTS AND DISCUSSION

Statistical analysis

CoMFA model obtained with 12 molecules in training set and 5 molecules in test set resulted in

a cross validated r2

of 0.445 with minimum standard error and optimum numbers of

components. The PLS statistics for CoMFA are shown in Table C2. This analysis was used for

final non-cross validated run, giving a correlation coefficient value of 0.999 with a low standard

error of estimate giving a good linear correlation between the observed and computed affinities

of the compounds in the training set. Contributions of steric and electrostatic fields are in the

ratio 76.6:23.4. A plot of predicted (CoMFA) versus actual activity for training and test set

molecules is shown in Figure CB. The actual, predicted and residual values of training and test

set for CoMFA are given in Tables C3 and C4 respectively. Contour maps were generated as

scalar product of coefficients and standard deviation associated with each CoMFA column. The

CoMFA contour maps revealing the contribution of steric and electrostatic fields are shown in

figures C8a and C9.

Contour analysis:

Steric Contours

The steric contours have been shown figure C9 with two most active molecules (1,13 of Table

C4) inside. The steric contours mainly appear around the 4-chlorophenyl ring of the template.

Though a fine green line appears above the middle part of the molecule, the contribution is so

less that it can be ignored. A green contour on the left hand side is observed near the 4- position

of the phenyl ring covering the region from front and back side strongly indicating the

requirement of bulky groups in the region favorable for the activity (Figure C9a and C9b).

Placing larger groups like chloro, bromo and cyano in stead of keeping sterically small

substituents in this position or keeping the positon unsubstituted in the designed molecues is

accounted for by this contour. There is though a very small yellow contour in front of the 4-

position indicating preference of smaller bulk but it is far away from that particular position.

Other yellow contours can be seen around the C3 and C3-C4 positions of the phenyl ring. Two

yellow contours pose face to face around the C3. The first yellow rhombohedral map can be

noticed near the C3 position in the front view image and the other on the opposite side near to

the same position that is clearly visible in the rear view image. The presence of two yellow

125

contours near this region boldly supports the fact that even small bulk in this position is

detrimental for the activity. So this position needs to be kept unsubstituted while designing

NCEs. The third yellow map is positioned in front of the C3-C4 bond of the phenyl ring

providing further support to the above reasoning for the design of new molecules.

Electrostatic Contours

The electrostatic contours have been shown figure C9 and C10 with two most active molecules

(1, 13 of Table C4) inside. These contours can be seen spreading around almost the whole

template. A blue sleek contour ranges from the C4-C3 bond to a region near to C2 position of the

phenyl ring. This also explains the need of small substituent in those positions as already

indicated by the yellow steric maps. A small blue cubic contour can be observed near the bond

connecting the phenyl ring with the triazole ring and a small trigonal blue contour is seen near to

the C3 position of the phenyl ring indicating the sites required to impose electropositive

character. These positions were kept unsubstituted to avoid undesired bulk in the designed

molecules. A large hood shaped blue contour can be noticed covering the amino alkyl group in

both the template molecules (i.e., for both cyclic and acyclic systems) on the right hand side

indicating a site where electron deficiency will favor the activity. So placing N-tBu group in the

acyclic and N-Me groups in the cyclic systems resulted in better predicted activities of the

theoretically designed molecules. A cap shaped blue contour can also be seen over the triazole

ring and far away from the thiocarbonyl group. A rhombohedral red contour can be observed

near the triazole ring ‘N’ and the neighbouring amino group indicating a very important feature

that electron rich character at these sites will favor the activity. This has been specially found to

be so in the designed molecules in reference to the suitability of the heterocyclic rings thus

chosen to occupy this region to satisfy the electronegative requirement.

The FlexS Alignment and CoMFA studies carried out for generating alignment scores

(Total and Normalized) and activity predictions (% eosinophil inhibition) of all the 84

theoretically designed molecules lead to certain conclusions to select molecules for further

synthesis:

All the molecules of the imidazoline based series were deleted as a result of Fit

Atom Alignment Studies (Figure C12).

126

The n-propyl and iso-propyl substituted molecules were deleted from all the series

except imidazole, on the basis of FlexS Alignment Scores and CoMFA generated

Activity Prediction Studies (Figure C12, Tables C5-10).

Ethyl substituted molecules were also deleted as is recommended in the literature43

.

Some of the N-Methycarboxamide and N-tert-Butyl-carboxamide containing

molecules have shown better activity then the corresponding N-

Alkylcorbothioamide analogs and hence were selected for synthesis (Tables C5-10).

Molecules selected for synthesis on the basis of all these computational studies

(virtual screening) are highlighted in Green colour (carbothioamide based and

Orange colour (carboxamide based) in various Tables C5-10.

STRUCTURE OF THE MOST ACTIVE MOLECULES

INHIBITING INTERLEUKIN-5 INDUCES SURVIVAL OF

EOSINOPHILS

127

N

N

NH2

Cl

NHS

CH3

5

32

4

7

6

1

8

THE TEMPLATE (1, Table CI)

N

N

N

N

N

Cl

S CH3

THE CYCLIC ANALOG (2, Table CI)

Figure CA

Figure CB

12

8

FlexS is a computer program for predicting ligand superpositions. For a given template

molecule and set of ligands, FlexS predicts conformation(s) and orientation(s) of the ligands

relative to the template. FlexS combines conformational search with alignment. The

conformational search generates new conformations in two steps: (a) By selection and placement

of a base fragment (of the current ligand) on the template; (b) By incremental build up of the

remaining parts of the ligand. As the incremental build proceeds torsion angles are perturbed to

generate new conformations. Each conformation is then scored for superposition quality.

After sketching and minimising the sketched molecules they are subjected to FlexS alignment

job. FlexS produces a database and a spreadsheet for each ligand it successfully screened. The

results are displayed in terms of 'total score' and 'normalised scores'. The Total Scores are similar

to energies only in that the more negative the value, the better the alignment. The Normalized

Score is the total score divided by the score for the current test ligand aligned to itself. Because

the score is highly volume dependent, a set of ligands with a wide range of volumes is

problematic to compare just based on Total Score values. The Normalized score tries to correct

for this kind of difference. On the other hand, a large ligand with a region that aligns well with

the template may score relatively poorly in Normalized Score. Therefore, it is useful to look at

both score orders when considering which ligands to look at more closely451-452

.

130

Table CI: Data Set – 64 Compounds reported in literature for

(% Inhibition of eosinophilia)*

S. No.

Structures

% Inhibition

c = 30 mg/kg

d = 3 mg/kg

S. No.

Acc. to

Reference

paper

No. of moles

1 N

N

N

NH2

NH

S

93c 1a .000191

2 N

N

N

NH2

NH

S

CH3

93c 1b .000162

3 N

N

N

NH2

NH

S

CH3

21c 1c .000150

4 N

N

N

NH2

NH

S (CH2)3OEt

1c 1e .000131

5 N

N

N

NH2

NH

S Cyclohexyl

31c 1f .000132

6 N

N

N

NH2

NH

S Bn

40c 1h .000136

131

7 N

N

N

NH2

NH

SCH3

CH3

34c 3 .000162

8 N N

NH2

NH

S

27c 4 .000192

9

N

N

N

N

NH2

NH

S

53c 5 .000189

10 NH

N

NH2

NH

S

5c 7b .000192

11 S

NH2

NH

S

4c 7c .000174

12

NH2

S

NH

47c 7d .000180

13

N NH2

S

NH

27c 7e .000179

14 N

NH2

S

NH

9c 7f .000179

15 N

N NH2

S

NH

27c 7g .000178

132

16 N

N

N

NH2

NH

O

40c 8 .000212

17 N

N

N

NH2

S

S

36c 9 .000172

18 N

N

N

NH

S

NH2

25c 13 .000191

19 N

N

N

NH2

NH

S

NH2

53c 14 .000174

20 N

N

N

NH

S

CH3

13c 18 .000175

21 N

N

N

NH

S

SCH3

54c 19 .000159

22 N

N

N

NH

S

54c 23a .000137

23 N

N

N

NH

S

CH3

65c 23b .000129

133

25 N

N

N

NH

S

Cl

88d,c

23c

.000118

And

.0000118

26 N

N

N

NH

S

NC

87c 23d .000123

27 N

N

N

NH

S

MeO

70c 23e .000120

28 N

N

N

NH

S

Ethoxalylamino

49c 23h

.000142

29 N

N

N

NH

S

3-Methylthiouriedo

31c 23i

.000145

30

N

N

N

NH

S

Cl

54d 23j .0000118

31 N

N

N

NH

S

Cl

18d 23k .0000118

134

32 N

N

N

NH

S

F

73d 25a .0000127

33 N

N

N

NH

S

Br

88d 25b .0000101

34 N

N

N

NH

S

F3C

91d

25c .0000102

35 N

N

N

NH

S

Cl Cl

91d 25d .0000104

36

N

N

N

NH

S

Cl

Cl

85d 25e .0000104

37 N

N

N

NH

S

Cl OMe

68d 25f .0000106

38 N

N

N

NH

S

2-Furyl

18d 2a .0000144

135

39 N

N

N

NH

S

4-Chloro

-1

-naphthyl

14d 2e .00000911

40 N

N

N

NH

S

Cl

25d 2g .0000107

41

N

N

N

NH

S

58d 2i .0000102

42 N

N

N

NH

S

N

37d 2k .0000122

43 N

N

N

NH

S

S

CH3

54c 12 .000159

44 N

N

NN

S

N

69c 3a .000179

45 N

H

N

N

S

N

9c 3b .000178

46

N

S

N

22c 3d .000170

136

47 N

N

NN

O

N

17c 8 .000198

48 N

N

NNH

EtO

N

6c 9 .000178

49 N

N

NNH

NH

N NH

CH3

CH3

38c 11a .000166

50 N

N

NNH

NH

N NH

Cyclohexyl

Cyclohexyl

1c 11b .0000949

51 N

N

NN

S

N

69c 3a .000222

52 N

N

NN

S

N

CH3

49c 3e .000184

53 N

N

NN

S

N

CH3

23c 3f .000169

54 N

N

NN

S

N CH3

18c 4a .00020

55 N

N

NN

S

N

29c 5a .000142

137

56 N

N

NN

S

NCl

90d,c

3h

.000121

And

.0000121

57 N

N

NN

S

NF

38d 3i .0000131

58 N

N

NN

S

NBr

84d 3j .0000103

59 N

N

NN

S

NNC

79c 3k .000132

60 N

N

NN

S

NF3C

87d 3l .0000108

61 N

N

NN

S

NCl

Cl

90d 3m .0000107

62

N

N

NN

S

NCl

Cl

89d 3n .0000107

63 N

N

NN

S

NCl CH3

60d 4b .000115

138

64 N

N

NN

S

NCl

5d 5b .0000093

S No. 1-37 compounds43

J. Med. Chem. 1996, 39, 3019-3029

S No. 38-64 compounds44

J. Med. Chem. 1998, 41, 2985-2993

139

Table CII: All 43 Compounds reported in literature for

(% Inhibition of eosinophilia)* at 30 mg/kg dose

S. No.

Structures

% Inhibition

c = 30 mg/kg

d = 3 mg/kg

S. No.

Acc. to

Reference

paper

No. of moles

1 N

N

N

NH2

NH

S

93c 1a .000191

2 N

N

N

NH2

NH

S

CH3

93c 1b .000162

3 N

N

N

NH2

NH

S

CH3

21c 1c .000150

4 N

N

N

NH2

NH

S (CH2)3OEt

1c 1e .000131

5 N

N

N

NH2

NH

S Cyclohexyl

31c 1f .000132

6 N

N

N

NH2

NH

S Bn

40c 1h .000136

140

7 N

N

N

NH2

NH

SCH3

CH3

34c 3 .000162

8 N N

NH2

NH

S

27c 4 .000192

9

N

N

N

N

NH2

NH

S

53c 5 .000189

10 NH

N

NH2

NH

S

5c 7b .000192

11 S

NH2

NH

S

4c 7c .000174

12

NH2

S

NH

47c 7d .000180

13

N NH2

S

NH

27c 7e .000179

14 N

NH2

S

NH

9c 7f .000179

15 N

N NH2

S

NH

27c 7g .000178

141

16 N

N

N

NH2

NH

O

40c 8 .000212

17 N

N

N

NH2

S

S

36c 9 .000172

18 N

N

N

NH

S

NH2

25c 13 .000191

19 N

N

N

NH2

NH

S

NH2

53c 14 .000174

20 N

N

N

NH

S

CH3

13c 18 .000175

21 N

N

N

NH

S

SCH3

54c 19 .000159

22 N

N

N

NH

S

54c 23a .000137

23 N

N

N

NH

S

CH3

65c 23b .000129

142

24 N

N

N

NH

S

Cl

88d,c

23c .000118

25 N

N

N

NH

S

NC

87c 23d .000123

26 N

N

N

NH

S

MeO

70c 23e .000120

27 N

N

N

NH

S

Ethoxalylamino

49c 23h .000142

28 N

N

N

NH

S

3-Methylthiouriedo

31c 23i .000145

29 N

N

N

NH

S

S

CH3

54c 12 .000159

30 N

N

NN

S

N

69c 3a .000179

31 N

H

N

N

S

N

9c 3b .000178

143

32

N

S

N

22c 3d .000170

33 N

N

NN

O

N

17c 8 .000198

34 N

N

NNH

EtO

N

6c 9 .000178

35 N

N

NNH

NH

N NH

CH3

CH3

38c 11a .000166

36 N

N

NNH

NH

N NH

Cyclohexyl

Cyclohexyl

1c 11b .0000949

37 N

N

NN

S

N

69c 3a .000222

38 N

N

NN

S

N

CH3

49c 3e .000184

39 N

N

NN

S

N

CH3

23c 3f .000169

40 N

N

NN

S

N CH3

18c 4a .00020

144

41 N

N

NN

S

N

29c 5a .000142

42 N

N

NN

S

NCl

90d,c

3h

.000121

43 N

N

NN

S

NNC

79c 3k .000132

S No. 1-28 compounds43

J. Med. Chem. 1996, 39, 3019-3029

S No. 29-43 compounds44

J. Med. Chem. 1998, 41, 2985-2993

145

Table CIII: All 22 Compounds reported in literature for

(% Inhibition of eosinophilia)* at 3 mg/kg dose

S. No.

Structures

% Inhibition

c = 30 mg/kg

d = 3 mg/kg

S. No.

Acc. to

Reference

paper

No. of moles

1 N

N

N

NH

S

Cl

88d,c

23c

.000118

And

.0000118

2

N

N

N

NH

S

Cl

54d 23j .0000118

3 N

N

N

NH

S

Cl

18d 23k .0000118

4 N

N

N

NH

S

F

73d 25a .0000127

5 N

N

N

NH

S

Br

88d 25b .0000101

146

6 N

N

N

NH

S

F3C

91d

25c .0000102

7 N

N

N

NH

S

Cl Cl

91d 25d .0000104

8

N

N

N

NH

S

Cl

Cl

85d 25e .0000104

9 N

N

N

NH

S

Cl OMe

68d 25f .0000106

10 N

N

N

NH

S

2-Furyl

18d 2a .0000144

11 N

N

N

NH

S

4-Chloro

-1

-naphthyl

14d 2e .00000911

12 N

N

N

NH

S

Cl

25d 2g .0000107

147

13

N

N

N

NH

S

58d 2i .0000102

14 N

N

N

NH

S

N

37d 2k .0000122

15 N

N

NN

S

NCl

90d,c

3h

.000121

And

.0000121

16 N

N

NN

S

NF

38d 3i .0000131

17 N

N

NN

S

NBr

84d 3j .0000103

18 N

N

NN

S

NF3C

87d 3l .0000108

19 N

N

NN

S

NCl

Cl

90d 3m .0000107

20

N

N

NN

S

NCl

Cl

89d 3n .0000107

148

21 N

N

NN

S

NCl CH3

60d 4b .000115

22 N

N

NN

S

NCl

5d 5b .0000093

Table CIIa:

All 43 Compounds which showed % Inhibition at 30 mg/kg dose

and average of no. of moles is 0.000161(average value calculated taking 39

compounds only)

S. No.

Structures

%

Inhibition

c = 30

mg/kg

d = 3 mg/kg

S. No.

Acc. to

Reference

paper

No. of

moles

Value within

10% of

0.000161

(0.000145–

0.000177)

Value within

15% of

0.000161

(0.000137–

0.000185)

Value within

20% of

0.000161

(0.000129–

0.000193)

1 N

N

N

NH2

NH

S

93c 1a .000191 No No Yes

2 N

N

N

NH2

NH

S

CH3

93c 1b .000162 Yes Yes Yes

150

3 N

N

N

NH2

NH

S

CH3

21c 1c .000150 Yes Yes Yes

4 N

N

N

NH2

NH

S (CH2)3OEt

1c 1e .000131 No No Yes

5 N

N

N

NH2

NH

S Cyclohexyl

31c 1f .000132 No No Yes

6 N

N

N

NH2

NH

S Bn

40c 1h .000136 No No Yes

7 N

N

N

NH2

NH

SCH3

CH3

34c 3 .000162 Yes Yes Yes

8 N N

NH2

NH

S

27c 4 .000192 No No Yes

151

9

N

N

N

N

NH2

NH

S

53c 5 .000189 No No Yes

10 NH

N

NH2

NH

S

5c 7b .000192 No No Yes

11 S

NH2

NH

S

4c 7c .000174 Yes Yes Yes

12

NH2

S

NH

47c 7d .000180 No Yes Yes

13

N NH2

S

NH

27c 7e .000179 No Yes Yes

152

14 N

NH2

S

NH

9c 7f .000179 No Yes Yes

15 N

N NH2

S

NH

27c 7g .000178 No Yes Yes

16 N

N

N

NH2

NH

O

40c 8 .000212 Delete Delete Delete

17 N

N

N

NH2

S

S

36c 9 .000172 Yes Yes Yes

18 N

N

N

NH

S

NH2

25c 13 .000191 No No Yes

153

19 N

N

N

NH2

NH

S

NH2

53c 14 .000174 Yes Yes Yes

20 N

N

N

NH

S

CH3

13c 18 .000175 Yes Yes Yes

21 N

N

N

NH

S

SCH3

54c 19 .000159 Yes Yes Yes

22 N

N

N

NH

S

54c 23a .000137 No Yes Yes

23 N

N

N

NH

S

CH3

65c 23b .000129 No No Yes

154

24 N

N

N

NH

S

Cl

88d,c

23c .000118 No No No

25 N

N

N

NH

S

NC

87c 23d .000123 No No No

26 N

N

N

NH

S

MeO

70c 23e .000120 No No No

27 N

N

N

NH

S

Ethoxalylamino

49c 23h .000142 No Yes Yes

28 N

N

N

NH

S

3-Methylthiouriedo

31c 23i .000145 Yes Yes Yes

155

29 N

N

N

NH

S

S

CH3

54c 12 .000159 Yes Yes Yes

30 N

N

NN

S

N

69c 3a .000179 No Yes Yes

31 N

H

N

N

S

N

9c 3b .000178 No Yes Yes

32

N

S

N

22c 3d .000170 Yes Yes Yes

33 N

N

NN

O

N

17c 8 .000198 No No No

156

34 N

N

NNH

EtO

N

6c 9 .000178 No Yes Yes

35 N

N

NNH

NH

N NH

CH3

CH3

38c 11a .000166 Yes Yes Yes

36 N

N

NNH

NH

N NH

Cyclohexyl

Cyclohexyl

1c 11b .0000949 Delete Delete Delete

37 N

N

NN

S

N

69c 3a .000222 Delete Delete Delete

38 N

N

NN

S

N

CH3

49c 3e .000184 No Yes Yes

39 N

N

NN

S

N

CH3

23c 3f .000169 Yes Yes Yes

157

40 N

N

NN

S

N CH3

18c 4a .00020 Delete Delete Delete

41 N

N

NN

S

N

29c 5a .000142 No Yes Yes

42 N

N

NN

S

NCl

90d,c

3h .000121 No No No

43 N

N

NN

S

NNC

79c 3k .000132 No No Yes

158

Table CIIIa: All 22 Compounds which showed % Inhibition with 3 mg/kg dose

and average of no. of moles is 0.0000113 (average value calculated taking 20

compounds only)

S. No.

Structures

%

Inhibition

c = 30

mg/kg

d = 3

mg/kg

S. No.

Acc. to

Reference paper

No. of moles

Value within

10% of

0.0000113

(0.0000101–

0.0000127)

Value within

15% of

0.0000113

(0.0000096–

0.0000131)

Value within

20% of

0.0000113

(0.00000904–

0.0000136)

1 N

N

N

NH

S

Cl

88d,c

23c

.000118

And

.0000118

Yes Yes Yes

2 N

N

N

NH

S

Cl

54d 23j .0000118 Yes Yes Yes

159

3 N

N

N

NH

S

Cl

18d 23k .0000118 Yes Yes Yes

4 N

N

N

NH

S

F

73d 25a .0000127 Yes Yes Yes

5 N

N

N

NH

S

Br

88d 25b .0000101 Yes Yes Yes

6 N

N

N

NH

S

F3C

91d

25c .0000102 Yes Yes Yes

7 N

N

N

NH

S

Cl Cl

91d 25d .0000104 Yes Yes Yes

160

8

N

N

N

NH

S

Cl

Cl

85d 25e .0000104 Yes Yes Yes

9 N

N

N

NH

S

Cl OMe

68d 25f .0000106 Yes Yes Yes

10 N

N

N

NH

S

2-Furyl

18d 2a .0000144 No No No

11 N

N

N

NH

S

4-Chloro

-1

-naphthyl

14d 2e .00000911 Delete Delete Delete

12 N

N

N

NH

S

Cl

25d 2g .0000107 Yes Yes Yes

13

N

N

N

NH

S

58d 2i .0000102 Yes Yes Yes

161

14 N

N

N

NH

S

N

37d 2k .0000122 Yes Yes Yes

15 N

N

NN

S

NCl

90d,c

3h

.000121

And

.0000121

Yes Yes Yes

16 N

N

NN

S

NF

38d 3i .0000131 No Yes Yes

17 N

N

NN

S

NBr

84d 3j .0000103 Yes Yes Yes

18 N

N

NN

S

NF3C

87d 3l .0000108 Yes Yes Yes

19 N

N

NN

S

NCl

Cl

90d 3m .0000107 Yes Yes Yes

162

20 N

N

NN

S

NCl

Cl

89d 3n .0000107 Yes Yes Yes

21 N

N

NN

S

NCl CH3

60d 4b .0000115 Yes Yes Yes

22 N

N

NN

S

NCl

5d 5b .0000093 Delete Delete Delete

Total

Compounds

=18

Total

Compounds=19

Total

Compounds=

19

TABLE C1a: Data Set used for CoMFA analysis with their % inhibition of eosinophilia (3

mg/kg)

S. No. Structures Code Ref. code

%

Inhibition

3 mg/kg

No. of moles

1 N

N

N

NH

S CH3

Cl

M 1 23c 88 .0000118

2 N

N

NN

SCH3

NCl

M 2 23j 90 .0000121

3 N

N

N

NHS CH3

F3C

M 3 23k 91 .0000102

4 N

N

N

NHS CH3

Cl Cl

M 4 25a 91d .0000104

5 N

N

NN

SCH3

NCl

Cl

M 5 25b 90 .0000107

6 N

N

NN

SCH3

NCl

Cl

M 6

25c 89 .0000107

7 N

N

N

NHS CH3

Br

M 7 25d 88 .0000101

8 N

N

NN

SCH3

NF3C

M 8 25e 87 .0000108

164

9 N

N

N

NHS CH3

Cl

Cl

M 9 25f 85 .0000104

10 N

N

NN

SCH3

NBr

M 10 2a 84 .0000103

11 N

N

N

NHS CH3

F

MAR 11 2e 73 .0000127

12 N

N

N

NHS CH3

Cl OMe

MAR 12 2g 68 .0000106

13 N

N

NN

SCH3

NCl CH3 MAR 13 2i 60 .0000115

14 N

N

N

NHS CH3

MAR 14 2k 58 .0000102

15 N

N

N

NHS CH3

Cl

MAR 15 3h 54 .0000118

16 N

N

N

NHS CH3

N

MAR 16 3i 37 .0000122

17 N

N

N

NH

S

CH3

Cl

MAR 17 3j 25 .0000107

18 N

N

N

NHS CH3

Cl

MAR 18 3l 18 .0000118

TABLE C1b: Data Set used for CoMFA analysis with their % inhibition of eosinophilia (3

mg/kg)

S. No. Structures Code Ref. code

% Inhibition No. of moles

165

3 mg/kg

1 N

N

N

NH

S CH3

Cl

NH2

M 1 23c 88 .0000118

2 N

N

NN

SCH3

NCl

M 2 23j 90 .0000121

3 N

N

N

NHS CH3

F3C

NH2

M 3 23k 91 .0000102

4 N

N

N

NHS CH3

Cl Cl

NH2

M 4 25a 91d .0000104

5 N

N

NN

SCH3

NCl

Cl

M 5 25b 90 .0000107

6 N

N

NN

SCH3

NCl

Cl

M 6

25c 89 .0000107

7 N

N

N

NHS CH3

Br

NH2

M 7 25d 88 .0000101

8 N

N

NN

SCH3

NF3C

M 8 25e 87 .0000108

9 N

N

N

NHS CH3

Cl

Cl

NH2

M 9 25f 85 .0000104

10 N

N

NN

SCH3

NBr

M 10 2a 84 .0000103

11 N

N

N

NHS CH3

F

NH2

M 11 2e 73 .0000127

166

12 N

N

N

NHS CH3

Cl OMe

NH2

M 12 2g 68 .0000106

13 N

N

NN

SCH3

NCl CH3 M 13 2i 60 .0000115

14 N

N

N

NHS CH3

NH2

M 14 2k 58 .0000102

15 N

N

N

NHS CH3

Cl

NH2

M 15 3h 54 .0000118

16 N

N

N

NHS CH3

NNH2

M 16 3i 37 .0000122

17 N

N

N

NH

S

CH3

Cl

NH2

M 17 3j 25 .0000107

167

TABLE C2a : 3D QSAR MODEL 1 - CoMFA Results

Parameter

CoMFA

r2

cv (q2

)

0.196

r2

ncv

0.985

SEE

3.301

F

107.833

Component

4

Electrostatic Contribution 0.566

Steric Contribution 0.214

168

TABLE C2b1 : 3D QSAR MODEL 2 - CoMFA Results

Parameter

CoMFA

r2

cv (q2

)

0.373

r2

ncv

0.993

SEE

2.458

F

163.350

Component

6

Electrostatic Contribution 0.889

Steric Contribution 0.329

169

TABLE C2b2 : 3D QSAR MODEL 3 - CoMFA Results

Parameter

CoMFA

r2

cv (q2

)

0.445

r2

ncv

0.999

SEE

1.299

F

556.846

Component

6

Electrostatic Contribution 0.766

Steric Contribution 0.234

170

TABLE C3: Actual (Act) and Predicted (Pred) % Inhibition of eosinophilia

(3 mg/kg) and the Residuals (Δ) of the Training set molecules

No.

Structures Code Ref.

Code Act Pred Δ

2 N

N

NN

SCH3

NCl

M 2 23j 90 87.996 2.00

4 N

N

N

NHS CH3

Cl Cl

NH2

M 4 25a 91d 91.396 -0.40

5 N

N

NN

SCH3

NCl

Cl

M 5 25b 90 90.657 -0.66

6 N

N

NN

SCH3

NCl

Cl

M 6

25c 89 88.620 0.38

7 N

N

N

NHS CH3

Br

NH2

M 7 25d 88 87.675 0.33

10 N

N

NN

SCH3

NBr

M 10 2a 84 85.740 -1.74

11 N

N

N

NHS CH3

F

NH2

M 11 2e 73 72.608 0.39

12 N

N

N

NHS CH3

Cl OMe

NH2

M 12 2g 68 68.515 -0.51

171

14 N

N

N

NHS CH3

NH2

M 14 2k 58 58.066 -0.07

15 N

N

N

NHS CH3

Cl

NH2

M 15 3h 54 53.674 0.33

16 N

N

N

NHS CH3

NNH2

M 16 3i 37 36.927 0.07

17 N

N

N

NH

S

CH3

Cl

NH2

M 17 3j 25 25.127 -0.13

172

TABLE C4: Actual (Act) and Predicted (Pred) % Inhibition of eosinophilia

(3 mg/kg) and the Residuals (Δ) of the Test set molecules

No.

Structures Code Ref.

Code Act Pred Δ

1 N

N

N

NHS CH3

Cl

NH2

M 1 23c 88 78 10

3 N

N

N

NHS CH3

F3C

NH2

M 3 23k 91 78 13

8 N

N

NN

SCH3

NF3C

M 8 25e 87 90 -3

9 N

N

N

NHS CH3

Cl

Cl

NH2

M 9 25f 85 70 15

13 N

N

NN

SCH3

NCl CH3

M 13 2i 60 86 26

173

TABLE C5 : FlexS Alignment Scores and Prediction (% Inhibition of eosinophilia) for the

Imidazole Based Designed Set of Molecules

S. No. Code Compound Total

Score

Norm.

Score

Prediction

1

Ie1 N

NNH

S

NH2

Cl

CH3

-511.9 1.124

124.95

N

NNH

O

NH2

Cl

CH3

75.802

2

Ie2 N

NNH

S

NH2

Cl

CH3

- -

75.959

N

NNH

O

NH2

Cl

CH3

77.145

3 Ie3 N

NNH

S

NH2

Cl

CH3

- - 76.602

4 Ie4 N

NNH

S

NH2

Cl

CH3

CH3

- - 75.370

5

Ie5 N

NNH

S

NH2

Cl

CH3

CH3CH3

- - 81.008

N

NNH

O

NH2

Cl

CH3

CH3CH3

80.122

174

6 If1 N

NNH

S

NH2

Br

CH3

-512.5 1.121 89.747

7 Ig1 N

NNH

S

NH2

CH3

N

- - 88.168

8

IC1 N

N

N

N

Cl

SCH3

-412.5 0.814

111.304

N

N

N

N

Cl

OCH3

97.767

9

IC2 N

N

N

N

Cl

SCH3

-419.5 0.758 81.136

N

N

N

N

Cl

OCH3

105.947

10 IC3 N

N

N

N

Cl

S CH3

- - 75.957

11 IC4 N

N

N

N

Cl

SCH3

CH3

- - 74.665

12

IC5 N

N

N

N

Cl

SCH3

CH3CH3

-422.4 0.630 72.465

N

N

N

N

Cl

OCH3

CH3CH3

102.077

175

13 ICf1 N

N

N

N

Br

SCH3

- - 118.292

14 ICg1 N

N

N

N

SCH3

N

-419.1 0.788

116.214

176

TABLE C6 : FlexS Alignment Scores and Prediction (% Inhibition of eosinophilia) for

the Triazole Based Designed Set of Molecules

S. No. Code Compound Total Score Norm.

Score

Prediction

15

Te1 N

N NNH

S

NH2

Cl

CH3

-455.8 0.892 77.316

N

N NNH

O

NH2

Cl

CH3

78.861

16

Te2 N

N NNH

S

NH2

Cl

CH3

-491.4 0.878 79.591

N

N NNH

O

NH2

Cl

CH3

79.856

17 Te3 N

N NNH

S

NH2

Cl

CH3

-491.2 0.808 61.136

18 Te4 N

N NNH

S

NH2

Cl

CH3

CH3

- - 61.757

19

Te5 N

N NNH

S

NH2

Cl

CH3

CH3CH3

-497.6 0.733 87.729

N

N NNH

O

NH2

Cl

CH3

CH3CH3

82.141

177

20 Tf1 N

N NNH

S

NH2

Br

CH3

-457.7 1.019 86.093

21 Tg1 N

N NNH

S

NH2

CH3

N

-469.4 0.893 85.859

22

TC1 N

NN

N

N

Cl

SCH3

-391.6 0.805

122.103

N

NN

N

N

Cl

OCH3

107.545

23

TC2 N

NN

N

N

Cl

SCH3

- - 93.567

N

NN

N

N

Cl

OCH3

109.429

24 TC3 N

NN

N

N

Cl

S CH3

-379.9 0.791 95.773

25 TC4 N

NN

N

N

Cl

SCH3

CH3

- - 93.951

26

TC5 N

NN

N

N

Cl

SCH3

CH3CH3

-399.2 0.614 100.171

N

NN

N

N

Cl

OCH3

CH3CH3

112.197

178

27 TCf1 N

NN

N

N

Br

SCH3

-391.9 0.803 105.353

28 TCg1 N

NN

N

N

SCH3

N

-396.7 0.775 102.345

179

TABLE C7 : FlexS Alignment Scores and Prediction (% Inhibition of eosinophilia) for the

Imidazoline Based Designed Set of Molecules

S. No. Code Compound Total Score Norm.

Score

Prediction

29

IMe1 N

NNH

S

NH2

Cl

CH3

- - 65.864

N

NNH

O

NH2

Cl

CH3

69.714

30

IMe2 N

NNH

S

NH2

Cl

CH3

- - 69.909

N

NNH

O

NH2

Cl

CH3

58.621

31 IMe3 N

NNH

S

NH2

Cl

CH3

- - 67.398

32 IMe4 N

NNH

S

NH2

Cl

CH3

CH3

-407.0 0.562 66.833

33

IMe5 N

NNH

S

NH2

Cl

CH3

CH3CH3

- - 69.703

N

NNH

O

NH2

Cl

CH3

CH3CH3

46.233

180

34 IMf1 N

NNH

S

NH2

Br

CH3

- - 92.753

35 IMg1 N

NNH

S

NH2

CH3

N

- - 108.659

36

IMC1 N

N

N

N

Cl

SCH3

- - 71.018

N

N

N

N

Cl

OCH3

73.820

37

IMC2 N

N

N

N

Cl

SCH3

- - 70.746

N

N

N

N

Cl

OCH3

72.820

38 IMC3 N

N

N

N

Cl

S CH3

- - 58.121

39 IMC4 N

N

N

N

Cl

SCH3

CH3

- - 57.226

40

IMC5 N

N

N

N

Cl

SCH3

CH3CH3

-371.0 0.532 73.916

N

N

N

N

Cl

OCH3

CH3CH3

78.061

41 IMCf1 N

N

N

N

Br

SCH3

- - 105.872

181

42 IMCg1 N

N

N

N

SCH3

N

-369.1 0.659 70.104

182

TABLE C 8 : FlexS Alignment Scores and Prediction (% Inhibition of eosinophilia) for

the Benzene(Biphenyl) Based Designed Set of Molecules

S.No. Code Compound Total

Score

Norm.

Score

Prediction

43

Bf1 NH2

Cl

S

NH

CH3

-459.2 0.892 77.572

Be1 NH2

Cl

O

NH

CH3

54.322

44

Bf2 NH2

Cl

S

NHCH3

- - 77.540

Be2 NH2

Cl

O

NHCH3

79.651

45 Bf3 NH2

Cl

S

NH

CH3

- - 82.858

46 Bf4 NH2

Cl

S

NHCH3

CH3

- - 90.136

47

Bf5 NH2

Cl

S

NHCH3

CH3CH3

-449.9 0.615 69.293

Be5 NH2

Cl

O

NHCH3

CH3CH3

85.025

183

48 Bg1 NH2

Br

S

NH

CH3

-432.2 0.875 66.861

49 Bh1 NH2

S

NH

CH3

N

- - 71.299

50

BC1 N

N

CH3

S

Cl

-397.5 0.724 88.636

N

N

CH3

O

Cl

72.715

51

BC2 N

N

S

Cl

CH3

- - 85.362

N

N

O

Cl

CH3

55.381

52 BC3 N

N

S

Cl

CH3

-406.7 0.629 67.837

53 BC4 N

N

S

Cl

CH3

CH3

- - 86.379

54

BC5 N

N

S

Cl

CH3

CH3CH3

-411.8 0.579 68.939

N

N

O

Cl

CH3

CH3CH3

45.900

184

55 BCg1 N

N

CH3

S

Br

-397.7 0.722 66.874

56 BCh1 N

N

CH3

S

N

- - 78.804

185

TABLE C 9 : FlexS Alignment Scores and Prediction (% Inhibition of eosinophilia) for the

Pyridine Based Designed Set of Molecules

S.No. Code Compound Total

Score

Norm.

Score

Prediction

57

Pi1 N NH2

Cl

S

NH

CH3

-428.6 0.895 85.826

Ph1 N NH2

Cl

O

NH

CH3

53.309

58

Pi2 N NH2

Cl

S

NHCH3

-444.5 0.738 72.821

Ph2 N NH2

Cl

O

NHCH3

74.658

59 Pi3 N NH2

Cl

S

NH

CH3

- - 81.249

60 Pi4 N NH2

Cl

S

NHCH3

CH3

- - 91.581

61 Pi5 N NH2

Cl

S

NHCH3

CH3CH3

-425.4 0.887 66.133

186

Ph5 N NH2

Cl

O

NHCH3

CH3CH3

83.958

62 Pj1 N NH2

Br

S

NH

CH3

- - 69.122

63 Pk1 N NH2

S

NH

CH3

N

- - 75.389

64

PC1 N

N

N

CH3

S

Cl

-414.8 0.898 94.569

N

N

N

CH3

O

Cl

67.899

65

PC2 N

N

N

S

Cl

CH3

- - 91.558

N

N

N

O

Cl

CH3

46.300

66 PC3 N

N

N

S

Cl

CH3

- - 92.960

67 PC4 N

N

N

S

Cl

CH3

CH3

- - 93.011

68 PC5 N

N

N

S

Cl

CH3

CH3CH3

-393.4 0.566 73.062

187

N

N

N

O

Cl

CH3

CH3CH3

49.940

69 PCj1 N

N

N

CH3

S

Br

-381.7 0.715 70.281

70 PCk1 N

N

N

CH3

S

N

-385.7 0.692 70.745

188

TABLE C 10 : FlexS Alignment Scores and Prediction (% Inhibition of eosinophilia) for

the Pyrimidine Based Designed Set of Molecules

S.No. Code Compound Total

Score

Norm.

Score

Prediction

71

PMi1 N

N

NH2

Cl

S

NH

CH3

-415.4 0.904 81.041

PMh1 N

N

NH2

Cl

O

NH

CH3

47.429

72

PMi2 N

N

NH2

Cl

S

NHCH3

-414.6 0.701 71.088

PMh2 N

N

NH2

Cl

O

NHCH3

75.188

73 PMi3 N

N

NH2

Cl

S

NH

CH3

-424.6 0.788 64.209

74 PMi4 N

N

NH2

Cl

S

NHCH3

CH3

-410.9 0.638 70.113

75

PMi5 N

N

NH2

Cl

S

NHCH3

CH3CH3

-415.8 0.587 77.380

PMh5 N

N

NH2

Cl

O

NHCH3

CH3CH3

85.006

189

76 PMj1 N

N

NH2

Br

S

NH

CH3

-429.5 0.926 68.058

77 PMk1 N

N

NH2

S

NH

CH3

N

- - 71.773

78

PMC1 N

N N

N

CH3

S

Cl

-372.7 0.714 88.966

N

N N

N

CH3

O

Cl

70.415

79

PMC2 N

N N

N

S

Cl

CH3

- - 77.847

N

N N

N

O

Cl

CH3

72.406

80 PMC3 N

N N

N

S

Cl

CH3

- - 89.257

81 PMC4 N

N N

N

S

Cl

CH3

CH3

- - 91.567

82

PMC5 N

N N

N

S

Cl

CH3

CH3CH3

-384.5 0.561 67.835

N

N N

N

O

Cl

CH3

CH3CH3

70.055

190

83 PMCj1 N

N N

N

CH3

S

Br

- - 103.471

84 PMCk1 N

N N

N

CH3

S

N

- - 83.253

191

Figure C1: Database alignment of designed set of molecules 1 to 84

(Table C5 to C10)

a b

c d

Figure C2: Fit atom alignment of designed set of molecules

(a) five membered acyclic and cyclic (1 to 42, Table C5 to C7) (b) six membered acyclic

and cyclic (43 to 84, Table C8 to C10) (c) all acyclic (methyl amino thiocarbonyl

substituted (1, 15, 29, 43, 57, 71;Table C 5 to C 10) (d) all cyclic (methyl substituted (8,

22, 36, 50, 64, 78;Table C5 to C10)

193

a

b

Figure C3: FlexS alignment of designed set of molecules (a) 1 to 42 (Table C5 to C7)

(b) 43 to 84 (Table C8 to C10)

194

a b

Figure C4: Structural alignment of the compounds in the training set of 12 molecules for

constructing 3D-QSAR CoMFA (a) Wire Frame (b) Ball and Stick, Hydrogens are not shown

(Table C3)

a b

Figure C5 : Structural alignment of the compounds in the test set of 5 molecules for

constructing 3D-QSAR CoMFA (a) Wire Frame (b) Ball and Stick, Hydrogens are not shown

(Table C4)

195

a b

Figure C6: Structural alignment of the most active molecules

(1 and 2 of the data set Table C1b) (a) colour by atom type (b) colour by molecule

(acyclic: 1 orange and cyclic: 2 magenta)

a b

Figure C7: Structural alignment of carbonyl analogs of (a) 43, 44, 47, 50, 51, 54 (Table C8 all six

membered molecules), 57, 58, 61, 64, 65, 68 (Table C9 all six membered molecules), 71, 72, 75,

78, 79, 82 (Table C10 all six membered molecules) (b) synthesized six membered molecules

43(Be1)44(Be2), 47(Be5) (Table C8), 57 (Ph1) 58(Ph2), 61(Ph5) (Table C9), 71(PMh1)72(PMh2),

75(PMh5) (Table C10)

a b

Figure C8: 3D Scatter Plot of the predicted vs actual activities of the training set of 12 molecules

(a) along with Electostatic and Steric contours housing both the most active molecules (1 and 2 of the data set

Table C1) (b) 3D Scatter Plot only

19

6

a

b

Figure C9: Most active molecules (1 and 2 of the data set Table C1) placed into the CoMFA

contour maps electrostatic and stearic (a)front view (b) rear view

a b

Figure C10: Most active molecules (1 and 2 of the data set Table C1) placed into the CoMFA

contour maps (a) electrostatic (b) steric

of the training set of molecules (front view)

b c

Figure C10a: Most active molecules (1 and 2 of the data set Table C1) placed into the

CoMFA contour maps (a) electrostatic (b) steric

of the training set of molecules (rear view)

199

a

b c

Figure C11: CoMFA contour maps (a) electrostatic and stearic (b) electrostatic (c) steric of

training set of 12 molecules housing all the 12 molecules (Table C3),

(front view)

200

a

b c

Figure C11a: CoMFA contour maps (a) electrostatic and stearic (b) electrostatic (c) steric of

training set of 12 molecules housing all the 12 molecules (Table C3),

(rear view)

201

a b

Figure C12: Most, intermediate and least active molecules (1, 4, 6 and 9, 12, 13 acyclic and

cyclic respectively) of the designed Imidazole based series (Table C5) placed into the CoMFA

contour maps (a) electrostatic and (b) stearic of the training set of molecules (front view)

a b

Figure C12a: Most, intermediate and least active molecules (1, 4, 6 and 9, 12, 13 acyclic and

cyclic respectively) of the designed Imidazole based series ( Table C5) placed into the

CoMFA contour maps (a) electrostatic and (b) stearic of the training set of molecules (rear

view)

The most active molecules (1 and 2 of the data set Table C 1, acyclic: orange and cyclic: magenta)

are shown in all these snapshots for comparison

202

a b

Figure C13: Most, intermediate and least active molecules (15, 17, 19 and 22, 23, 27 acyclic

and cyclic respectively) of the designed Triazole based series (Table C6) placed into the

CoMFA contour maps (a) electrostatic and (b) stearic of the training set of molecules (front

view)

a b

Figure C13a: Most, intermediate and least active molecules (15, 17, 19 and 22, 23, 27 acyclic

and cyclic respectively) of the designed Triazole based series (Table C 6) placed into the

CoMFA contour maps (a) electrostatic and (b) stearic of the training set of molecules (rear

view)

The most active molecules (1 and 2 of the data set Table C 1, acyclic: orange and cyclic: magenta)

are shown in all these snapshots for comparison

203

a b

Figure C14: Most, intermediate and least active molecules (29, 34, 35and 39, 40, 41 acyclic

and cyclic respectively) of the designed Imidazoline based series (Table C7) placed into the

CoMFA contour maps (a) electrostatic and (b) stearic of the training set of molecules (front

view)

a b

Figure C14a: Most, intermediate and least active molecules (29, 34, 35and 39, 40, 41 acyclic

and cyclic respectively) of the designed Imidazoline based series (Table C7) placed into the

CoMFA contour maps (a) electrostatic and (b) stearic of the training set of molecules (rear

view)

The most active molecules (1 and 2 of the data set Table C 1, acyclic: orange and cyclic: magenta)

are shown in all these snapshots for comparison

204

a b

Figure C15: Alkyl variants of N-Alkyl (43, 44, 47 and 50, 51, 54 acyclic and cyclic respectively)

of the designed Benzene (Biphenyl) based series (Table C8) placed into the CoMFA contour

maps (a) electrostatic and (b) stearic of the training set of molecules (front view)

a b

Figure C15a: Alkyl variants of N-Alkyl (43, 44, 47 and 50, 51, 54 acyclic and cyclic

respectively) of the designed Benzene (Biphenyl) based series ( Table C8) placed into the

CoMFA contour maps (a) electrostatic and (b) stearic of the training set of molecules (rear

view)

The most active molecules (1 and 2 of the data set Table C 1, acyclic: orange and cyclic: magenta)

are shown in all these snapshots for comparison

205

a b

Figure C16: Alkyl variants of N-Alkyl (57, 58, 61and 64, 65, 68 acyclic and cyclic respectively)

of the designed Pyridine based series (Table C9) placed into the CoMFA contour maps (a)

electrostatic and (b) stearic of the training set of molecules (front view)

a b

Figure C16a: Alkyl variants of N-Alkyl (57, 58, 61and 64, 65, 68 acyclic and cyclic

respectively) of the designed Pyridine based series ( Table C9) placed into the CoMFA

contour maps (a) electrostatic and (b) stearic of the training set of molecules (rear view)

The most active molecules (1 and 2 of the data set Table C 1, acyclic: orange and cyclic: magenta)

are shown in all these snapshots for comparison

206

a b

Figure C17: Alkyl variants of N-Alkyl (71, 72, 75and 78, 79, 82 acyclic and cyclic respectively)

of the designed Pyrimidine based series (Table C10) placed into the CoMFA contour maps

(a) electrostatic and (b) stearic of the training set of molecules (front view)

a b

Figure C17a: Alkyl variants of N-Alkyl (71, 72, 75and 78, 79, 82 acyclic and cyclic

respectively) of the designed Pyrimidine based series (Table C10) placed into the CoMFA

contour maps (a) electrostatic and (b) stearic of the training set of molecules (rear view)

The most active molecules (1 and 2 of the data set Table C 1, acyclic: orange and cyclic: magenta)

are shown in all these snapshots for comparison

207

a

b c

Figure C18: Six membered acyclic carbonyl analogs of 43, 44, 47 (Table C8) 57, 58, 61 (Table

C9), 71, 72, 75 (Table C10) placed into the CoMFA contour maps electrostatic and stearic (b)

electrostatic (c) stearic of the training set of molecules (front view)

The most active molecules (1 of the data set Table C 1, acyclic: orange) is shown in all these

snapshots for comparison

208

a

b c

Figure C18a: Six membered acyclic carbonyl analogs of 43, 44, 47 (Table C8) 57, 58, 61

(Table C9), 71, 72, 75 (Table C10) placed into the CoMFA contour maps electrostatic and

stearic (b) electrostatic (c) stearic of the training set of molecules (rear view)

The most active molecules (1 of the data set Table C 1, acyclic: orange) is shown in all these

snapshots for comparison

209

a

b c

Figure C18’: Six membered cyclic carbonyl analogs of 50, 51, 54 (Table C8) 64, 65, 68 (Table

C9) 78, 79, 82 (Table C10) placed into the CoMFA contour maps (a) electrostatic and stearic

(b) electrostatic (c) stearic of the training set of molecules (front view)

The most active molecules (2 of the data set Table C1, cyclic: magenta) is shown in all these

snapshots for comparison

210

a

b c

Figure C18’a: Six membered cyclic carbonyl analogs of 50, 51, 54 (Table C8) 64, 65, 68 (Table

C9) 78, 79, 82 (Table C10) placed into the CoMFA contour maps (a) electrostatic and stearic

(b) electrostatic (c) stearic of the training set of molecules (rear view)

The most active molecules (2 of the data set Table C 1, cyclic: magenta) is shown in all these

snapshots for comparison

211

a

b c

Figure C19: Synthesised six membered acyclic carbonyl analogs of 44, 47 (Table C8) 58, 61

(Table C9) 72, 75 (Table C10) placed into the CoMFA contour maps (a) electrostatic and

stearic (b) electrostatic (c) stearic of the training set of molecules (front view)

The most active molecules (1 of the data set Table C 1, acyclic: orange) is shown in all these

snapshots for comparison

212

a

b c

Figure C19a: Synthesised six membered acyclic carbonyl analogs of 44, 47 (Table C8) 58, 61

(Table C9) 72,75 (Table C10) placed into the CoMFA contour maps (a) electrostatic and

stearic (b) electrostatic (c) stearic of the training set of molecules (rear view)

The most active molecules (1 of the data set Table C 1, acyclic: orange) is shown in all these

snapshots for comparison

213

a b

Figure C20: Molecule 19 (Table C6) placed into the CoMFA contour maps (a) electrostatic (b)

stearic of the training set molecules along with the template1

(Table C1) (front view)

a b

Figure C20a: Molecule 19 (Table C6) placed into the CoMFA contour maps (a) electrostatic

(b) stearic of the training set molecules along with the template1

(Table C1) (rear view)

214

a b

Figure C21 : Molecule 26 (Table C6) placed into the CoMFA contour maps (a) electrostatic

(b) stearic of the training set molecules along with the template 2

(Table C1) (front view)

a b

Figure C21a : Molecule 26 (Table C6) placed into the CoMFA contour maps (a) electrostatic

(b) stearic of the training set molecules along with the template 2

(Table C 1) (rear view)

215

a b

Figure C22 : Molecule 51 (Table C8) placed into the CoMFA contour maps (a) electrostatic

(b) stearic of the training set molecules along with the template 2

(Table C1) (front view)

a b

Figure C22a : Molecule 51 (Table C8) placed into the CoMFA contour maps (a) electrostatic

(b) stearic of the training set molecules along with the template 2

(Table C1) (rear view)

216

a b

Figure C23 : Molecule 65 (Table C9) placed into the CoMFA contour maps (a) electrostatic

(b) stearic of the training set molecules along with the template 2

(Table C1) (front view)

a b

Figure C23a : Molecule 65 (Table C9) placed into the CoMFA contour maps (a) electrostatic

(b) stearic of the training set molecules along with the template 2

(Table C1) (rear view)

EXPERIMENTAL

The Melting/Boiling Points reported here were recorded using an open conc. sulphuric acid

bath and are uncorrected.

The Infrared Spectra of all the compounds reported here, were recorded on Perkin-Elmer

Spectrum RX FTIR Spectrophotometer, at the Department of Chemistry, Punjabi University,

Patiala and at SAIF, Panjab University, Chandigarh.

1H NMR Spectra were recorded on AC300F, 300MHz and AC400F, 400MHz Bruker

Spectrometer at SAIF, Panjab University Chandigarh. 1H NMR spectra of some of the

compounds has also been recorded on JEOL FT-NMR AL-300MHz at Department of

Chemistry, Guru Nanak Dev University, Amritsar.

.

Mass Spectral Analysis was performed on GCMS-QP 5000 and LCMS LCQ Finnigan Matt

(APCI +ve mode) at Central Instrumentation Laboratory, NIPER, SAS Nagar, Mohali, Punjab.

Mass Spectral Analysis of some of the compounds has also been recorded on Micromass Q-Tof

Micro (TOF MS ES+ve) and Shimadzu GCMS-QP2010 Plus at SAIF, Panjab University

Chandigarh and at department of Chemistry, Punjabi University, Patiala respectively.

218

3.2. SYNTHESIS

A. MONOCYCLICS: 5-MEMBERED RINGS WITH 2 AND 3 NITROGENS

I. 2-AMINO-4-(4-SUBSTITUTEDPHENYL-N-ALKYL-1H-IMIDAZOLE-1-

CARBOTHIOAMIDE:

1. Preparation of alkylisothiocyanates (A1-A5)

A1. Methylisothiocyanate

A mixture solution of methylamine (40%; 7.750g, 0.25mol) and triethylamine (22.250g,

0.25mol) taken in a dropping funnel was added dropwise with stirring to carbon disulphide

(40mL, placed in a 250mL RBF surrounded by an ice salt cooling bath) in about 15 min. Stirring

continued for another 2 h at 00C and the heavy solid separated during this time was filtered,

washed with ether and dried. It was then dissolved in water and to this solution lead (II) nitrate

(82.750g, 0.25mol) was added in installments .The contents of the flask was distilled with steam

into a receiving RBF (500mL, containing 5-6mL of 1N-H2SO4 ) as long as any oil comes out.

The oil was extracted with ether, washed with distilled water, dried over anhydrous Na2SO4,

filtered and the filtrate upon evaporation gave Methylisothiocyanate A1 25% as yellow oil, bp,

118-1190C.

IR (Neat)Vmax cm-1

2983 and 2874 C-H stretch of methyl, antisymmetric and symmetric

2070 N=C=S stretch

1445 and 1366 C-H bend of methyl, antisymmetric and symmetric

PMR (CDCl3) δppm

3.325 (s, 3H, -CH3)

A2. Ethylisothiocyanate

Ethylisothiocyanate A2 was prepared (5.655g, 26%) as per procedure described for the

preparation of A1, starting from ethylamine (70%; 11.250g, 0.25mol) as yellow oil, bp, 134-

1350C.

219

IR (Neat)Vmax cm-1

2970 and 2907 C-H stretch of methyl, antisymmetric and symmetric

2938 and 2877 C-H stretch of methylene, antisymmetric and symmetric

2098 N=C=S stretch

1463 C-H bend of methylene

1448 and 1388 C-H bend of methyl, antisymmetric and symmetric

PMR (CDCl3) δppm

3.582 (q, 2H, -CH2CH3)

1.425 (t, 3H, -CH2CH3)

A3. n-Propylisothiocyanate

n-Propylisothiocyanate A3 was prepared (8.585g, 34%) as per procedure described for the

preparation of A1, starting from n-Propylamine (14.750g, 0.25mol) as yellow oil, bp, 155-1570C.

IR (Neat)Vmax cm-1

2970 and 2907 C-H stretch of methyl, antisymmetric and symmetric

2938 and 2877 C-H stretch of methylene, antisymmetric and symmetric

2096 N=C=S stretch

1463 C-H bend of methylene

1450 and 1385 C-H bend of methyl, antisymmetric and symmetric

PMR (CDCl3) δppm

3.445 (t, 2H, -CH2CH2CH3)

1.708 (sextet, 2H, -CH2CH2CH3)

1.007 (t, 3H, -CH2CH2CH3)

220

A4. iso-Propylisothiocyanate

iso-Propylisothiocyanate A4 was prepared (10.100g, 40%) as per procedure described for the

preparation of A1, starting from iso-Propylamine (14.740g, 0.25mol) as yellow oil, bp, 135-

1370C.

IR (Neat)Vmax cm-1

2983 and 2875 C-H stretch of methyl, antisymmetric and symmetric

2091 N=C=S stretch

1387 and 1370 C-H bend of methyl, antisymmetric and symmetric

PMR (CDCl3) δppm

3.926 (septet, 1H, -CH(CH3)2)

1.426 (d, 6H, -CH(CH3)2)

A5. tert-Butylisothiocyanate

tert-Butylisothiocyanate A5 was prepared (12.937g, 45%) as per procedure described for the

preparation of A1, starting from tert-Butylamine (18.250g, 0.25mol) as yellow oil, bp, 144-

1460C.

IR (Neat)Vmax cm-1

2982 and 2874 C-H stretch of methyl, antisymmetric and symmetric

2004 N=C=S stretch

1397 and 1369 C-H bend of methyl, antisymmetric and symmetric

PMR (CDCl3) δppm

1.436 (s, 9H, -C(CH3)3)

221

2. 2-amino-4-(4-chlorophenyl)-N-alkyl-1H-imidazole-1-carbothioamide (Ie1-5)

Preparation of 2-Phenylazoimidazole: Ib

A two-step synthetic sequence involving:

Preparation of Benzene diazoniumchloride Ia and

Coupling of Benzene diazoniumchloride Ia with Imidazole was followed.

a. Benzene diazoniumchloride: Ia

To (9.800g, 0.10mol) of aniline taken in a 500mL RBF was added dilute acid (30mL of

concentrated HCl in 30mL of water). The clear solution obtained was stirred and chilled to -5°C.

To this was added an ice cold solution of sodium nitrite (7.245g, 0.10mol) in 45mL of water

from a dropping funnel (The stem of which extends below the surface of liquid) at a rate such

that the temperature should not rise above 00C. The solution stirred for additional 5 min after the

addition of sodium nitrite and then a mixture of 20mL of ice cold water, 0.2 g of urea and 20 g

of crushed ice was added successively.

b. 2-Phenylazoimidazole: Ib

To a vigorously stirred and cold (00C) solution of Imidazole (6.800 gm, 0.10mol) in 10%

NaHCO3 (45mL) taken in a 250mL RBF immersed in an ice salt bath (assisted by direct addition

of 25g of crushed ice) was added cold diazonium salt solution Ia prepared above very slowly.

When all the diazonium salt solution was added the contents of the flask were allowed to stand

in an ice bath for about 30 min. The reddish brown solid separated was filtered, washed with

water, dried and which upon recrystallization from glacial acetic acid gave 6.880g, (40%) of Ib,

mp, 115-1200C.

IR (KBr)Vmax cm-1

3600- 2200 N-H stretch of Imidazole, bonded

3406 N-H stretch of Imidazole, free

3056 and 3139 C-H stretch of imidazole

1527, 1490, 1316,

222

1584, 1468 Aromatic/Heteroaromatic skeletal stretch

1442 N=N stretch of diazo

934 and 836 2-substituted imidazole out of plane deformations

762 and 682 5-adjacent aromatic hydrogens

PMR (CDCl3) δppm

13.098 br. s, 1H, -NH of imidazole

7.822 m, 2H, aromatic

7.423 m, 3H, aromatic

7.095 d, 1H, -HC=CH- of imidazole, J=8.85 Hz

6.942 d, 1H, -HC=CH- of imidazole, J=8.76 Hz

LCMS

m/z % intensity

345(2M+H+) 100

c. 2-Amino-4-(4-aminophenyl) imidazole dihydrochloride: Ic

A warm solution of tin (II) chloride (18.900g, 0.10mol) in 40mL of concentrated HCl was

added to gently boiling solution of Ib (8.600g, 0.05 mol) in rectified spirit (80mL) taken in a

250mL RBF provided with a reflux condenser. The contents of the flask were refluxed for 1.5 h,

the reaction mixture was allowed to cool and then chilled to -50C during this time crystallisation

took place. Solid separated was filtered, washed with ether, dried and upon recrystallization

from 20% hot alcohol gave 20.825g, 85% of Ic, mp, 202-2050C Decomp.

IR (KBr)Vmax cm-1

3547 N-H stretch of Imidazole

3385-3250 Multiple band, N-H stretch of NH3+

3136 and 3060 C-H stretch of imidazole

1569, 1513 1489, 1406, 1331 Aromatic/Heteroaromatic skeletal stretch

223

829 2-adjacent aromatic hydrogens

PMR (D2O) δppm

7.376 d, 2H, aromatic, p-disubtitued, J=8.613 Hz

7.253 d, 2H, aromatic,p-disubtitued, J=8.667 Hz

6.886 s, 1H, -CH of amino imidazole

LCMS: free base

m/z % intensity

175(M+H+) 100

d. Synthesis of 2-Amino-4-(4-aminophenyl) -1H-1,3-diazol-1-yl-alkylamino

methanethiones (Id1-5)

Id1. 2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-methylaminomethanethione

To a solution of sodium hydroxide (3.000g, 0.75mol) in 10ml of water taken in a RBF (100 mL)

was added a solution of 2-amino-4-(4-aminophenyl) imidazole dihydrochloride (6.175g,

0.025mol) Ic in 10 mL of water. Methylisothiocyanate A1 (1.898g, 0.026mol) in THF (10mL)

was added to the above mixture solution while stirring. The reaction mixture solution was stirred

at reflux for 4h, neutralised with in 1N-HCl and extracted with ethyl acetate (3x50mL). The

combined ethyl acetate extract washed with distilled water, dried over anhydrous sodium

sulphate, filtered and solvents removed by distillation. The residue thus left upon

recrystallization from methanol gave 1.480g, (24%) of Id1, mp, 185-1900C.

IR (KBr)Vmax cm-1

3400-3100 (3245) Multiple band, N-H stretch of NH2

2958 and 2870 C-H stretch of methyl, antisymmetric and symmetric

1600-1325 Aromatic/Heteroaromatic skeletal stretch

224

1540 C-N-H bend of thiomide II

1071 C=S stretch

835 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

9.287 br.s, 1H, -NH, thiomide

7.45 br.s, 2H, -NH2, imidazole

7.65 d, 2H, aromatic, J=8.4 Hz

7.23 d, 2H, aromatic, J=8.4 Hz

6.81 s, 1H, -CH of amino imidazole

4.67 br.s, 2H, -NH2, aromatic

1.97 s, 3H, -CH3

LCMS

m/z % intensity

249(M+2H+) 100

247(M+) 36.75

217 67.25

175 34.50

173 7.25

Id2. 2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-ethylaminomethanethione

225

2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-ethylaminomethanethione Id2 was prepared

(1.570g, 24%) as per procedure described for the preparation of Id1 starting from

ethylisothiocyanate A2 (2.262g, 0.025mol), mp, 190-1950C.

IR (KBr)Vmax cm-1

3450-3100 Multiple band, N-H stretch of NH2

2971 and 2871 C-H stretch of methyl, antisymmetric and symmetric

2938 and 2877 C-H stretch of methylene, antisymmetric and symmetric

1600-1355 Aromatic/Heteroaromatic skeletal stretch

1541 C-N-H bend of thiomide II

1092 C=S stretch

836 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

7.96 br.s, 1H, -NH, thiomide

7.75 br.s, 2H, -NH2, imidazole

7.45 d, 2H, aromatic, J=7.5 Hz

7.13 d, 2H, aromatic, J=8.5 Hz

6.62 s, 1H, -CH of amino imidazole

5.35 br.s, 2H, -NH2, aromatic

3.72 q, 2H, -CH2CH3

1.30 t, 3H, -CH2CH3

LCMS

226

m/z % intensity

242 7.75

261(M+) 5.25

232 15.50

202 100

Id3. 2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-n-propylaminomethanethione

2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-n-propylaminomethanethion Id3 was prepared

(1.720g, 25%) as per procedure described for the preparation of Id1 starting from n-

propylisothiocyanate A3 (2.626g, 0.026mol), mp, 195-2000C.

IR (KBr)Vmax cm-1

3400-3100 Multiple band, N-H stretch of NH2

2958 and 2870 C-H stretch of methyl, antisymmetric and symmetric

2928 and 2877 C-H stretch of methylene, antisymmetric and symmetric

1600-1350 Aromatic/Heteroaromatic skeletal stretch

1541 C-N-H bend of thiomide II

1071 C=S stretch

835 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

7.96 br.s, 1H, -NH, thiomide

7.55 br.s, 2H, -NH2, imidazole

7.22 d, 2H, aromatic, J=7.3 Hz

7.04 d, 2H, aromatic, J=8.4 Hz

227

6.84 s, 1H, -CH of amino imidazole

5.60 br.s, 2H, -NH2, aromatic

3.56 t, 2H, -CH2CH2CH3

1.62 sextet, 2H, -CH2CH2CH3

0.96 t, 3H, -CH2CH2CH3

Id4. 2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-iso-propylaminomethanethione

2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-iso-propylaminomethanethione Id4 was

prepared (1.720g, 25 %) as per procedure described for the preparation of Id1 starting from iso-

propylisothiocyanate A4 (2.626g, 0.026mol), mp, 185-1880C.

IR (KBr)Vmax cm-1

3550-3150 Multiple band, N-H stretch of NH2

2970 and 2870 C-H stretch of methyl, antisymmetric and symmetric

1650-1300 Aromatic/Heteroaromatic skeletal stretch

1540 C-N-H bend of thiomide II

1050 C=S stretch

840 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

8.13 br.s, 1H, -NH, thiomide

7.43 d, 2H, aromatic, J=9.9 Hz

7.23 d, 2H, aromatic, J=8.4 Hz

7.15 br.s, 2H, -NH2, imidazole

6.93 s, 1H, -CH of amino imidazole

228

5.35 br.s, 2H, -NH2, aromatic

3.79 septet, 1H, -CH(CH3)2

1.32 d, 6H, -CH(CH3)2

Id5. 2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-tert-butylamino methanethione

2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-tert-butylamino methanethione Id5 was

prepared (1.730g, 24 %) as per procedure described for the preparation of Id1 starting from tert-

butylisothiocyanate A5 (2.990g, 0.026mol), mp, 200-2040C.

IR (KBr)Vmax cm-1

3575-3175 Multiple band, N-H stretch of NH2

2980 and 2875 C-H stretch of methyl, antisymmetric and symmetric

1675-1300 Aromatic/Heteroaromatic skeletal stretch

1526 C-N-H bend of thiomide II

1075 C=S stretch

845 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

8.30 br.s, 1H, -NH, thiomide

7.60 d, 2H, aromatic, J=9.3 Hz

7.19 d, 2H, aromatic, J=9.3 Hz

6.95 br.s, 2H, -NH2, imidazole

6.86 s, 1H, -CH of amino imidazole

5.39 br.s, 2H, -NH2, aromatic

229

1.53 s, 9H, -C(CH3)3

e. 2-amino-4-(4-chlorophenyl)-N-alkyl-1H-imidazole-1-carbothioamide (Ie1-5)

Ie1. 2-amino-4-(4-chlorophenyl)-N-methyl-1H-imidazole-1-carbothioamide

Dissolved the (6.175g, 0.025mol) of 2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-yl-

methylaminomethanethione Id1 in a solution of 20mL of concentrated hydrochloric acid and

50mL of water, cool to about 50C, and diazotized by the gradual addition of cold solution of

(2.070g, 0.03mol) of sodium nitrite in 25mL of water to an end-point with starch-potassium

iodide paper. In the meantime prepared the solution of copper (I) chloride. Dissolved (2.705g,

0.0304mol) cuprous chloride in 8mL of concentrated hydrochloric acid and cooled in ice, and

then added the cold diazonium solution slowly and with shaking. The reaction proceeds rapidly

and with frothing. Allowed the mixture to stand for 2-3 hours with frequent shaking. The solid

separated was filtered, washed and dried and which upon recrystallization from hot water

containing a little alcohol gave 2.780g (42%) of pure Ie1, m p, 201-2050C.

IR (KBr)Vmax cm-1

3500-3200 Multiple band, N-H stretch of NH2

2961 and 2852 C-H stretch of methyl, antisymmetric and symmetric

1629-1325 Aromatic/Heteroaromatic skeletal stretch

1540 C-N-H bend of thiomide II

1094 C=S stretch,

865 2-adjacent aromatic hydrogens

801 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

8.03 br.s, 1H, -NH, thiomide

7.43 br.s, 2H, -NH2, imidazole

230

7.83 d, 2H, aromatic, J=8.20 Hz

7.30 d, 2H, aromatic, J=8.12 Hz

7.52 s, 1H, -CH of amino imidazole

2.15 s, 3H, -CH3

GCMS

m/z % intensity

281 1.08

207 22.88

193 1.23

191 1.69

73 100

Ie2. 2-amino-4-(4-chlorophenyl)-N-ethyl-1H-imidazole-1-carbothioamide

2-amino-4-(4-chlorophenyl)-N-ethyl-1H-imidazole-1-carbothioamide Ie2 was prepared (3.130g,

45%) as per procedure described for the preparation of Ie1 starting from 2-Amino-4-(4-

aminophenyl)-1H-1,3-diazol-1-yl-ethylaminomethanethione, Id2 (6.525g, 0.025mol), mp, 204-

2070C.

IR (KBr)Vmax cm-1

3400-3200 Multiple band, N-H stretch of NH2

2971 and 2871 C-H stretch of methyl, antisymmetric and Symmetric,

2938 and 2877 C-H stretch of methylene, antisymmetric and symmetric,

1664-1321 Aromatic/Heteroaromatic skeletal stretch

1523 C-N-H bend of thiomide II

231

1090 C=S stretch,

833 2-adjacent aromatic hydrogens

762 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

9.40 br.s, 1H, -NH, thiomide

8.17 br.s, 2H, -NH2, imidazole

7.96 d, 2H, aromatic, J=8.48 Hz

7.39 d, 2H, aromatic, J=8.56 Hz

7.79 s, 1H, -CH of amino imidazole

3.72 q, 2H, -CH2CH3

1.30 t, 3H, -CH2CH3

Ie3. 2-amino-4-(4-chlorophenyl)-N-propyl-1H-imidazole-1-carbothioamide

2-amino-4-(4-chlorophenyl)-N-propyl-1H-imidazole-1-carbothioamide Ie3 was prepared

(2.850g, 39%) as per procedure described for the preparation of Ie1 starting from, 2-Amino-4-(4-

aminophenyl)-1H-1,3-diazol-1-yl-n-propylamino methanethione, Id3 (6.875g, 0.025mol), mp,

205-2090C.

IR (KBr)Vmax cm-1

3400-3150 Multiple band, N-H stretch of NH2

2960 and 2865 C-H stretch of methyl, antisymmetric and symmetric

2938 and 2853 C-H stretch of methylene, antisymmetric and symmetric,

232

1681-1318 Aromatic/Heteroaromatic skeletal stretch

1541 C-N-H bend of thiomide II

1099 C=S stretch

833 2-adjacent aromatic hydrogens

790 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

9.64 br.s, 1H, -NH, thiomide

8.06 br.s, 2H, -NH2, imidazole

7.89 m ,3H, aromatic

7.46 s ,1H, aromatic

6.73 s, 1H, -CH of amino imidazole

3.53 t, 2H, -CH2CH2CH3

1.63 sextet, 2H, -CH2CH2CH3

0.96 t, 3H, -CH2CH2CH3

Ie4. 2-amino-4-(4-chlorophenyl)-N-(propan-2-yl)-1H-imidazole-1-carbothioamide

2-amino-4-(4-chlorophenyl)-N-(propan-2-yl)-1H-imidazole-1-carbothioamide Ie4 was prepared

(2.920g, 40 %) as per procedure described for the preparation of Ie1 starting from 2-Amino-4-(4-

aminophenyl)-1H-1,3-diazol-1-yl-iso-propylamino methanethione, Id4 (6.875g, 0.025mol), mp,

203-2060C.

IR (KBr)Vmax cm-1

3300-3200 Multiple band, N-H stretch of NH2

2953 and 2869 C-H stretch of methyl, antisymmetric and symmetric

233

1596-1572 Aromatic/Heteroaromatic skeletal stretch

1503 C-N-H bend of thiomide II

1092 C=S stretch

1052 C=S stretch

831 2-adjacent aromatic hydrogens

802 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

8.86 br.s, 1H, -NH, thiomide

7.95 d, 2H, aromatic, J=9.9 Hz

7.58 d, 2H, aromatic, J=8.4 Hz

7.23 br.s, 2H, -NH2, imidazole

6.93 s, 1H, -CH of amino imidazole

2.58 septet, 1H, -CH(CH3)2

1.40 d, 6H, -CH(CH3)2

Ie5. 2-amino-N-tert-butyl-4-(4-chlorophenyl)-1H-imidazole-1-carbothioamide

2-amino-N-tert-butyl-4-(4-chlorophenyl)-1H-imidazole-1-carbothioamide Ie5 was prepared

(5.500g, 76 %) as per procedure described for the preparation of Ie1 starting from, 2-Amino-4-

(4-aminophenyl)-1H-1,3-diazol-1-yl-tert-butylamino methanethione, Id5 (7.225g, 0.025mol),

mp, 208-2110C.

IR (KBr)Vmax cm-1

3400-3200 Multiple band, N-H stretch of NH2

2966 and 2871 C-H stretch of methyl, antisymmetric and symmetric

234

1684-1316 Aromatic/Heteroaromatic skeletal stretch

1524 C-N-H bend of thiomide II

1090 C=S stretch

833 2-adjacent aromatic hydrogens

793 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

7.94 d, 2H, aromatic, J=7.56 Hz

7.48 d, 2H, aromatic, J=8.68 Hz

7.30 br.s, 2H, -NH2, imidazole

7.27 s, 1H, -CH of amino imidazole

7.10 br.s, 1H, -NH, thiomide

2.58 s, 9H, -C(CH3)3

If1. 2-amino-4-(4-bromophenyl)-N-methyl-1H-imidazole-1-carbothioamide

Prepared the solution of (6.650g, 0.025mol) of 2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-

methylaminomethanethione Id1 in 10mL of 40 percent w/w hydrobromic acid in a 100 mL RBF,

cooled to 50C by immersion in a bath of ice and salt. Diazotised by the gradual addition of a

solution of (2.070g, 0.03mol) of sodium nitrite in 20mL of water, stoppered the flask after each

addition and shake until all red fumes are absorbed. Kept the tempreature between 5 and 100C.

When the diazotisation was complete, added the 2g of copper powder and copper bronze,

attached the reflux condenser to the flask and heat very cautiously on a water bath. Immediately

evolution of gas occurred, cooled the flask in crushed ice; unless the flask was rapidly removed

from the water bath, the reaction may become so violent that the contents may be shot out of the

flask. When the vigorous evolution of nitrogen moderated, heated the flask on a water bath for

30 min. Then diluted with 10mL of water, brown colour precipitate was formed, filtered the

precipitate and dried it at 178-1810C. The yield of pure If1 is 3.710g (48%), mp, 213-216

0C.

235

IR (KBr)Vmax cm-1

3550-3100 Multiple band, N-H stretch of NH2

2961 and 2853 C-H stretch of methyl, antisymmetric and symmetric,

1611-1340 Aromatic/Heteroaromatic skeletal stretch

1533 C-N-H bend of thiomide II

1094 C=S stretch,

863 2-adjacent aromatic hydrogens

741 Aromatic C-Br stretch

PMR (DMSO-d6) δppm

8.85 br.s, 1H, -NH, thiomide

8.24 d, 2H, aromatic, J=9.2 Hz

8.18 s, 1H, -CH of amino imidazole

7.82 d, 2H, aromatic, J=9.2 Hz

7.75 br.s, 2H, -NH2, imidazole

2.67 s, 3H, -CH3

Ig1. 2-amino-4-(4-cyanophenyl)-N-methyl-1H-imidazole-1-carbothioamide

Copper (I) cyanide (2.705g, 0.0304mol) was taken in a 100mL RBF and dissolved it in a

solution of (1.976g, 0.0304mol) of potassium cyanide in 125ml of water. Diazotize (6.650g

,0.025mol) of 2-Amino-4-(4-aminophenyl)-1H-1,3-diazol-1-methylaminomethanethione Id1,

following the method given above, whilst keeping the solution cold, carefully add to it about

2.0g of powdered anhydrous sodium carbonate with constant stirring until the solution is neutral

to litmus. Then warmed the copper (I) cyanide solution on a water bath to about 60 0C, and add

the cold neutralized diazonium salt solution in small quantities at a time, shaking vigorously

236

after each addition and taking care to maintain the temperature of the mixture to 60-700C Attach

a reflux condenser to the flask and heat on a boiling water bath for 15-20 min. in order to

compete the reaction. Equip the flask for steam distillation and pass steam into the mixture until

no more yellow oil passes over; if the oil solidifies in the condenser tube, turn off the condenser

water, and, after the material melts and flows through, slowly turn on the water again. Cooled

the distillate in ice-water. The solid separated was filtered, washed and dried and which upon

recrystallization from hot water containing a little alcohol gave 2.940g (46%) of pure Ig1, mp,

203-2050C.

IR (KBr)Vmax cm-1

3400-3100 Multiple band, N-H stretch of NH2

2961 and 2854 C-H stretch of methyl, antisymmetric and symmetric

2091 Cyanide C-N stretch

1635-1325 Aromatic/Heteroaromatic skeletal stretch

1595 C-N-H bend of thiomide II

1098 C=S stretch

864 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

9.39 br.s, 1H, -NH, thiomide

7.62 br.s, 2H, -NH2, imidazole

7.57-7.16 m, 4H, aromatic

6.97 s, 1H, -CH of amino imidazole

3.01 s, 3H, -CH3

GCMS

m/z % intensity

237

257(M+) 4.58

256 13.39

II. 5-AMINO-3-(4-SUBSTITUTEDPHENYL-N-ALKYL-1H-1,2,4-TRIAZOLE-1-

CARBOTHIOAMIDE:

3. 5-amino-3-(4-chlorophenyl)-N-alkyl-1H-1,2,4-triazole-1-carbothioamide (Te1-2)

Preparation of 3-Phenylazo-1, 2, 4-triazole: (Tb)

A two-step synthetic sequence involving:

Preparation of Benzene diazoniumchloride Ia/Ta and

Coupling of Benzene diazoniumchloride Ia/Ta with 1,24-triazole was followed.

a. Benzene diazoniumchloride: Ia/Ta

To (9.800g, 0.1mol) of aniline taken in a 500mL RBF was added dilute acid (30 mL of

concentrated HCl in 30mL of water). The clear solution obtained was stirred and chilled to -

50C.To this was added an ice cold solution of sodium nitrite (7.245g, 0.1mol) in 45mL of water

from a dropping funnel (The stem of which extends below the surface of liquid) at a rate such

that the temperature should not rise above 00C. The solution stirred for additional 5 min after the

addition of sodium nitrite and then a mixture of 20mL of ice cold water, 0.2 g of urea and 20 g

of crushed ice was added successively.

b. 3-Phenylazo-1, 2, 4-triazole: Tb

The cold diazonium chloride solution thus prepared was added very slowly to a vigorously

stirred and cold (00C) solution of 1,2,4-triazole (6.900g, 0.10mol) in 10% of sodium bicarbonate

(45mL) taken in a 250mL RBF immersed in an ice salt bath (assisted by direct addition of 25 g

of crushed ice. When all the diazonium salt solution was added the contents of the flask were

stirred at room temperature for 0.5 h and allowed to stand at room temperature for 0.5 h. The

reddish brown solid separated was filtered, washed with water, dried and which upon

recrystallization from glacial acetic acid gave 4.370g, (25%) of Tb, mp, 85-880C.

238

IR (KBr)Vmax cm-1

3691- 2565 N-H stretch of Triazole, bonded

3458 N-H stretch of Triazole, free

3064 and 3140 C-H stretch of Triazole

1598, 1525 1484, 1314 Aromatic/Heteroaromatic skeletal stretch

1460 N=N stretch of diazo

914 2-substituted Triazole out of plane deformations

769 and 686 5-adjacent aromatic hydrogens

PMR (CDCl3) δppm

13.24 (13.30) br. s, 1H, -NH of triazole

7.88-7.85 m, 2H, aromatic

7.51-7.40 m, 3H, aromatic

7.19 d, 1H, -HC=CH- of triazole, J=8.85 Hz

LCMS

m/z % intensity

347 (2M+H+) 18.25

346(2M+) 95.50

345(2M-H+) 100

c. 3-Amino-5-(4-aminophenyl)-1, 2, 4-triazole dihydrochloride: Tc

A warm solution of tin (II) chloride (18.900 g, 0.10mol) in 40 ml of conc. HCl was added to gently

boiling solution (8.650g, 0.05mol) of 3-phenylazo-1,2,4-triazole Tb in rectified spirit (8 mL) taken

in a RBF 250mL provided with a reflux condenser. The contents of the flask were refluxed for 1.5 h

and the reaction mixture was allowed to cool and then chilled to -50C, during this time

crystallisation took place. Solid separated was filtered, washed with ether, dried and upon

239

recrystallization from 20% hot absolute alcohol gave 16.120g (65%) of Tc, mp, 200- 2050C

Decomp.

IR (KBr)Vmax cm-1

3410 N-H stretch of Triazole

3395-2831 Multiple band, N-H stretch of NH3+

3110 and 3055 C-H stretch of Triazole

1665, 1616 1520, 1478, 1340 Aromatic/Heteroaromatic skeletal stretch

857 2-adjacent aromatic hydrogens

PMR (D2O) δppm

7.40 d, 2H, aromatic, p-disubtitued, J=8.613 Hz

7.27 d, 2H, aromatic, p-disubtitued, J=8.667 Hz

LCMS

m/z % intensity

175(M+) 100

176(M+H+) 10.75

174(M-H+) 15.25

d. Synthesis of 5-amino-3-(4-aminophenyl)-N-alkyl-1H-1,2,4-triazole-1-

carbothioamide (Td1-2)

Td1. 5-amino-3-(4-aminophenyl)-N-methyl-1H-1,2,4-triazole-1-carbothioamide

To a solution of sodium hydroxide (3.000g, 0.75mol) in 10mL of water taken in a RBF (100

mL) was added a solution of 3-Amino-5-(4-aminophenyl)-1, 2, 4-triazole dihydrochloride Tc

(6.175g, 0.025mol) in 10 ml of water. Methylisothiocyanate A1 (1.898g, 0.026mol) in THF

(10mL) was added to the above mixture solution while stirring. The reaction mixture solution

240

was stirred at reflux for 4h, neutralised with in 1N-HCl and extracted with ethyl acetate

(3x50mL). The combined ethyl acetate extract washed with distilled water, dried over anhydrous

sodium sulphate, filtered and solvents removed by distillation. The residue thus left upon

recrystallization from methanol gave 2.780 (45%) of Td1, mp, 202-2050C.

IR (KBr)Vmax cm-1

3400-3200 Multiple band, N-H stretch of NH2

2928 and 2850 C-H stretch of methyl, antisymmetric and symmetric

1683-1356 Aromatic/Heteroaromatic skeletal stretch

1564 C-N-H bend of thiomide II

1037 C=S stretch,

830 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

8.00 br.s, 1H, -NH, thiomide

7.90 d, 2H, aromatic, J=8.68 Hz

6.67 d, 2H, aromatic, J=8.64 Hz

6.04 br.s, 2H, -NH2, aromatic

2.80 s, 3H, -CH3

Td2. 5-amino-3-(4-aminophenyl)-N-ethyl-1H-1,2,4-triazole-1-carbothioamide

5-amino-3-(4-aminophenyl)-N-ethyl-1H-1,2,4-triazole-1-carbothioamide Td2 was prepared

(2.870g, 44%) as per procedure described for the synthesis of Td1 starting from

ethylisothiocyanate A2 (2.262g, 0.025mol), mp, 206-2090C.

IR (KBr)Vmax cm-1

3450-3200 Multiple band, N-H stretch of NH2

2965 and 2856 C-H stretch of methyl, antisymmetric and symmetric

241

2927 and 2877 C-H stretch of methylene, antisymmetric and symmetric

1650-1340 Aromatic/Heteroaromatic skeletal stretch

1555 C-N-H bend of thiomide II

1093 C=S stretch,

863 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

8.35 br.s, 1H, -NH, thiomide

7.21 d, 2H, aromatic, J=8.28 Hz

7.03 br.s, 2H, -NH2, triazole

6.87 d, 2H, aromatic, J=8.68 Hz

6.53 br.s, 2H, -NH2, aromatic

3.50 q, 2H, -CH2CH3

1.90 t, 3H, -CH2CH3

e. 5-amino-3-(4-chlorophenyl)-N-methyl-1H-1,2,4-triazole-1-carbothioamide (Te1-2)

Te1. 5-amino-3-(4-chlorophenyl)-N-methyl-1H-1,2,4-triazole-1-carbothioamide

Dissolved (6.200g, 0.025mol) of 5-Amino-3-(4-chlorophenyl)-1H-1,2,4-

triazol-1-yl-methylaminomethanethione Td1 in a solution of 20mL of concentrated hydrochloric

acid and 50ml of water, cooled to about 50C, and diazotised by the gradual addition of cold

solution of (2.070g, 0.03mol) of sodium nitrite in 25mL of water to an end-point with starch-

potassium iodide paper. In the meantime prepared the solution of copper(I) chloride. Dissolved

(2.600g, 0.030mol) cuprous chloride in 8 mL of concentrated hydrochloric acid and cooled in

ice, and then added the cold diazonium solution slowly and with shaking. The reaction proceeds

rapidly and with frothing: allow the mixture to stand for 2-3 h with frequent shaking. The solid

242

separated was filtered, washed and dried and which upon recrystallization from hot water

containing a little alcohol gave 2.320g (35%) of Te1, mp, 203-2050C.

IR (KBr)Vmax cm-1

3400-3150 Multiple band, N-H stretch of NH2

2925 and 2852 C-H stretch of methyl, antisymmetric and symmetric

1638-1321 Aromatic/Heteroaromatic skeletal stretch

1590 C-N-H bend of thiomide II

1091 C=S stretch

835 2-adjacent aromatic hydrogens

807 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

9.68 br.s, 1H, -NH, thiomide

7.95 d, 2H, aromatic, J=8.28 Hz

7.41 d, 2H, aromatic, J=8.68 Hz

7.03 br.s, 2H, -NH2, triazole

3.02 s, 3H, -CH3

LCMS

m/z % intensity

270 1.75

269 1.25

268 (M+H+) 4.25

267(M+) 3.50

243

240 8.75

238 16.75

224 49.25

222 82.50

Te2. 5-amino-3-(4-chlorophenyl)-N-ethyl-1H-1,2,4-triazole-1-carbothioamide

5-amino-3-(4-chlorophenyl)-N-ethyl-1H-1,2,4-triazole-1-carbothioamide Te2 was prepared

(2.310g, 33%) as per procedure described for the synthesis of Te1 starting from 5-amino-3-(4-

aminophenyl)-N-ethyl-1H-1,2,4-triazole-1-carbothioamide Td2, (6.55g, 0.025mol), mp, 205-

2070C.

IR (KBr)Vmax cm-1

3400-3200 Multiple band, N-H stretch of NH2

2969 and 2871 C-H stretch of methyl, antisymmetric and symmetric

2940 and 2877 C-H stretch of methylene, antisymmetric and symmetric

1665-1361 Aromatic/Heteroaromatic skeletal stretch

1525 C-N-H bend of thiomide II

1089 C=S stretch,

834 2-adjacent aromatic hydrogens

761 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

9.13 br.s, 1H, -NH, thiomide

7.91 d, 2H, aromatic, J=9.12 Hz

7.52 br.s, 2H, -NH2, triazole

244

7.36 d, 2H, aromatic, J=9.23 Hz

3.71 q, 2H, -CH2CH3

1.28 t, 3H, -CH2CH3

LCMS

m/z % intensity

282(M+H+) 100

284 21.50

255 10.75

253 16.75

225 5.50

223 14.25

245

B. MONOCYCLICS: 6-MEMBERED RINGS WITHOUT AND WITH 1 AND 2

NITROGENS

III. 3-AMINO-4’-SUBSTITUTED-N-ALKYL-1,1’-BIPHENYL-4-CARBOTHIOAMIDE:

4. 3-amino-4'-chloro-N-alkylbiphenyl-4-carbothioamide (Bf)

Preparation of 4'-chloro-4-methyl-3-nitrobiphenyl: (Bb)

A two-step synthetic sequence involving:

Preparation of 4-Chlorobenzenediazoniumsulphate Ba and

Coupling of 4-Chlorobenzenediazoniumsulphate Ba with 2-nitrotoluene was

followed.

a. 4-Chlorobenzenediazonium sulphate: Ba

4-chloroaniline (12.750g, 0.10mol) was added with stirring to 100mL of water containing 14mL

of Conc. sulphuric acid and the contents were cooled to 0-50C diazotized by the gradual addition

of (7.450g, 0.101mol) of sodium nitrite in 20mL of water with continuous stirring to en end

point with starch iodide paper.

b. 4'-chloro-4-methyl-3-nitrobiphenyl: Bb

The above diazotized solution was filtered and transferred to a 2L conical flask surrounded by

ice water, (41.100g, 0.30mol) of 2-nitrotoluene, 120 mL of carbon tetra chloride and 40 mL of

acetonitrile as co-solvent were added to this. The contents were stirred vigorously and to stirred

solution was added an ice cold aqueous solution of 80g of sodium acetate trihydrate in 200mL of

water in installments while maintaining the temperature 5-100C. The stirring continued for 24h,

after first 3h the reaction was allowed to proceed at room temperature. The organic layer was

separated, washed with water, dried over anhydrous sodium sulphate and excess carbon tetra

chloride was removed in vacuum. The unreacted 2-Nitrotoluene was also removed by vacuum.

Then product was separated by column chromatography yielded 10.250g (41%) of Bb, as

viscous mass.

IR (KBr)Vmax cm-1

246

2963 and 2830 C-H stretch of methyl, antisymmetric and symmetric

1530 N=O stretch

1261 C-N stretch

828 2-adjacent aromatic hydrogens

800 1,2,4 trisubstituted aromatic hydrogens

756 Aromatic C-Cl stretch

PMR (CDCl3) δppm

9.89 s,1H, aromatic

8.00 d, 1H, aromatic, J=7.76 Hz

7.85 d, 2H, aromatic, J=9.44 Hz

7.01 d,2H, aromatic, J=9.52 Hz

6.91 d,1H, aromatic, J=9.00 Hz

3.89 s, 3H, -CH3

LCMS

m/z % intensity

250 1.25

248(M+H) 3.75

249 2.50

247(M+) 6.50

235 4.25

233 6.25

211 100

247

204 3.50

202 7.25

Oxidation of methyl group and reduction of nitro group of compound Bb to generate Bd:

c. 4'-chloro-3-nitrobiphenyl-4-carboxylic acid: Bc

Placed the (18.340g, 0.07mole) of sodium di chromate in 250mL RBF with the further addition

of 40mL water. Shaked the flask properly so that sodium di chromate may get dissolved in

water. Further added (12.375 g, 0.05mole) of 6-(4-chlorophenyl)-2-nitrotoluene to the solution

and shake the flask. To the solution added the 40ml of Conc. sulphuric acid over a time span of

25 min. with continuous shaking when addition was completed, cooled the contents of flask

properly and mixed the 20mL of glacial acetic acid to the solution. Now refluxed the reaction

mixture on water bath for 4 h. and further on direct flame for 30 min. cooled the RBF under

running tap water and poured the reaction mixture into a beaker containing crushed ice. Shaked

the solution and precipitation occurred with greenish tinch. Filtered the solid on water pump and

washed with water at least three times so that sodium sulphate was washed. Further transferred

the precipitates into 100mL beaker containing 30mL 0f 5% sulphuric acid. Digested the

precipitates on water bath for about 15 min with continuous shaking. Filtered the solution on

water pump. Washed the precipitates with water and transferred this solid in a beaker containing

50mL of sodium hydroxide by continuous shaking. Filtered the solution and preserved the

filtrate. Acidified the filtrate with 5% sulphuric acid till it was acidic. The solid separated was

filtered, washed with cold water and dried and which upon recrystallization from acetic acid

gave 8.020g (58%), of Bc mp, 225-2280C and simultaneously this compound was subjected to

reduction of nitro group to generate Bd.

3-amino-4'-chlorobiphenyl-4-carboxylic acid: Bd

248

(13.850g, 0.05mol) 6-(4-chlorophenyl)-2-nitrobenzoic acid was placed in a RBF fitted with a

condenser added to it (17.700g, 0.15mol) powdered tin and 100mL Conc. HCl. The mixture was

gently heated until the reaction commences and removed the flame. The flask was frequently

shaked so that the insoluble acid adhering to the sides of the flask is transferred to the reaction

mixture. Occasional gentle warming may be necessary. After 2h most of the tin have been

reacted and clear solution remained allow cooling some what and the liquid was decanted into a

1L beaker. The residual tin was washed by decantation with 100mL of water, and added the

washings to the contents of beaker. Conc. Ammonia solution was added until the solution is just

alkaline to litmus and digested the suspension of precipitate hydrated tin oxide on a steam bath

for 20min. Now 10g of filter aid was added, stirred well filter at the pump and washed with hot

water. Transferred the filter cake to a beaker, heat on a water bath with 200 mL of water to

ensure extraction of product and refiltered, concentrated the combined filtrate and washing until

the volume has been reduced to 175-200mL filter off any solid which separates. Acidified the

liquid to litmus with glacial acetic acid and evaporated on a water bath until crystal commences

to separate and cooled in ice bath. Filtered the crystals and dried. The yield of pure Bd is 8.750g

(71%), mp, 240-2450C.

IR (KBr)Vmax cm-1

3365 N-H stretch of NH2

1701 C=O stretch

1236 C-N stretch

810 2-adjacent aromatic hydrogens

801 1,2,4 trisubstituted aromatic hydrogens

PMR (CDCl3) δppm

7.90 d,1H, aromatic, J=8.60 Hz

7.34 d, 2H, aromatic, J=8.56 Hz

7.21 s,1H,aromatic

7. 13 d, 2H, aromatic, J=8.32 Hz

249

6.63 d,1H, aromatic, J=8.24 Hz

3.23 br. s, 2H, -NH2

LCMS

m/z % intensity

249 5.25

247(M+) 19.50

233 7.50

231 21.25

215 33.25

213 77.50

187 12.51

e. 3-amino-4'-chloro-N-alkylbiphenyl-4-carboxamide (Be1 and 5)

Be1. 3-amino-4'-chloro-N-methylbiphenyl-4-carboxamide

To a solution of (2.470 g, 0.010mol) 3-amino-4'-chlorobiphenyl-4-carboxylic acid Bd in 10mL

of dry THF was added (1.620g, 0.010mol) of N,N'-carbonyldiimidazole. One-half hour later,

when effervescence of CO2 gas was stopped then (5mL, 0.310 g, 0.010mole) of methylamine

solution in 2M methanol was added. Then reaction mixture was stirred 3h on magnetic stirrer.

Now reaction mixture was placed in dark for overnight. After standing overnight, the THF was

removed by an air stream and 50mL of 1N hydrochloric acid was added. Cooling of the solution

gave a solid. This was washed with water, triturated with 20 mL of 5% sodium bicarbonate

solutions, filtered and again washed with water, dried and which upon recrystallization from

50% ethanol gave 1.250g (48%) of Be1, mp, 280-2820C.

IR (KBr)Vmax cm-1

3151 N-H stretch of NH2

250

2929 and 2854 C-H stretch of methyl, antisymmetric and symmetric

1699 C=O stretch, amide

1257 C-N stretch

827 2-adjacent aromatic hydrogens

801 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO-d6) δppm

10.03 s,1H, NH

8.32 s, 1H, aromatic

7.89 d, 2H, aromatic, J=8.24 Hz

7.81 d, 1H, aromatic, J=7.64 Hz

7. 39 d, 2H, aromatic, J=7.48 Hz

7.04 d, 1H, aromatic, J=7.92 Hz

4.35 s, 1H, -CH3

LCMS

m/z % intensity

263 3.25

261(M+H) 9.25

262 7.25

260 (M+) 15.75

248 7.50

246 23.50

251

233 5.25

232 5.25

231 20.50

230 18.25

225 53.25

Be5. 3-amino-N-tert-butyl-4'-chlorobiphenyl-4-carboxamide

3-amino-N-tert-butyl-4'-chlorobiphenyl-4-carboxamide Be5 was prepared (1.390g, 46%) as per

procedure desscribed for the synthesis of Be1 by treating (2.470 g, 0.010mol) 3-amino-4'-

chlorobiphenyl-4-carboxylic acid Bd with tert.butylamine solution (5ml, 0.730g, and 0.0mol),

mp, 285-2860C

IR (KBr)Vmax cm-1

3300-3090 N-H stretch of NH2

2966 and 2854 C-H stretch of methyl, antisymmetric and symmetric

1687 C=O stretch, amide

1279 C-N stretch

830 2-adjacent aromatic hydrogens

752 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO-d6) δppm

8.10 d, 2H, aromatic, J=9.00 Hz

8.00 s, 1H, NH

7.59 d, 2H, aromatic, J=9.00 Hz

7.52 2H, aromatic

7.23 s, 1H, aromatic

252

1.36 s, 9H, -C(CH3)3

f. 3-amino-4'-chloro-N-alkylbiphenyl-4-carbothioamide (Bf1)

Bf1. 3-amino-4'-chloro-N-methylbiphenyl-4-carbothioamide

A mixture of 3-amino-4'-chloro-N-methylbiphenyl-4-carboxamide Be1 2.595g (0.01mol) and

Lawesson’s reagent (2.020g, 0.005 mmol), was taken in a glass tube and mixed thoroughly with

a spatula. The glass tube was then placed in an alumina bath inside the microwave oven (900 W)

and irradiated for 3 min. On completion of the reaction, followed by TLC examination, the

colored material was dissolved in dichloromethane and adsorbed on silica gel and purified by

silica gel column chromatography, which afforded the pure Bf1 1.740g, (63%) as viscous mass.

IR (KBr)Vmax cm-1

3271 N-H stretch of NH2

2930 and 2854 C-H stretch of methyl, antisymmetric and symmetric

1090 C=S stretch, thioamide

1275 C-N stretch

833 2-adjacent aromatic hydrogens

801 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO-d6) δppm

10.03 s,1H, NH

8.32 s, 1H, aromatic,

7.89 d, 2H, aromatic, J=8.24 Hz

7.81 d, 1H, aromatic, J=7.64 Hz

7. 39 d, 2H, aromatic, J=7.48 Hz

7.04 d, 1H, aromatic, J=7.92 Hz

253

4.35 s, 1H, -CH3

LCMS

m/z % intensity

280 12.50

278 (M+2H) 26.25

276 8.75

248 5.25

246 12.25

247 6.50

245 16.25

206 10.25

204 30.25

IV. 2-AMINO-6-(4-SUBSTITUTEDPHENYL) -N-ALKYLPYRIDINE -3-CARBO

THIOAMIDE:

5. 2-amino-6-(4-chlorophenyl)-N-alkylpyridine-3-carbothioamide (Pi)

a. Sodium formyl 4-chloroacetophenone: Pa

In a 1L three necked flask fitted with a hershberg stirrer sealed by a lubricated rubber sleeve, a

dropping funnel, and a reflux condenser attached to a calcium chloride drying tube was placed

(5.400 g, 0.10mol) of sodium methoxide and 150mL dry ether (dried over sodium wire). The

flask was cooled in an ice bath and a mixture of (15.400g, 0.10mol) 4-chloro acetophenone and

(7.400 g, 0.10mol) of ethyl formate is added through the dropping funnel at a rate of about 2

drops per second, with stirring, during a period of about 1 h. Stirring was continued 15 min.

longer with the ice bath in place and then 1 h after it was removed. Then the reflux condenser

was replaced by a condenser set for distillation, and the ether was distilled by heating the

mixture in a water bath at a temperature which was not allowed to rise above 700C. The last

254

traces of the ether were removed by distillation under reduced pressure with the aid of water

aspirator. The solid residue of sodium formyl 4-chloroacetophenone thus obtained was

immediately used in the next step without any further purification.

b. 6-(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile: Pb

To the solid residue of sodium formyl 4-chloroacetophenone Pa remaining in the flask was

added a solution of (8.400g, 0.10mol) of cyanoacetamide in 40mL of water and piperidine

acetate (prepared by adding piperidine to 1mL of glacial acetic acid in 2.5mL of water until the

solution was just basic to litmus.) The flask was equipped with a reflux condenser, and the

mixture was heated under reflux for 2 h. At the end of this time 20mL of water was added and

the solution was acidified (to litmus) with acetic acid, causing separation of the product as a

voluminous yellow precipitate. The mixture was cooled in an ice bath for 2 h, and the product

was collected on a suction filter, washed on the filter with three 100mL portions of ice water,

dried, which upon recrystalisation from rectified spirit gave 18.000g (78%) of Pb, mp, 175-

1800C.

IR (KBr)Vmax cm-1

3447 N-H stretch

2222 Cyanide C-N stretch

1638 C=O stretch

820 2-adjacent aromatic hydrogens

770 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

7.91 d,1H, aromatic, J=7.56 Hz

7.70 d, 2H, aromatic, J=8.64 Hz

7.45 d, 2H, aromatic, J=8.64 Hz

6.54 d, 1H, aromatic, J=7.52 Hz

255

GCMS

m/z % intensity

233 0.74

231(M+H) 3.24

232 10.99

230(M+) 26.09

c 6-(4-chlorophenyl)-2-methoxypyridine-3-carbonitrile: Pc

Silver carbonate (21.920 g, 0.08mol) and 6-(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3-

carbonitrile Pb (11.540 g, 0.05mol) was stirred with methyliodide (11.324 g, 0..008mol) for 72 h

in 60 mL of benzene at room temperature in the dark. The reaction mixture was cooled and

filtered. The filtrate was washed with 30 mL of 10% sodium bicarbonate solution and then

washed twice with 30 mL of water. The benzene solution was dried over magnesium sulfate,

filtered, and evaporated under reduced pressure. The solid separated was dried and upon

recrystalization from rectified spirit gave 10.200g (87%) of Pc, mp, 230-2320C.

IR (KBr)Vmax cm-1

2955 and 2850 C-H stretch of methyl, antisymmetric and symmetric

2224 C-N stretch

1260 and 1028 C-O-C stretch of ether, antisymmetric and symmetric

1600-1449 C=C, C=N stretch of heteroaromatic

822 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

8.00 d, 2H, aromatic, J=8.64 Hz

7.93 d, 1H, aromatic, J=7.88 Hz

256

7.46 d, 2H, aromatic, J=8.64 Hz

7.30 d, 1H, aromatic, J=8.48 Hz

4.15 s, 1H, -OCH3

LCMS

m/z % intensity

248 7.25

246(M+H) 21.25

247 34.25

245(M+) 100

d. 6-(4-chlorophenyl)-2(1)-pyridone-3-carboxylic acid: Pd

In a 250mL RBF fitted with a reflux condenser are placed (11.475g, 0.050mol) 3-cyano-6-(4-

chlorophenyl)-2(1)-pyridone Pb, 50% 20mL Conc. sulfuric acid and 20mL glacial acetic acid.

The mixture was refluxed for 6 h on rota mental at room temperature. Then it was poured into

the ice-cold water. The compound 6-(4-chlorophenyl)-2(1)-pyridone-3-carboxylic acid (Pd) was

not characterized.

e. 6-(4-chlorophenyl)-2-methoxypyridine-3-carboxylic acid: Pe

From: 6-(4-chlorophenyl)-2(1)-pyridone-3-carboxylic acid

Silver carbonate (21.920 g, 0.08mol) and 6-(4-chlorophenyl)-2(1)-pyridone-3-carboxylic acid

Pd (12.425 g, 0.05mol) was stirred with methyliodide (11.324 g, 0..008mol) for 72 h in 60 mL

of benzene at room temperature in the dark. The reaction mixture was cooled and filtered. The

filtrate was washed with 30mL of 10% sodium bicarbonate solution and then washed twice with

30mL of water. The benzene solution was dried over magnesium sulfate, filtered, and

evaporated under reduced pressure gave a crude viscous mass, which could not be characterized.

From: 6-(4-chlorophenyl)-2-methoxypyridine-3-carbonitrile

257

In a 250mL RBF fitted with a reflux condenser are placed (12.150g, 0.050mol) 6-(4-

chlorophenyl)-2-methoxypyridine-3-carbonitrile Pc, 50% 20mL Conc. sulfuric acid and 20mL

glacial acetic acid. The mixture was refluxed for 6 h on rota mental at room temperature, the

reaction mixture got coagulated when quenched into ice cold water and hence also could not be

characterized.

f. 2-amino-6-(4-chlorophenyl)nicotinonitrile: Pf

The Lewis acid BF3.Et2O (1 equiv) was stirred with (12.150g, 0.050mol) of 6-(4-chlorophenyl)-

2-methoxypyridine-3-carbonitrile Pc in dry THF (0.05 mol/ L) for 10 min at 0 0C under

nitrogen atmosphere, and then a alcoholic ammonia (1.1 equiv) was added over a period of 15

min. After being stirred for 2 h at 300C, the mixture was quenched by adding ammonium

chloride solution (5mL), extracted with ethylacetate, washed with brine, and dried over sodium

sulfate. The solid product obtained after evaporation which upon recrystallization from rectified

spirit gave 5.960g (52%) of Pf, mp, 220-2210C.

IR (KBr)Vmax cm-1

3132 N-H stretch of NH2

2223 C-N stretch of cynide

1600-1490 C=C, C=N stretch of heteroaromatic

823 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

8.01 d, 2H, aromatic, J=9.20 Hz

7.93 d, 1H, aromatic, J=7.76 Hz

7.46 d, 2H, aromatic, J=8.68 Hz

7.41 d, 1H, aromatic, J=7.84 Hz

1,73 br. s, 2H NH2

258

LCMS

m/z % intensity

272(M+23) 10.10

270 50.00

232 6.09

230 15.28

204 10.09

202 51.01

186 55.76

184 100

g. 2-amino-6-(4-chlorophenyl)nicotinic acid : Pg

In a 250ml RBF fitted with a reflux condenser are placed (11.475g, 0.050mol) 2-amino-6-(4-

chlorophenyl)nicotinonitrile Pf, 50% 20mL Conc. sulfuric acid and 20mL glacial acetic acid.

The mixture was refluxed for 6h on rota mental at room temperature. Then it was poured into the

ice-cold water. The compound 2-amino-6-(4-chlorophenyl)nicotinic acid Pg was precipitate out

and the product is collected on a suction filter, washed on the filter with three 100-mL portions

of ice water, dried and upon recrystalization from 80% rectified spirit and 20% hot water gave

5.590g (45%), mp, 218-2190C

IR (KBr)Vmax cm-1

3408 N-H stretch of NH2

1735 C=O stretch of acid

1600-1458 C=C, C=N stretch of heteroaromatic

824 2-adjacent aromatic hydrogens

259

PMR (DMSO-d6) δppm

8.50 d, 1H, aromatic, J=7.64 Hz

7.72 d, 2H, aromatic, J=8.68 Hz

7.50 d, 2H, aromatic, J=8.64 Hz

6.77 d, 1H, aromatic, J=7.60 Hz

LCMS

m/z % intensity

251 2.50

249(M+H) 7.25

250 11.75

248 (M+) 36.75

234 5.50

232 12.50

206 5.75

204 12.75

h. 6-(4-chlorophenyl)-2-amino-N-alkylpyridine-3-carboxamide :( Ph1and5)

Ph1. 6-(4-chlorophenyl)-2-amino-N-methylpyridine-3-carboxamide

To a solution of (12.480 g, 0.050mol) of 2-amino-6-(4-chlorophenyl)nicotinic acid Pg in

10mL of dry THF was added (8.100 g, 0.050mol) of N,N'-carbonyldiimidazole. One-half hour

later, when effervescence of CO2 gas was stopped then (5mL, 0.310 g, 0.010mole) of

methylamine solution in 2M methanol was added. Then reaction mixture was stirred 3h on

260

magnetic stirrer. Now reaction mixture was placed in dark for overnight. After standing

overnight, the THF was removed by an air stream and 50mL of 1N hydrochloric acid was added.

Cooling of the solution gave a solid. This was washed with water, triturated with 20 mL of 5%

sodium bicarbonate solutions, filtered and again washed with water, dried and which upon

recrystallization from 50% ethanol gave 5.600g (43%) of Ph1, mp, 276-2770C.

IR (KBr)Vmax cm-1

3419 N-H stretch of NH2

2928 and 2827 C-H stretch of methyl, antisymmetric and symmetric

1640 C=O stretch, amide

1600-1480 C=C, C=N stretch of heteroaromatic

1259 C-N stretch, amide

824 2-adjacent aromatic hydrogens

795 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO-d6) δppm

8.80 s, 1H, NH (overlapping)

8.48 d,1H, aromatic, J=7.48 Hz

7.55 d, 2H, aromatic, J=8.36 Hz

7.40 d, 2H, aromatic, J=8.44 Hz

6.55 d,1H, aromatic, J=7.44 Hz

7.38 br, s, 2H, -NH2

3.55 s, 1H, -CH3

LCMS

m/z % intensity

261

266 22.75

264(M+3) 75.25

248 30.50

246 90.77

234 5.25

232 14.25

205 1.25

203 4.25

Ph5. 6-(4-chlorophenyl)-2-amino-N-tertbutylpyridine-3-carboxamide

6-(4-chlorophenyl)-2-amino-N-tertbutylpyridine-3-carboxamide Ph5 was prepared (6.050g,

40%) as per procedure desscribed for synthesis of Ph1 by treating (12.480 g, 0.050mol) of 2-

amino-6-(4-chlorophenyl)nicotinic acid Pg with tert.butylamine solution (5mL, 0.730g, and

0.01mol), mp, 283-2850C

IR (KBr)Vmax cm-1

3403 N-H stretch of NH2

2930 and 2871 C-H stretch of methyl, antisymmetric and symmetric

1640 C=O stretch of amide

1600-1480 C=C, C=N stretch of heteroaromatic

1258 C-N stretch of amide

824 2-adjacent aromatic hydrogens

794 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO-d6) δppm

262

8.85 s, 1H, NH

8.45 d,1H, aromatic, J=7.32 Hz

8.10 br, s, 2H, -NH2

7.56 d, 2H, aromatic, J=8.20 Hz

7.48 d ,2H, aromatic, J=8.36 Hz

6.59 d,1H, aromatic, J=7.40 Hz

1.35 s, 9H, -C(CH3)3

i. Synthesis of 2-amino-6-(4-chlorophenyl)-N-alkylpyridine-3-carbothioamide (Pi1)

Pi1. 2-amino-6-(4-chlorophenyl)-N-methylpyridine-3-carbothioamide

A mixture of 6-(4-chlorophenyl)-2-amino-N-methylpyridine-3-carboxamide Ph1 (2.600g,

0.01mol) and Lawesson’s reagent (2.020g, 0.005mmol), was taken in a glass tube and mixed

thoroughly with a spatula. The glass tube was then placed in an alumina bath inside the

microwave oven (900 W) and irradiated for 3 min on completion of the reaction, followed by

TLC examination, the colored material was dissolved in dichloromethane and adsorbed on silica

gel and purified by silica gel column chromatography, which afforded the pure Pi11.656g, (60%)

as viscous mass.

IR (KBr)Vmax cm-1

3403 N-H stretch of NH2

2924 and 2853 C-H stretch of methyl, antisymmetric and symmetric

1261 C-N stretch

1101 C=S stretch

801 2-adjacent aromatic hydrogens

263

795 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO) δppm

8.43 d,1H, aromatic, J=7.00 Hz

8.04 s, 1H, NH

7.82 d, 2H, aromatic, J=7.24 Hz

7.54 d, 2H, aromatic, J=7.52 Hz

6.92 d,1H, aromatic, J=6.40 Hz

3.68 s, 1H, -CH3

LCMS

m/z % intensity

281 3.25

279(M+2H) 0.94

266 25.25

264 46.25

248 30.50

246 80.25

205 1.25

203 5.25

264

V. 2-Amino-6-(4-substitutedphenyl)-N-alkylpyrimidine-3-carbothioamide:

6. 4-amino-2-(4-chlorophenyl)-N-alkylpyrimidine-5-carbothioamide (PMi)

a. Ethyl 4-chlorobenzenecarboximidoate hydrochloride: PMa

4-chlorobenzonitrile (13.700g, 0.10mol) dissolved in 30mL of dry chloroform was cooled to 40C

and treated with 5.4mL of absolute ethanol which was 6.6 M with respect to dry hydrogen

chloride (20% excess hydrogen chloride). The solution was held at 40C overnight and then at

250C for 2 h. At this point the imino ether hydrochloride had separated as crystals. The solid

separated was dried and upon recrystalization from 50% chloroform gave 20.160g (80%) of

PMa, mp, 193-1970C Decomp.

IR (KBr)Vmax cm-1

3400-3200 Multiple band, N-H stretch of NH2

2985 and 2871 C-H stretch of methyl, antisymmetric and symmetric

2906 and 2877 C-H stretch of methylene, antisymmetric and symmetric

1283 and 1007 C-O-C stretch of ether, antisymmetric and symmetric

821 2-adjacent aromatic hydrogens

741 Aromatic C-Cl stretch

PMR (CDCl3) δppm

7.60 d, 2H, aromatic, J=8.60 Hz

7.47 d, 2H, aromatic, J=8.56 Hz

6.24 br.s, 2H,

4.94 q, 2H, -CH2CH3

1.62 t, 3H, -CH2CH3

b. 4-chlorobenzenecarboximidamide hydrochloride: PMb

265

The solvents were removed under reduced pressure at 250C and the residue was dissolved in

10mL of absolute ethanol at 00C. A saturated solution of dry ammonia in absolute ethanol

(20mL) was added and the mixture was held in a tightly stoppered flask for 4 days at 250C. The

solvents were removed under reduced pressure and the residue was dissolved in the system

methanol-chloroform-water, 2: 2: 1; the top layer was adjusted to pH 3 with hydrochloric acid

and a countercurrent distribution was performed. The solid separated was dried and upon

recrystalization from chloroform gave 15.920g (84%) of PMb, mp, 210-2110C Decomp.

IR (KBr)Vmax cm-1

3460 N-H stretch of NH2

1677 and 1595 Amidinium I and II band

1285 C-N stretch,

1087 Aromatic C-Cl stretch

844 2-adjacent aromatic hydrogens

PMR (D2O) δppm

7.60 d, 2H, aromatic, J=8.72 Hz

7.48 d, 2H, aromatic, J=8.76 Hz

Or

A1-L round-bottom flask was charged with 50 mL of CH3OH (dried over Mg metal), (13.7g,

0.1) mol of the 4-chlorobenzonitrile and (.54g, 0.01 mol) of sodium methoxide. The contents of

the flask were protected from moisture and stirred magnetically for 48 h. Then, 0.1 mol of

NH4CI was added and stirring was continued for 24 h. Unreacted NH4Cl was filtered, and

methanol was stripped from the filtrate to afford the crude products. These were washed free of

unreacted arylnitriles with ether. The recovered nitrile was recycled, Small samples were

crystallized from ethanol for melting point determinations.

c. 2-cyanopropanoic acid: PMc

266

Placed the (9.900g, 0.10mol) 2-chloropropionic acid and crushed ice in a large beaker and

neutralized it accurately to litmus with a cold solution of NaOH the temperature did not allow to

rise above 300C during the neutralization. Then prepared the solution of potassium cyanide in

water in a flask in the fuming cupboard. Heated to about 550C for rapid solution and finally to

boiling. Added to the resulting hot solution a portion of the solution of sodium chloro acid and

removed the flame immediately the reaction commences. When the vigorous reaction has been

subsided somewhat, added another portion, followed by the remainder when the temperature

commences to fall again. Boiled the mixture for 5 min. but no longer (otherwise some HCN may

be lost and sodium glycolate may form) and then cooled with running water for 30 min., and

then filtered the solution as it was not clear.

Liberated the cyano acid by cautiously adding with vigorous stirring 100mL of Conc. HCl,

evaporated the solution under reduced pressure using a rotary evaporator, do not heat above

750C as considerable loss may result owing to the decomposition of 2-cyanopropionic acid.

Added 250 ml of rectified spirit to the residue, filtered at the pump from the sodium chloride and

wash the residue with another rectified spirit, evaporation of the alcoholic solution gave 7.420g

(75%) of PMc, bp, 145-1460C.

IR (KBr)Vmax cm-1

2985 and 2941 C-H stretch of methyl, antisymmetric and symmetric

2247 C-N stretch of cyanide

1731 C=O stretch

d. Ethyl 2-cyanopropanoate : PMd

Added the mixture of 250mL of absolute ethanol and 4.5 mL of conc. Sulphuric acid into above

prepared cyanoacid and reflux on a water bath for 3 h. Removed the excess of alcohol and some

of the water formed by distillation under reduced pressure. Heated the residue again with 125mL

of absolute ethanol and 2mL of Conc. sulphuric acid for 2 h. and removed the excess of alcohol

under reduced pressure. Allowed the ester to cool to room temptature and neutralized the

sulphuric acid with a Conc. solution of sodium carbonate. Separated the upper layer of ester and

extracted the aqueous layer with ether. Dried the combined products with anhyd. sodium

sulphate. Removation of the solvent by distillation gave 8.500g (67%) of PMd, bp, 152-1540C.

267

IR (KBr)Vmax cm-1

2983 and 2865 C-H stretch of methyl, antisymmetric and symmetric

2943 and 2860 C-H stretch of methylene, antisymmetric and symmetric

2203 C-N stretch of cyanide

1744 C=O stretch

PMR (CDCl3) δppm

4.12 q, 2H, -CH2CH3

3.41 q, 1H, -CHCH3

1.32 d, 3H, -CHCH3

1.20 t, 3H, -CH2CH3

e. 6-amino-2-(4-chlorophenyl)-5-methylpyrimidin-4-ol: PMe

4-chloroBenzamidinehydrochloride (19.000 g., 0.10mol) and 2-cyanopropionicester (12.700 g.,

0.10mol) in 100 ml. of methanol cooled in an ice-bath was added to (22.000g, 0.40mol) of

sodium methoxide dissolved in 100mL of methanol. The mixture was refluxed for 2 h, taken

down to dryness under reduced pressure, and dissolved in 80mL of warm water. The cooled

solution was filtered from a trace of precipitate' and diluted to twice its volume with water. Upon

acidification to pH 5 with glacial acetic acid, a voluminous white powdery precipitate appeared

The solid separated was filtered, washed with water and dried and upon recrystalization from hot

water gave 18.990g (81%) of PMe, mp, 245-2460C.

IR (KBr)Vmax cm-1

3366 O-H stretch of phenolic H

3179 N-H stretch of NH2

2985 and 2871 C-H stretch of methyl, antisymmetric and symmetric

1600-1492 C=C, C=N stretch of heteroaromatic

845 2-adjacent aromatic hydrogens

268

789 1,2,3,5 tetrasubstituted aromatic hydrogens

PMR (DMSO-d6) δppm

11.88 br.s, 1H, -OH

7.95 d, 2H, aromatic, J=8.48 Hz

7.37 d, 2H, aromatic, J=8.52 Hz

1.88 s, 3H, -CH3

LCMS

m/z % intensity

239 2.25

237 (M+2H) 5.25

205 4.25

203 23.00

156 100.00

158 33.25

f. 2-(4-chlorophenyl)-5-methylpyrimidin-4-amine: PMf

(11.750g, 0.05mol) of 6-amino-2-(4-chlorophenyl)-5-methylpyrimidin-4-ol PMe and (6.800g,

0.10mol) Zn dust taken in dry THF in a 100 mL RBF. Then reaction mixture was stirred and

refluxed at high temp. for 8 h. The reaction mixture was filtered and solvent was evaporated.

Upon evaporation gave 4.690g (43%) of PMf, mp, 247-2480C.

IR (KBr)Vmax cm-1

3159 N-H stretch of NH2

2962 and 2871 C-H stretch of methyl, antisymmetric and symmetric,

269

1600-1490 C=C, C=N stretch of heteroaromatic

1088 Aromatic C-Cl stretch

846 2-adjacent aromatic hydrogens

800 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO) δppm

7.90 d, 2H, aromatic, J=9.15 Hz

7.87 d, 2H, aromatic, J=8.68 Hz

7.82 s, 1H, pyrimidine

2.14 s, 3H, -CH3

LCMS

m/z % intensity

204 7.25

202 31.25

192 4.25

190 12.25

157 18.75

155 45.50

g. 4-amino-2-(4-chlorophenyl)pyrimidine-5-carboxylic acid :PMg

Placed the (18.340g, 0.07mole) of sodium di chromate in 250mL of RBF with the further

addition of 100mL water shake the flask properly so that sodium di chromate may get dissolved

in water. Further added (10.950g, 0.05mol) of 2-(4-chlorophenyl)-5-methylpyrimidin-4-amine

270

PMf to the solution and shake the flask. to the solution added the 24mL of Conc. sulphuric acid

over a time span of 25 min. with continuous shaking when addition was completed, cooled t he

contents of flask properly and mixed the 12mL of glacial acetic acid to the solution. Now

refluxed the reaction mixture on water bath for 4 h. and further on direct flame for 30 min.

cooled the RBF under running tap water and poured the reaction mixture into a beaker

containing crushed ice. Shake the solution and precipitation occurred with greenish tinch.

Filtered the solid on water pump and washed with water at least three times so that sodium

sulphate was washed. Further transferred the precipitates into 100mL beaker containing 30mL 0f

5% sulphuric acid. Digested the precipitates on water bath for about 15 min. with continuous

shaking. Filtered the solution on water pump. Washed the precipitates with water and transferred

this solid in a beaker containing 50mL of sodium hydroxide by continuous shaking. Filtered the

solution and preserved the filtrate. Acidified the filtrate with 5% sulphuric acid till it was acidic.

The solid separated was filtered, washed with water and dried and upon recrystalization with

acetic acid gave 5.460g, (44%) of PMg, mp, 245-2470C.

IR (KBr)Vmax cm-1

3460 O-H stretch of -COOH

3092 N-H stretch of NH2

1683 C=O stretch

1600-1423 C=C, C=N stretch of heteroaromatic

851 2-adjacent aromatic hydrogens

806 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO-d6) δppm

12.30 s, 1H, carboxylic acid

7.97 d, 2H, aromatic, J=8.56 Hz

271

7.56 s, 1H, pyrimidine

7.41 d, 2H, aromatic, J=8.52 Hz

2.91 br.s, 2H, NH2

LCMS

m/z % intensity

252 1.75

250(M+H) 3.75

249(M+) 7.50

251 2.50

232 6.25

234 2.75

212 25.25

210 100

211 35.25

209 55.25

188 7.00

190 2.50

152 6.25

4-amino-2-(4-chlorophenyl)pyrimidine-5-carboxylic acid :PMg

h. 4-amino-2-(4-chlorophenyl)-N-alkylpyrimidine-5-carboxamide: (PMh1 and5)

PMh1. 4-amino-2-(4-chlorophenyl)-N-methylpyrimidine-5-carboxamide

272

To a solution of (2.490 g, 0.010 mol) 4-amino-2-(4-chlorophenyl)pyrimidine-5-carboxylic acid

PMg in 10mL of dry THF was added (1.620 g, 0.010) of N,N'-carbonyldiimidazole. One-half

hour later, when effervescence of CO2 gas was stopped then (5mL, 0.310g, 0.010mole) of

methylamine was added. Then reaction mixture was stirred 3 h on magnetic stirrer. Now

reaction mixture was placed in dark for overnight. After standing overnight, the THF was

removed by an air stream and 50mL of 1N hydrochloric acid was added. Cooling of the solution

gave a solid. This was washed with water, triturated with 20mL of 5% sodium bicarbonate

solutions, filtered and again washed with water, dried and which upon recrystallization from

50% ethanol gave 0.889g (34%) of PMh1, mp, 275-277 0C

IR (KBr)Vmax cm-1

3295 N-H stretch

2938 and 2857 C-H stretch of methyl, antisymmetric and symmetric

1653 C=O stretch of amide

1600-1449 C=C, C=N stretch of heteroaromatic

1256 C-N stretch of amide

834 2-adjacent aromatic hydrogens

778 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO-d6) δppm

8.19 br.s, 1H, -NH, thiomide

7.99 d, 2H, aromatic, J=8.52 Hz

7.70 s, 1H, pyrimidine

7.35 d, 2H, aromatic, J= 8.48 Hz

2.52 s, 3H, -CH3

LCMS

m/z % intensity

273

265 3.25

263(M+H+) 9.25

249 2.25

247 5.25

191 4.75

189 7.75

152 8.25

PMh5. 4-amino-N-tert-butyl-2-(4-chlorophenyl)pyrimidine-5-carboxamide

4-amino-N-tert-butyl-2-(4-chlorophenyl)pyrimidine-5-carboxamide (PMh5) was prepared

(1.550g, 60%) as per procedure described for synthesis of PMh1 by treating 4-amino-2-(4-

chlorophenyl)pyrimidine-5-carboxylic acid PMg (2.490 g, 0.010 mol) with tert.butylamine

solution (5mL, 0.730g, 0.01mol), mp, 282-2840C

IR (KBr)Vmax cm-1

3352 N-H stretch

2966 and 2827 C-H stretch of methyl, antisymmetric and symmetric

1637 C=O stretch of amide

1600-1446 C=C, C=N, stretch of heteroaromatic

1257 C-N stretch of amide

829 2-adjacent aromatic hydrogens

751 1,2,4 trisubstituted aromatic hydrogens

PMR (DMSO-d6) δppm

8.02 d, 2H, aromatic, J=8.64 Hz

274

7.51 s, 1H, pyrimidine

7.40 d, 2H, aromatic, J=8.64 Hz

7.30 br.s, 1H, -NH, thiomide

1.47 s, 9H, -C(CH3)3

e. 4-amino-2-(4-chlorophenyl)-N-alkylpyrimidine-5-carbothioamide (PMi1)

PMi1. 4-amino-2-(4-chlorophenyl)-N-methylpyrimidine-5-carbothioamide

A mixture of 4-amino-2-(4-chlorophenyl)-N-methylpyrimidine-5-carboxamide PMh1 (2.620g,

0.01mol) and Lawesson’s reagent (2.02g, 0.005mmol), was taken in a glass tube and mixed

thoroughly with a spatula. The glass tube was then placed in an alumina bath inside the

microwave oven (900 W) and irradiated for 3 min on completion of the reaction, followed by

TLC examination, the colored material was dissolved in dichloromethane and adsorbed on silica

gel and purified by silica gel column chromatography, which afforded the pure PMi11.385g,

(50%), as viscous mass.

IR (KBr)Vmax cm-1

3301 N-H stretch

2941 and 2854 C-H stretch of methyl, antisymmetric and symmetric,

1092 C=S stretch, thioamide

1258 C-N stretch

835 2-adjacent aromatic hydrogens

801 1,2,4 trisubstituted aromatic hydrogens

755 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

8.19 br.s, 1H, -NH, thiomide

7.99 d,2H,aromatic, J= 8.52Hz

275

7.70 s, 1H, pyrimidine

7.35 d, 2H, aromatic, J= 8.48Hz

2.52 3H, -CH3

LCMS

m/z % intensity

266 10.50

248 24.50

246 16.25

238 9.50

236 20.75

226 52.25

224 100

225 7.25

223 18.25

167 11.25

264 20.25

276

C. BICYCLIC RINGS WITH RING JUNCTION NITROGEN

VI. 7-(4-SUBSTITUTED PHENYL)-3-ALKYLHETEROCYCLIC-4(3H)-THIONES

IC1. 7-(4-chlorophenyl)-3-methylimidazo[1,2-a][1,3,5]triazine-4(3H)-thione

A mixture of 2-amino-4-(4-chlorophenyl)-N-methyl-1H-imidazole-1-carbothioamide Ie1 2.640g,

(0.010mol) and diethoxymethylacetate (12 mL) was stirred at 80°C for 2 h. After cooling to

room temperature, the precipitated solid was collected by filtration to give white crystals (0.688

g, 25%) of IC1, mp, 289-2900C.

IR (KBr)Vmax cm-1

2961 and 2852 C-H stretch of methyl, antisymmetric and symmetric

1629-1325 Aromatic/Heteroaromatic skeletal stretch

1260 C-N stretch

1094 C=S stretch

802 2-adjacent aromatic hydrogens

740 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

7.69 d, 2H, aromatic, J=8.60 Hz

7.59 d, 2H, aromatic, J=9.24 Hz

7.43 s, 1H, -CH of triazine

7.21 s, 1H, -CH of imidazole

4.17 s, 3H, -CH3

LCMS

277

m/z % intensity

279 6.25

277(M+H) 13.25

278 4.25

276(M+) 8.25

263 8.75

261 16.25

225 55.25

165 8.75

ICf1. 7-(4-bromophenyl)-3-methylimidazo[1,2-a][1,3,5]triazine-4(3H)-thione

A mixture of 2-amino-4-(4-bromophenyl)-N-methyl-1H-imidazole-1-carbothioamide If1

(3.090g, 0.010mol) and diethoxymethylacetate (12 mL) was stirred at 80 °C for 2 h. After

cooling to room temperature, the precipitated solid was collected by filtration to give white

crystals (1.39 g, 88%) of ICf1, mp, 289-2900C

IR (KBr)Vmax cm-1

2924 and 2855 C-H stretch of methyl, antisymmetric and symmetric,

1611-1340 Aromatic/Heteroaromatic skeletal stretch

1262 C-N stretch

1106 C=S stretch,

807 2-adjacent aromatic hydrogens

746 Aromatic C-Br stretch

PMR (DMSO-d6) δppm

278

7.94 d, 2H, aromatic, J=8.32 Hz

7.42 d, 2H, aromatic, J=7.16 Hz

7.35 s, 1H, -CH of triazine

7.29 s, 1H, -CH of imidazole

2.22 s, 3H, -CH3

LCMS

m/z % intensity

307 7.50

305(M+-CH

3) 8.50

294 34.25

292 100

266 7.25

264 12.25

1Cg1. 4-(3-methyl-4-thioxo-3,4-dihydroimidazo[1,2-a][1,3,5]triazin-7-yl)benzonitrile

A mixture of 2-amino-4-(4-cyanophenyl)-N-methyl-1H-imidazole-1-carbothioamide Ig1

(2.560g, 0.010mol) and diethoxymethylacetate (12 mL) was stirred at 80°C for 2 h. After

cooling to room temperature, the precipitated solid was collected by filtration to give white

crystals (1.39 g, 88%) of ICg1, mp, 289-2900C.

IR (KBr)Vmax cm-1

2966 and 2854 C-H stretch of methyl, antisymmetric and symmetric

2225 C-N stretch of cyanide

1635-1325 Aromatic/Heteroaromatic skeletal stretch

1257 C-N stretch

279

1092 C=S stretch,

831 2-adjacent aromatic hydrogens

PMR (DMSO-d6) δppm

8.40 s, 1H, -CH of triazine

7.92 d, 2H, aromatic, J=8.32 Hz

7.41 d, 2H, aromatic, J=9.28 Hz

7.36 s, 1H, -CH of imidazole

2.63 s, 3H, -CH3

LCMS

m/z % intensity

268(M+H) 18.50

267(M+) 100

239 23.25

TC1. 6-Methyl-1,2,4-triazolo[1,5-a]-1,3,5-triazine-7(6H)-thione

A mixture of 5-amino-1-[(methylamino)thiocarbonyl]-1H-1,2,4-triazole Te1 (2.650g, 0.010mol)

and diethoxymethylacetate (12 mL) was stirred at 80°C for 2 h. After cooling to room

temperature, the precipitated solid was collected by filtration to give white crystals (0.774 g,

28%), of TC1, mp, 280-2810C.

IR (KBr)Vmax cm-1

2925 and 2852 C-H stretch of methyl, antisymmetric and symmetric

1638-1321 Aromatic/Heteroaromatic skeletal stretch

1258 C-N stretch

280

1091 C=S stretch

835 2-adjacent aromatic hydrogens

802 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

8.63 s, 1H, -CH of triazine

8.28 d,2H,aromatic, J=8.64 Hz

7.47 d,2H,Aromatic, J=7.72 Hz

3.98 s, 3H, -CH3

LCMS

m/z % intensity

280 3.25

278(M+H) 9.25

279 6.75

277(M+) 11.75

265 5.50

263 12.50

225 38

223 100

167 4.75

166 2.75

D. BICYCLIC RINGS WITHOUT RING JUNCTION NITROGEN

281

VII: 7-(4-SUBSTITUTED PHENYL)-3-ALKYLHETEROCYCLIC-4(3H)-THIONES

BC1. 7-(4-chlorophenyl)-3-methylquinazoline-4(3H)-thione

A mixture of 3-amino-4'-chloro-N-methylbiphenyl-4-carbothioamide Bf1 (2.890g, 0.010mol)

and diethoxymethylacetate (12 mL) was stirred at 80°C for 2 h. After cooling to room

temperature and evaporation of solvent the viscous mass was obtained. (1.39 g, 88%) of BC1,

mp, 289-2900C.

IR (KBr)Vmax cm-1

2934 and 2856 C-H stretch of methyl, antisymmetric and symmetric

1246 C-N stretch

1093 C=S stretch

1045 C=S stretch

829 2-adjacent aromatic hydrogens

801 1,2,4 trisubstituted aromatic hydrogens

753 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

8.03 s, 1H, -CH of pyramidine

7.67 d, 2H, aromatic, J=7.92 Hz

7.43 m, 3H, aromatic

6.91 d, 2H, aromatic, J=8.48 Hz

3.78 s, 3H, -CH3

LCMS

282

m/z % intensity m/z % intensity

288 16.75

286(M+) 90.25

273 9.75

271 18.75

178 100

PC1. 7-(4-chlorophenyl)-3-methylpyrido[2,3-d]pyrimidine-4(3H)-thione

A mixture of 2-amino-6-(4-chlorophenyl)-N-methylpyridine-3-carbothioamide Pi1 (2.760g,

0.010mol) and diethoxymethylacetate (12 mL) was stirred at 80°C for 2 h. After cooling to room

temperature and evaporation of solvent the viscous mass was obtained. (1.39 g, 88%) of PC1,

mp, 289-2900C.

IR (KBr)Vmax cm-1

2978 and 2842 C-H stretch of methyl, antisymmetric and symmetric

1257 C-N stretch

1092 C=S stretch

833 2-adjacent aromatic hydrogens

808 1,2,4 trisubstituted aromatic hydrogens

790 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

8.04 s, 1H, -CH of pyramidine

7.83-7.72 m, 5H, aromatic

6.89 d, 1H, aromatic, J=8.28 Hz

3.85 s, 3H, -CH3

283

LCMS

m/z % intensity

289 2.25

287(M+) 5.25

243 20.50

242 100

178 14.25

177 4.25

PMC1. 7-(4-chlorophenyl)-3-methylpyrimido[4,5-d]pyrimidine-4(3H)-thione

A mixture of 4-amino-2-(4-chlorophenyl)-N-methylpyrimidine-5-carbothioamide PMi1

(2.770g, 0.010mol) and diethoxymethylacetate (12 mL) was stirred at 80°C for 2 h. After

cooling to room temperature and evaporation of solvent the viscous mass was obtained. (1.39 g,

88%) of PMC1, mp, 289-290 0C.

IR (KBr)Vmax cm-1

2980 and 2870 C-H stretch of methyl, antisymmetric and symmetric

1274 C-N stretch,

1092 C=S stretch Aromatic C-Cl stretch

850 2-adjacent aromatic hydrogens

803 1,2,4 trisubstituted aromatic hydrogens

761 Aromatic C-Cl stretch

PMR (DMSO-d6) δppm

284

7.59 s, 1H, -CH of pyramidine

7.98 d, 2H, aromatic, J=8.64 Hz

7.41 d, 2H, aromatic, J=8.60 Hz

6.93 s, 1H, -CH of pyramidine

3.98 s, 3H, -CH3

LCMS

m/z % intensity

290 5.25

288(M+) 10.25

275 6.75

273 17.75

180 100

179 80