Synthesis And Characterisation Of Sulphonamide Drugs Biology Essay

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Chemotherapy, the term most commonly used for anticancer therapy nowadays, was first used by a German doctor Paul Ehlrich in 1900's for the treatment of bacterial infections with antibacterial. Ehlrich found that harmful bacteria can be stained using certain dyes and developed the idea of these molecules as "Magic bullets". For medicine Gerhard Domagk was awarded Nobel prize in 1939 for finding the use of dye Prontosil as antibiotic. Prontosil breaks down to form sulphanilamide in vivo and this was found by research group headed by J.Trefouel. This discovery started the research of sulphanilamide derivatives which proved to be a vital milestone in development of antibiotics .

Sulphapyridine (treatment of Pneumonia), Sulphacetamide (treatment of Urinary tract infections, Succinoylsulphathiazole (treatment of gastrointestinal tract infections) and Sulphathiazole used for treating battle wounds in World War II).

Sulphonamide drugs are bacteriostatic, i.e. they suppress the growth of bacteria. Sulphanilamide is a competitive inhibitor of PABA (p-aminobenzoic acid), which is required by bacteria to synthesise folic acid (a cofactor). The competitive inhibition of PABA by sulphanilamide due to structural similarity, and inhibits the synthesis of nucleic acids that are required for growth by bacteria and hence, suppresses bacterial growth. The sulphonamides gained the status of "wonder drugs" in the World War II period, and were the main treatment against bacterial infections until penicillin came into general use, after its successful use in humans was established in 1941. Apart from penicillin, tetracycline derivatives like Aureomycin and Terramycin were discovered. The newly discovered antibiotics were more effective and had fewer side effects [1].

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p Amino benzoic acid Sulphanilamide

Folic acid

Figure 1. Structure of p-aminobenzoic acid, sulphanilamide and folic acid

Sulphonamides are synthetic antibacterial agent, active against number of infections. They are active against gram positive organisms mainly pneumococci and meningococci. [1]. They are considered as antibacterial, carbonic anhydrase inhibitor, anticancerous, anti inflammatory agents [2].

Structure activity relationships

Figure 2. General structure of sulphonamides.

The para amino group has to be unsubstituted (R1 should be either H or an acyl group).

The aromatic and sulphonamide group are both essential for activity.

The two groups, amino and sulphonamide, has to be directly attached to aromatic group.

Only para substituted aromatic ring exhibits activity, other substitution diminishes or eliminates the activity.

The sulphonamide nitrogen must be primary or secondary to produce the action.

Substitutions can only be made at R2 position [1].

Applications of sulphonamides:

The sulphonamides were the drug of choice before penicillin came in 1942. After the introduction of penicillin, the sulphonamides took a back seat due to the large number of side effects. With the introduction of less toxic analogues like sulphadoxine, the sulphonamides have again become the drug of interest. Presently the sulphonamides are prescribed in the following indication

Urinary tract infections

Eye lotions

Infections of mucous membrane

Gut infections [1].

The major side effect of sulphonamides is the crystalurea that occurs due precipitation of drug in liver and kidney. This limits their frequent use. However, the sulphonamide derivatives like sulphanylureas are still useful in the diabetes mellitus type II [3].

Some of the researches carried out with sulphonamides reveal that:

they are CAR agonists, a nuclear receptor which detects potentially toxic endo- and exogenous compounds and induces their elimination from the body [4].

They possess antitumor drugs due to their functionality to interact with cellular targets [5].

In this project work I will be preparing three sulphonamide drugs, sulphanilamide, sulphapyridine and sulphathiazole from a common precursor p-acetamidobenzenesulphonyl chloride and will find out its purity by spectroscopic methods like NMR, mass and IR spectroscopy. The p-acetamidobenzenesulphonyl chloride can be synthesised from acetanilide, by reacting the latter with chorosulphonic acid. Through electrophillic aromatic substitution reaction, the acetamido group directs totally to the para position. However in this experiment I have ordered the p-acetamidobenzenesulphonyl chloride from Alfa Aesar chemical supplier.

NUCLEAR MAGNETIC RESONANCE

Being the universal method for determination of intermolecular interaction and not requiring any target specific knowledge, NMR has lot of things to offer in structure determination. Moreover, the ligand-target interaction is screened in a straight forward manner to avoid any false leads. High sensitivity to weak interactions and detailed structural elucidation for ligand binding modes are the main advantages of NMR spectroscopy [6]. This technique is based on the magnetic properties of the atomic nuclei. Nuclear magnetic moment of nucleus and external magnetic field (B0) creates a nuclear energy level diagram. As per the quantum mechanics the magnetic energy of the nucleus is limited to discrete values, Ei. These are known as Eigenvalues. Eigenvalues are related to eigenstates which are the states in which particle can exist, also known as stationary states. Using the high frequency transmitter, the atoms can be made to shift through eigenstates within energy level diagram. The energy absorbed is detected, amplified and reproduced in the form of spectrum, known as resonance signal. We can obtain a spectrum like this for compounds having atomic nuclei with non zero magnetic moments, such as 1H, 19F, 14N, 15N, etc. 12C, which is so important in organic chemistry, has zero magnetic moment with even mass and even atomic number, therefore the NMR studies are only limited to 13C.

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Some key points to note in NMR spectrum are

1) Different protons in the compound produce different resonance signals, and these are separated by chemical shift.

2) The number of protons giving rise to particular signal is represented by area under signal, which is given by Integration.

3) The spectral lines appear as singlets, doublets, triplets, or quartets, depending on the spin-spin coupling.

Thus we can say that chemical shift tells us the chemical environment of the nucleus responsible for that signal and integration concludes the number of protons present. The spin-spin coupling relates to the positional relationship of the nuclei as the coupling constant is directly proportional to the number and type of bonds separating them. And the signal multiplicity is due to the number of protons on the neighbouring group. Also the NMR spectrum is temperature dependant for many compounds due to the presence of resonance forms (high barrier of rotations along the bonds). 13C NMR is also a vital technique in organic chemistry and biochemistry and also 19F, 15N, 31P yields important information.

Instrumentation: There are commonly 2 types of NMR instrument, continuous wave and Fourier transform. Earlier the continuous wave was used but after the introduction of FT in 1970, CW are mostly replaced by FT. In CW NMR the sample is introduced to the strong magnetic field and the source's frequency is scanned.

In FT-NMR the sensitivity of NMR is increased by recording large number of spectra and then adding them together. In this addition the noise increases ten folds but the signal intensity is increased by 100 folds, and thus the sensitivity is increased.

The typical FT-NMR system contains a 3 components:

Superconducting magnet and probe,

spectrometer, and

computer terminal.

The superconducting magnet has ports that are filled with liquid helium which creates an environment giving zero resistance.

Figure 3. Picture of NMR spectrometer

Applications:

1). A very important tool for structural elucidation of a compound.

2). Can be used to find out the purity of the compound.

3). Applied successfully in new drug discovery.

4). Can be used for reaction and process monitoring.

5). Used for studying cell metabolism non-invasively.

7). Used in Quality assurance.

8). Information about molecular motion.

9). Food analysis.

Strengths:

1). It is highly sensitive technique.

2). A non-invasive technique [7].

Infra Red Spectroscopy

Compounds with covalent bonds, organic and inorganic, absorb electromagnetic radiations in the infra red region of electromagnetic spectrum. Wavelength of this region is longer than visible light, ranging from 400 to 800 nm, however shorter than microwave radiations. From chemistry point of view, the vibrational portion of the infra red is of particular interest. The absorption of infra red radiation causes the molecule to excite to the higher state. Only certain frequencies of infra red radiation are absorbed by the molecule and correspond to change in energy from 8 to 40kJ/mole. The radiation in this range encompasses the stretching and vibrational frequencies of the covalent bonds. During absorption, only the frequencies of infra red radiations matching the vibrational frequency of the molecule are absorbed. The absorbed energy increases the amplitude of vibrational motions of the bonds in molecule. The matching of the frequency of radiation with the bond motion does not mean that the bonds will absorb energy. The bonds which have a dipole moment that changes as a function of time can absorb infra red radiations. Even the symmetric bonds do not absorb Infra red radiation.

Stretching and bending modes are the kind of vibrational motions that give rise to the absorption or in other words make the molecule infra red active. Any molecule having three or more atoms and at least two of which are identical, the stretching could be symmetric and asymmetric. The different bending modes are scissoring, wagging, rocking and twisting.

Combination band is the joining of two vibrational frequencies to give a new frequency within molecule, provided, such a vibration is infra red active.

Difference bands results from difference from two interacting frequencies.

Instrumentation:

Instrument used to measure the absorption spectrum is called the infra-red spectrometer. There are two types of spectrometers commonly in use, dispersive and Fourier transform instruments. Both these instruments measure the absorption spectrum in 4000 to 400 cm-1.

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FTIR spectrometer provides spectrum much faster than Dispersive type.

Dispersive infra red spectrometers: A beam of infra red radiation is produced from a hot wire, which is divided into two equal intensity parallel beams. In one beam, the sample is placed and reference in the other. Beams are then passed through the monochromator, and are dispersed in continuous spectrum of Infra red frequencies. Rapidly rotating sector (the beam chopper) alternately passes the two beams through diffraction gratings. The diffraction grating varies the wavelength of the radiation reaching the thermocouple detector. The detector senses the difference in the intensities of sample and reference beam. This signal is amplified and drawn by the recorder as spectrum of the sample.

Fourier transform spectrometers: the optical pathway produces interferogram, which is complex signal. However, its wave like pattern consists of all the frequencies making up the infra red spectrum. An interferogram is intensity versus time plot which is them converted by Fourier transform to a plot identical to intensity versus frequency. The plot made by Fourier transform is virtually identical to the plot received by Dispersive type spectrometer.

The main advantage of using FTIR spectrometer is its speed; it can obtain spectra in less than a second. Therefore the instrument can record number of spectrum of the same sample which are stored in the computer memory and later converted by Fourier transform. It generates a spectrum with better signal to noise ratio. Hence, FTIR is more fast and sensitive than dispersion instrument.

Uses:

As every bond has different absorption pattern and all the bonds exist in different environment in different compounds, no two compounds show the same spectrum. Hence, the infra red spectrum is used as fingerprint of a molecule.

Determination of molecule's structural information [8].

Mass Spectroscopy

It is the technique to determine the molecular mass of the compound. This is done by separating the ions according to mass/charge ratio.

Principle:

Figure 4. Principle of mass spectrometry

Adopted from M.Sc. OMED 0104 lecture notes, Dr. Birthe Neilson, 2010

The sample is introduced to the mass spectrometer through the inlet which could be HPLC, GC, or Sample plate. The sample is then ionized, and the ions are separated according to their mass/charge ratio. The separated ions are then detected by the detector and the signals are recorded in the data system.

Vacuum is created inside the spectrometer so that mean free path of molecules can be increased and the collision between the ions can be avoided.

Ionization: the molecules are ionized so that there path can be controlled using electric or magnetic fields. Some of the methods used to produce ionization are

Electron ejection M→M+ + e-

Electron capture M + e- →M-

Protonation M + H+ →MH+

Deprotonation MH → M- + H+

Cationizaion M + cation+ → M(cation)+

There are two types of ionization methods, soft and hard. Soft ionization is good for molecular weight determination and as the hard method causes fragmentation, is good for structural information [9].

Different ionization techniques used are:

Electron ionization: it is also known as electron impact. This technique is mostly useful for studying volatile organic molecules.

Chemical ionization: in this technique the reagent gas is ionised first and the charge is transferred to the sample molecule by chemical process. Commonly used reagent gas is methane.

Coupling gas chromatography to Mass spectroscopy: by coupling the gas chromatography to mass spec, more complex volatile sample mixtures can be analysed.

Field and Plasma desorption ionisation: these methods are not in use today but these two techniques were the first ones to be used for ionisation of non-volatile molecules.

Fast atom bombardment: secondary ion mass spectrometry, SIMS, is a technique used to study chemical nature of materials and surfaces.

Matrix assisted laser desorption ionization: In MALDI the sample is mixed with the matrix compound that absorbs the energy from the laser and assists in ionisations of the sample through electron transfer and chemical processes. MALDI is a well established method in studying polar high molecular weight compounds such as proteins and nucleic acids.

Electrospray ionization: The main advantage of this technique is that the molecular weight measurement of each multiply charged ion is made and average of all the values is taken, which gives the accuracy of +/- 0.01%.

Atmospheric pressure chemical ionization: an electrical discharge is developed around the gaseous sample molecule by vaporising the solution stream. Ionisation is done by chemical processes. This method cannot be applied for larger molecules [10].

After the ionization the charged ions are separated by mass analyzers. Some of the mass analyzers used are:

Time of flight (TOF)

Magnetic sector/ double focussing magnetic sector

Quadrupole mass analyzers

Quadrupole ion trap (QIT)

Ion cyclotron resonance (ICR)

EXPERIMENTAL

Aim: The aim of this project is to synthesise three sulphonamide drugs, sulphanilamide, sulphapyridine and sulphathiazole, and to characterise the synthesised compounds by NMR, mass and FTIR spectroscopies.

Instrumentation:

1). Infra red spectroscopy: Infra-red analysis was performed with KBr discs using Perkin Elmer Paragon 1000 FTIR spectrometer (range 4000 to 400 cm-1).

2). Mass spectroscopy: Analysis was performed using the ESI method and acetonitrile as solvent.

3). NMR analysis was performed by JEOL 500 MHz NMR spectrometer at a frequency of 270.16 MHz and at the pulse length of 3.76 micro-secs with the relaxation delay of 1sec.

p-acetamidobenzenesulphonyl chloride

Chemical structure:

Figure 5. Structure of p-acetamidobenzenesulphonyl chloride

IUPAC Name: 4 acetamidobenzenesulphonyl chloride

p-acetamidobenzenesulphonyl chloride was ordered from Alfa Aesar, lot no. 10148484. FTIR, Mass and NMR spectra were used to test the purity of the raw material as the purity of final product depends majorly on the purity of the raw material.

Physical properties: light brown powder.

Determination of melting point: The sample was loaded in the capillary tube and placed in the melting point apparatus, and the temperature was recorded when the sample melts.

Result: The melting point of the sample was 146.5oC

Literature Value: about 146oC [11].

RESULTS AND DISCUSSION:

FTIR spectroscopy:

Sample preparation- the mortar and pestle were washed with acetone to remove any traces of the water as potassium bromide dissolves in water. Approximately 100 mg of KBr was ground up to form fine powder. A small amount of sample was added to it. The dye set was also washed with the acetone, arranged and the sample loaded into it. The press was used to form the KBr disks to load in the slit. The vacuum was applied and the pressure applied for 3 min. The pressure was then released and the disc was taken out and loaded in the FTIR spectrometer.

Graph 1 - FTIR spectrum of p-acetamidobenzenesulphonyl chloride

The following table summarizes the results obtained by FTIR analysis for p-acetamidobenzenesulphonyl chloride

Table 1 - Table for FTIR analysis of p-acetamidobenzenesulphonyl chloride

No.

PEAK cm-1

ASSIGNMENT

1

3307

N-H stretch(20 amide)

2

1681.7

C=O stretch

3

1585.5

Aromatic ring C=C "breathing"

4

1402.6

-CH3 bend deformation

5

1369

S=O stretch

The peak at 3307 shows the presence of secondary amide, and the peak at 1681.7 is because of C=O stretching. The presence of benzene ring is confirmed by the peak at 1585.5. Medium -CH3 deformation has resulted into a peak at 1402.6. and presence of S=O stretch is evident from the peak at 1369.

1H NMR spectroscopy:

0.0013gm of sample was weighed and dissolved in DMSO D6 (premixed with TMS). The solution was loaded in the NMR tube, labelled and submitted for proton and C-13 NMR analysis.

Graph 2 - 1H NMR spectrum of p-acetamidobenzenesulphonyl chloride

Results derived from proton NMR spectrum:

Table 2 - Table for 1H NMR analysis of p-acetamidobenzenesulphonyl chloride

CHEMICAL SHIFT(ppm)

INTEGRATION

SPLITTING PATTERN

ASSIGNMENT

2.0

3

singlet

-CH3

7.55

4

multiplet

Ar-H

10.07

1

singlet

-NH

The proton nmr spectrum shows all the peaks as expected from the structure. The peak at around 7.5 is a multiplet due to non-equivalent protons attached to the ring. All the other peaks are at expected positions. The peak at 2.0 is the methyl group, which appears as singlet due to no adjacent hydrogen atoms. The peak at 10.07 is due to amide proton present in the molecule. The peak at 2.5 is due to DMSO-D6, the solvent used and the small peak at 0 is the Tetramethylsilane (TMS) , the reference standard used.

NMR C-13 spectroscopy:

Graph 3 - C-13 NMR spectrum of p-acetamidobenzenesulphonyl chloride

Table 3 - Table for C-13 NMR analysis of p-acetamidobenzenesulphonyl chloride

CHEMICAL SHIFT(ppm)

ASSIGNMEENT

21.0

-CH3

117.9

AROMATIC C

126.1

AROMATIC C

139.6

AROMATIC C

142.3

AROMATIC C

168.4

-C=O

The first peak a 21.0 is due to methyl carbon in the molecule. Since we have plane of symmetry in the molecule, therefore we will be getting only 4 peaks for the aromatic ring. The peaks at 117.9 and 126.1 are the carbon atoms with the hydrogen attached to them, at ortho and meta positions. The peaks at 139.6 and 142.3 are due to the carbons at position 1 and 4, as they do not have any protons attached to them therefore they will give a weak signal. And also due to attachment to nitrogen and sulphur they will be shifted more downfield. The peak at 40 is due to DMSO D6. The most downfield peak at 168.4 is due to -C=O, as oxygen is electronegative and strongly deshields the carbonyl carbon.

Mass spectroscopy:

Graph 4 - Mass spectrum of p-acetamidobenzenesulphonyl chloride

The mass spectrum of p-acetamidobenzenesulphonyl chloride has been obtained by negative ESI mode, therefore the expected mass of molecular ion is

[M+-1] = [233-1] = 232

The peak at 232 is due to the product, p-acetamidobenzenesulphonyl chloride.

The peak at 214 is the base peak, and it is due to the hydrolysis of the sample as the sample was kept in queue for analysis.

Ar-SO2Cl Ar-SO3H

The peak at 234 is due to the isotope (possibly Cl-37).

As we have received all the results as expected from the molecular structure, the sample is pure and can be used for further experiment.

SULPHANILAMIDE

Structure:

Figure 6. Structural representation of sulphanilamide synthesis

IUPAC Name: 4 aminobenzenesulphonamide

Synthesis of sulphanilamide:

5.006gm of p-acetamidobenzenesulphonyl chloride was added into a 150ml Erlenmeyer flask and 15ml of conc. ammonium hydroxide added to it in the hood. The mixture was stirred well with the stirring rod. The mixture was then heated on a heating mantle for 15 min., with frequent stirring. The material became a pasty suspension; the flask was removed and placed in the ice bath. The mixture was well cooled and 6M Hydrochloric acid was added until the mixture was acidic to litmus paper(blue litmus turned red). The mixture was further cooled and the product was filtered over the Buchner funnel with vacuum. The product is washed with 50ml of cold water and kept for drying overnight. The dried crude product was transferred to Round bottom flask. 3ml of conc. Hydrochloric acid, 6ml of water and anti-bumping stones were added to it. The solution is refluxed for 15min and then boiled for extra 10min. and cooled to room temperature. A clear solution was formed. To this 5ml of water and little decolourizing carbon was added. The solution was then filtered by gravity after shaking into a 200ml beaker. 4gm of sodium bicarbonate was dissolved in small amount of water and added to the filtrate with stirring until the solution became neutral to litmus. Sulphanilamide precipitated after the addition of sodium bicarbonate. The mixture was cooled on ice bath and filtered using Buchner funnel. The product was recrystallised by dissolving it in water, and then cooling it over ice bath. It was filtered using Buchner funnel. The product was allowed to dry.

Physical texture of sulphanilamide: White crystalline powder.

Determination of melting point: The sample was loaded in the capillary tube and placed in the melting point apparatus, and the temperature was recorded when the sample melts.

Result: The melting point of the sample was 163oC.

Literature value- melting point should be around 162oC-165oC [12].

Theoretical yield calculation:

P-Aceamidobenzenesulfonyl chloride → sulphanilamide

mol. wt. 233.67 mol. wt. 172.20

Moles of product:

5gm * 1mole = 0.021397 mol,

233.67

Therefore, mass of product will be

0.021397 * 172.20 = 3.6845gms

Theoretical yield of sulphanilamide is 3.6845gms

Practical yield of sulphanilamide is 2.130gms.

Percentage yield obtained for the product is 2.130/3.6845*100 = 57.81 %

Result and discussion for sulphanilamide:

FTIR spectroscopy: Sample preparation- the mortar and pestle were washed with acetone to remove any traces of the water as potassium bromide is dissolves in it. Approximately 100 mg of KBr was ground to form fine powder. Small amount of sample was added to it. The dye set was also washed with the acetone, arranged and the sample loaded in it. The press was used to form the KBr discs to load in the slit. The vacuum was applied and the pressure applied for 3 min. the pressure was released and the disk was taken out and loaded in the FTIR spectrometer.

Graph 5 - FTIR spectrum of sulphanilamide

Table 4 - Table for FTIR analysis of sulphanilamide

No.

PEAK cm-1

ASSIGNMENT

1

3478.6

-NH2 stretch (aniline), symmetric

2

3375.8

-NH2stretch (aniline), asymmetric

3

3366.2

-NH stretch(sulphonamide)

4

1595.7

Aromatic ring -C=C- stretch

5

1313.6

SO2 Stretch

The peaks at 3478.6 and 3375.8 is due to -NH2 stretch, the former one is the aniline NH2 and the latter is NH2 attached to SO2 . The aromatic ring is indicate by the [peak at 1595.7. the SO2 generated the peak at 1313.6.

1H NMR spectroscopy:

Sample preparation: 0.0011gm of sample was weighed and dissolved in DMSO D6 (premixed with TMS). The solution was loaded in the NMR tube, labelled and given for H1 and C-13 spectroscopy in the NMR laboratory.

Graph 6 - 1H NMR spectrum of sulphanilamide

The results from the 1H NMR analysis are as follows:

Table 5 - Table for 1H NMR analysis of sulphanilamide

CHEMICAL SHIFT(ppm)

INTEGRATION

SPLITTING PATTERN

ASSIGNMENT

5.8

2

singlet

Ar-NH2

6.6

2

doublet

Ar-H

6.9

2

singlet

-SO2NH2(amide)

7.4

2

doublet

Aromatic C

The peaks at 6.6 and 7.4 are due to the protons attached to the aromatic ring. Due to the plane of symmetry, we have received only two peaks and they are with roughly a doublet splitting pattern. Each of these protons is mainly coupled to a proton on an adjacent carbon. The peak at 5.8 is because of the -NH2 group attached to C-1.

The peak at 6.9 is also due to -NH2 group but it is shifted downfield being attached to a sulphonyl group. The hydrogen atoms of the amine and sulphonamide groups are exchangeable, so if my prediction about their signals is correct, the peaks should disappear with a D2O shake. The peak at 2.5 is of DMSO-D6.

1H NMR D2O shake: The deuterated H2O was added to the sample NMR tube and shaken well and the spectrum recorded again.

Graph 7 - 1H D2O shake NMR spectrum of sulphanilamide

As expected the two peaks due to amine and sulphonamide groups have disappeared, and this confirms that the sample is sulphanilamide. The extra peak generated at 3.9 in this spectrum is due to the water signal.

NMR C-13 spectroscopy:

Graph 8 - C-13 NMR spectrum of sulphanilamide

Table 6 - Table for C-13 NMR analysis of sulphanilamide

PEAKS

ASSIGNMENT

112.4

Aromatic C (ortho positions)

127.3

Aromatic C (meta positions)

130.0

Aromatic C (C-SO2NH2)

151.8

Aromatic C (C-NH2 position)

Due to plane of symmetry there are only four signals. The signal generated by carbon at 1 and 4 positions are downfield because of electronegative atoms attached to them. The peak at 40 is due to the solvent DMSO D6.

Mass spectroscopy:

Sample preparation: 0.00123 gm of sample was dissolved in 1ml (1000µl). 10 µl was taken from the solution and again diluted with 990µl solvent. The solvent used is Acetonitrile. The prepared sample was run with 30% H2O + 70% CH3OH in the mass spectrometer using ESI method for ionization.

Graph 9 - Mass spectrum of sulphanilamide

The spectrum was taken as positive ESI, so the peaks will show the mass as +1.

The peak at 173 shows the presence of the product, sulphanilamide.

The peak at 105.1 should be formed by the removal of SO2 group from the molecule.

The peak at 155.1 is due to loss of NH2.

The peak at 214 is due to the raw material left unreacted .This peak was also seen in the mass spectra of the raw material due to hydrolysis of p-acetamidobenzenesulphonyl chloride.

The peak at 255.0 is also an impurity.

SULPHAPYRIDINE:

Structure:

Figure 7. Structural representation of sulphapyridine synthesis

IUPAC Name: 4-amino-N-pyridine-2-ylbenzenesulphonamide

Synthesis:

2.4115gm of 2-aminopyridine was dissolved in 10ml of anhydrous pyridine (previously dried over KOH pellets) in a round bottom flask. 6.0018gm of p-acetamidobenzenesulphonyl chloride was added to the mixture. The solution was refluxed for 20min. in a heating mantle. The mixture was cooled slightly and 50ml of water added to it with some water to aid in the transfer. The mixture was stirred in the ice bath until the oil crystallised. The solid was filtered through the Buchner funnel. The crude product was transferred to the round bottom flask and 20ml of 10% sodium hydroxide is added. The solution was allowed to reflux for 40min, using stop cock grease to prevent the joints from freezing. The mixture was cooled and neutralized with 6M hydrochloric acid. The sulphapyridine precipitated out in the reaction is filtered using Buchner funnel. The sulphapyridine was recrystallised by dissolving in 100ml of 95% ethyl alcohol using hot plate for heating. The solution was then filtered, cooled and crystallised product separated by vacuum filtration.

Physical texture: yellowish white amorphous powder.

Determination of melting point: The sample was loaded in the capillary tube and placed in the melting point apparatus, and the temperature was recorded when the sample melts.

Result: The melting point of the sample was 191oC. literature value is between 190 nad 193oC [13].

Calculation of percentage yield:

p-acetamidobenzenesulphonyl chloride + 2-Aminopyridine → sulphapyridine

mol. wt. 233.67 mol.wt. 249.29

Theoretical yield:

6.0018gm * 1mole = 0.02568 mol,

233.67

Therefore, the expected mass is 0.02568 * 249.29 = 6.40gms

Practical yield =2.4115gm

Percentage yield =37.68%

RESULTS AND DISCUSSION:

IR spectroscopy: The sample was triturated with the potassium bromide and pressed to form the disks, which are then loaded in the FTIR spectrometer.

Graph 10 - FTIR spectrum of sulphapyridine

The results of the FTIR spectrum are as under:

Table 7 - Table for FTIR analysis of sulphapyridine

NO.

PEAK

ASSIGNMENT

1

3427.9 & 3349.1

-NH2 STRETCH

2

3254.1

-NH STRETCH (SULPHONAMIDE)

3

3036.5

AROMATIC RING,

C-H stretch

4

1596.3, 1531.0, 1499.4, 1461.3

CONFIRMS THE PRESENCE OF BENZENE RING "RING BREATHING"

5

1138.8

SO2 STRETCH

6

765.9

o-SUBSTITUTED PYRIDINE RING,

C-H BENDING

The IR spectrum of the product reveals the following details about the molecule:

The molecule contains NH2 stretch, evident by the peaks at 3427.9 and 3349.1.

The peak at 3254.1 is because of the NH stretch of the sulphonamide group in the molecule.

There is a peak at 3036.5 which is the characteristic of the aromatic ring C-H stretch, and presence of benzene ring is proven by the peaks present at 1596.3, 1531.0, 1499.4 and 1461.3 (typical of "ring breathing").

The SO2 group gives rise to the peak at 1138.8.

The peak observed at 765.9 is due to the ortho substituted pyridine ring,C-H bending.

Mass spectroscopy:

Sample preparation: 0.00115 gm of sample was dissolved in 1ml (1000µl). 10 µl was taken from the solution and again diluted with 990µl solvent. The solvent used is Acetonitrile. The prepared sample was run with 30% H2O + 70% CH3OH in the mass spectrometer using ESI method for ionization.

Graph 11 - Mass spectrum of sulphapyridine

The information gained from the mass spectrum analysis of sulphapyridine obtained by positive ESI is listed below:

Expected mass of molecular ion is M+1=249+1=250

The peak at 250.2 is the base peak and is due to the ionised molecule itself (M+1).

The peak at 251.2 and 252.2 could be due to the presence of and isotope, could be the deuterium atom or C-13.

The peak at 156 is due to loss of pyridine ring, and [H2N-Ar-SO2]+ is the fragmented molecule.

NMR Spectroscopy: 0.0014gm of sample was weighed and dissolved in acetone-d6 (premixed with TMS). The solution was loaded in the NMR tube, labelled and submitted for proton and C-13 spectroscopy in the NMR laboratory

RESULT AND DISCUSION:

NMR H1 spectrum:

Graph 12 - 1H NMR spectrum of sulphapyridine

The results from the H1 NMR spectrum for the product are as under:

Table 8 - Table for 1H NMR analysis of sulphapyridine

CHEMICAL SHIFT(ppm)

INTEGRATION

SPLITTING PATTERN

ASSIGNMENT

5.5

1

SINGLET

-NH2

6.6

2

DOUBLET

Ar-C

6.9

1

TRIPLET

H AT 4th POSITION IN PYRIDINE RING

7.6

2

DOUBLET

Ar-C

7.3

1

DOUBLET

H at 3rd POSITION IN PYRIDINE RING

8.1

1

DOUBLET

H at 6th POSITION ADJACENT TO N IN PYRIDINE RING

9.5

1

SINGLET

SO2-NH

NMR C-13 spectrum:

Graph 13 - C-13 NMR spectrum of sulphapyridine

The results obtained by C-13 NMR spectrum are listed below:

Table 9 - Table for C-13 NMR analysis of sulphapyridine

PEAKS

ASSIGNMENT

112.7

Ar-C

113.7

Ar-C

119.0

HETROAROMATIC C

119.3

HETROAROMATIC C

139.0

HETROAROMATIC C at position 2

130.1

HETROAROMATIC C at 3rd position

148.5

HETROAROMATIC C at 6th position

In this spectrum we should have received total of nine peaks. There are 11 carbon in the molecule but due to the presence of plane of symmetry in the aromatic ring we expect nine signals. Only seven peaks were observed. The carbons at positions 1 and 4 in the benzene ring would be expected to give weak signals (no hydrogen attached to them)

So these signals are probably obscured by the "noise" as the base line.

SULPHATHIAZOLE:

Structure:

Figure 8. Structural representation of sulphathiazole synthesis

IUPAC Name: 4-amino-N-(1,3-thiazol-2-yl)benzenesulfonamide

Synthesis:

2.5010gm of 2-aminothiazole was weighed. 6.5061gm of

p- acetamidobenzenesulphonyl chloride was weighed. 10ml of anhydrous pyridine (dried over KOH pellets) was added to 2-aminothiazole in round bottom flask.

p-acetamidobenzenesulphonyl chloride was added slowly and temperature was checked throughout the addition. The temperature was always kept below 40oC. Addition is done with continuous swirling. The mixture was allowed to reflux for 25 min. Mixture was cooled and poured into 75ml of water. The solution was stirred with glass rod. Crystals were isolated by vacuum filtration and washed with cold water, and left to dry overnight. Weight of the solid product was 5.6967gm. The solid was dissolved in 57ml of 10% NaOH. The solution was allowed to reflux for one hour. After reflux, solution was cooled and conc. HCl was added till the pH reached 6(litmus paper was used to adjust the pH). Solid sodium acetate was added till the litmus turned blue (red litmus was dipped in the solution). The solution was brought to boil and cooled in ice bath. The product was filtered by vacuum was filtration. The product obtained e dissolved in water and boiled. The mixture was filtered at Buchner funnel and filtrate was cooled slowly. The precipitated product was filtered by vacuum filtration and allowed to dry overnight.

Physical texture: Yellowish white powder.

Determination of melting point: The sample was loaded in the capillary tube and placed in the melting point apparatus, and the temperature was recorded when the sample melts.

Result: The melting point of the sample is 200.5oC.

Literature value- is between 200 and 204oC [14].

Calculation of percentage yield:

p-acetamidobenzenesulphonyl chloride + 2-Aminothiazole → sulphathiazole

mol. wt. 233.67 mol.wt. 255.31

Theoretical yield:

6.5061gm * 1mole = 0.02788 mol,

233.67

Therefore, theoretical mass is 0.02788 * 255.31 = 7.12gms

Practical yield =2.4095gm

Percentage yield =33.84%

RESULT AND DISCUSSION:

FTIR Spectroscopy:

Graph 14 - FTIR spectrum of sulphathiazole

Table 10 - Table for FTIR analysis of sulphathiazole

No.

PEAK

ASSIGNMENT

1

3464.8 & 3359.0

-NH2 stretch

2

3100.0

AROMATIC RING,

C-H STRETCH

3

1593.6, 1570.0, 1499.4, 1419.5

CONFIRMS BENZENE RING

"RING BREAHING"

4

1143.2

SO2 STRETCH

5

1088.3

C-S

6

830.9

Para DISUBSTITUTED RING, OUT OF PLANE C-H BENDING

The IR spectrum of sulphathiazole exhibits 2 sharp absorptions at 3464.8 and 3359.0, which are the characteristic of -NH2 stretch. The small peak at 3100.0 (its not marked) corresponds to the aromatic ring C-H stretch, and the benzene ring is confirmed by the presence of peaks at 1593.6, 1570.0 (not marked), 1499.4, 1419.5. in these the peaks at 1593.6 and 1499.4 are due to aromatic ring C=C vibration and other two peaks at 1570.0 and 1419.5 are the C=C bending modes. The 1143.2 peak is due to SO2 and the 1088.3 peak appears due to C-S bond in the thiazole ring. The peak at 830.9 is the indicative that the aromatic ring is para-disubstituted ring, with out of plane C-H bending.

1H NMR spectrum: 0.0015gm of sample was weighed and dissolved in DMSO D6 (premixed with TMS). The solution was loaded in the NMR tube, labelled and given for H1 and C-13 spectroscopy in the NMR laboratory

Graph 15 - 1H NMR spectrum of sulphathiazole

Table 11 - Table for 1H NMR analysis of sulphatiazole

CHEMICAL SHIFT(ppm)

INTEGRATION

SPLITTING PATTERN

ASSIGNMENT

5.8

2

SINGLET

-NH2

6.5

2

DOUBLET

AROMATIC H ortho positions

6.7

1

DOUBLET

H at position 4 in THIAZOLE RING

7.2

1

DOUBLET

H at position 3 in THIAZOLE RING

7.4

2

DOUBLET

AROMATIC H meta positions

12.4

1

SINGLET

-NH GROUP

The peak at 5.8ppm is due to the amine group in the molecule. The hydrogen atoms in the benzene ring give rise to the peaks at 6.5 and 7.4. The former is due to α hydrogen and due to plane of symmetry, so the integration is 2. The most downfield peak is due to the sulphonamide. The sulphonyl group and nitrogen are all electronegative atoms, so the sulphonamide H atom has shifted downfield more than expected.

NMR C-13 spectrum:

Graph 16 - C-13 NMR spectrum of sulphathiazole

C-13 NMR spectra of Sulphathiazole provides the following details:

Table 12 - Table for C-13 NMR analysis of sulphathiazole

PEAKS

ASSIGNMENT

107.3

HETROAROMATIC C at position 5

112.3

Ar-C

124.1

HETROAROMATIC C at position 2

127.6

Ar-C

134.0

C-NH2

152.1

HETROAROMATIC C at 4th position

In the above spectra we have received 6 peaks instead of 7. The signal of the remaining C-atom is possibly weak and is hidden within the vibrations.

Mass spectroscopy:

Sample preparation: 0.00112 gm of sample was dissolved in 1ml (1000µl). 10 µl was taken from the solution and again diluted with 990µl solvent. The solvent used is Acetonitrile. The prepared sample was run with 30% H2O + 70% CH3OH in the mass spectrometer using ESI method for ionization

Graph 17 - Mass spectrum of sulphathiazole

The information gained by the positive ESI mass spectrum of sulphathiazole is as under:

The peak at 256 is the base peak and is due to the ionised molecule of sulphathiazole.

The peaks at 257 and 258 could be due the presence of isotopes.

Peak at 258 is also due to the presence of isotopes.

CONCLUSION:

Sulphanilamide, sulphapyridine and sulphathiazole were synthesized from

p-acetamidobenzenesulphonyl chloride. The synthesized products were then assessed and analyzed by different spectroscopic techniques FTIR, NMR, and mass spectrometry.

In FTIR analysis all the expected characteristic peaks were observed and the structures of all compounds were confirmed.

In mass spectrometry using electrospray ionization method the products synthesized have shown the expected results. However, in the mass spectrum of Sulphanilamide, the peak at 214 m/z indicates the chances of some amount of starting material left unreacted as this peak is common in both, starting material and final product. The other two compounds gave the spectrum as expected and did not show any impurity. In NMR spectroscopic analysis, the C-13 spectrum of sulphapyridine and sulphathiazole showed less number of peaks than expected. The signals of the missing peaks were weak and hence remain hidden in the vibrational base line. All the peaks that were received were in correct position as expected, so the compound can be regarded as pure.

FURTHER WORK:

The C-13 spectrum of two compounds, sulphapyridine and sulphathiazole had some of the peaks missing. So, the further work involves the C-13 spectroscopy of the compounds at various parameters and understanding what are the different effects of these parameters on the spectrum and which are best for analysing these compounds.