QSAR Analysis Of Antibacterial Sulphonamides Biology Essay

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A QSAR method was used to investigate the lipophilicity of four sulphonamide drugs. Drug B ( Sulphacetamide) had the highest lipophilicity constant with a value of 1.343, followed by Drug D then C then A with values of 0.547, 0.264 and 0 respectively. Lipophilicity is the ability of a chemical compound to dissolve in fats, oils, lipids and non polar solvents.

Introduction

The quantitative structure-activity relationships (QSAR) method is a process in which biological activity of a drug is related to its physiochemical activities. A number of subunits are used in drug design to produce different derivatives with distinct physiochemical activities. The way these subunits affect the activity of the drug is then used to determine the derivative which would produce the most beneficial pharmacological response. Lipophilicity, electronic properties and steric effects are parameters used to determine the physiochemical properties of a drug. Each parameter is assigned a numerical value known as a constant which is used to determine the degree of the effect that the subunit has on the active site of the drug.

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There are two type of QSAR analysis; one is the Hansch and Free Wilson Analysis which considers 2D structures and the other is coMFA which considers 3D structures. In these analyses a number of derivatives (between 25-30) with different subunits and physiochemical properties are used in order to maximize the chances of finding the compound with the most beneficially effective activity. The activities of these derivatives are first determined, followed by the relationship between these activities and their QSAR parameters. The relationship is devised as a graph or an equation which can be used to predict the activity of the derivative.

Lipophilicity is the most important physiochemical property linked to biological activity of drugs. It is fundamental in assessing the bioavailability of a drug and it plays a very important role in the transverse of bioactive substances from body fluids across the biological membrane. It refers to the affinity of a drug for a lipophilic environment. It is measured by using the lipophilicity constant (Ï€). The partition coefficient of drugs between the 1-octanol phase and the aqueous phase is a very important parameter used to predict the biological activities of drugs. It is a measure of how lipophilic a drug is which in turn helps to indicate the ability of a drug to cross a membrane.

Sulphonamides are organic compounds with structures as shown in fig 1. They are antibacterial agents that have similar molecular structure to a natural substrate P-aminobenzoicacid (PABA). PABA is a substrate of the enzyme dihydropteroate synthetase which is needed by bacteria to synthetise tetrahydrofolic acid (THF). Sulphonamides work by blocking the activity of the enzyme.

The four sulphonamide drugs investigated in this experiment are: Sulphanilamide (drug A), Sulphacetamide (drug B), sulphapyridine (drug C) and Sulphamethazine (drug D).

The aim of the experiment was to determine the lipophilicity constant for the 4 drugs stated. And also to find out the relationship between lipophilicity and biological activity by using the partition coefficients, lipophilicity constant and Hansch analysis.

Fig 1: Chemical structures of the four sulphonamide drugs to be used in experimental determination of lipophilicity. The 'unsubstituted' drug, sulphanilamide (R = H) is the simplest of these drug molecules.

Method and Materials

Materials

1.5ml microcentrifuge tubes

Gilson p1000 pipette with disposable tips

0.1mM of the 4 sulphonamide drugs

Water

15ml screw-capped tubes

Spectrophotometer

Cuvettes and holding racks

1-octanol

Disposable bulb pipettes

Centrifuge

Part 1: Determining the concentration of sulphonamide in aqueous solution by UV spectroscopy; construction of a calibration curve.

Eleven 1.5 ml microcentrifuge (mcc) tubes were labeled as coded series A1-A11. A Gilson p1000 pipetter was used to make up sets of dilutions as shown in Table 1. The indicated volumes of water in Table 1 were added to the corresponding tubes and the position of each tube was moved in the rack after each addition. The requisite volumes of 0.1mM sulphanilamide as shown in Table 1 were added to each tube. The same pipette tip was used for each addition and once again the position of each tube in the rack was moved after each addition. The lids of the mcc tubes were secured then briefly vortexed using the mixer. The dilution series procedure was repeated for the three remaining drugs. The tubes were labeled appropriately, B1-B11 for sulphacetamide, C1-C11 for sulphapyridine and D1-D11 for sulphamethazine.

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Table 1a: Composition of dilution series of sulphonamide drugs to be used in calibration. The table shows the calculated final concentration of drug (mM) in each dilution with corresponding blank cells for entering recorded absorbance readings for each drug series.

Tube number

H2O (ml)

0.1mM drug (ml)

Final [drug] (mM)

A 258nm (sulphanilamide) (Drug A)

A 258nm (sulphacetamide) (Drug B)

A 261nm (sulphapyridine) (Drug C)

A 261 nm (sulphamethazine) (Drug D)

1

1.0

­

0

2

0.9

0.1

0.01

3

0.8

0.2

0.02

4

0.7

0.3

0.03

5

0.6

0.4

0.04

6

0.5

0.5

0.05

7

0.4

0.6

0.06

8

0.3

0.7

0.07

9

0.2

0.8

0.08

10

0.1

0.9

0.09

11

­

1.0

0.10

Using the Spectrophotometer

The machine was switched on and allowed to warm up for 10-15 minutes. The "go to" button was pressed and the wavelength required (258nm for sulphanilamide) was entered then the "E" button was pressed. The ready message was displayed on the display window. The spectrophotometer was calibrated to zero by putting a cuvette with the contents of the tube labeled A1 into the spectrometer, closing the lid and pressing the "zero" button. The display window read 0.000 which was an indication that the absorbance readings for each dilution series could commence. The water blank (A1) was removed from the machine and poured back into its tube. The contents of tube A2 was poured into the same cuvette, placed into the spectrophotometer, the lid was then closed and the absorbance reading appeared in the display window after 1-2 seconds. The reading was recorded in the appropriate blank cell in Table 1. The cuvette was removed and the contents poured back into its tube. The cuvette was rinsed with water and tapped unto paper towel to remove any droplets left inside. This procedure was repeated with the remaining tubes A3-A11 using the same cuvette, rinsing and drying it in between each addition.

The procedure was repeated for each remaining drug B-D. The wavelength was kept at 258nm for drug B (sulphacetamide) and changed to 261nm for the absorbance readings of drug C (sulphapyridine) and drug D (sulphamethazine). A graph as constructed for absorbance versus concentration for each of the 4 drugs.

Part 2: Determination of the partition coefficients of sulphonamide drugs

Eight 15 ml screw-capped tubes were labeled A1-D1, A2-D2 and placed on a rack. 2.0ml of each of the 0.1mM sulphonamide drug solution was added to the tubes as shown in Table 2 and the screw caps were replaced. The rack of tubes was taken to the fume cupboard for the addition of 1-octanol.

Table 2a: Composition of screw-capped tubes for the determination of partition coefficient of sulphonamide drugs.

Tube name

Sulphonamide Drug (0.1mM)

Drug (ml)

1­octanol (ml)

λ max (nm)

Absorbance at λmax

Absorbance at λmax of replicate

Ave. value of Absorbance at λmax

A1/A2

Sulphanilamide (A)

2.0

2.0

B1/B2

Sulphacetamide (B)

2.0

2.0

C1/C2

Sulphapyridine (C)

2.0

2.0

D1/D2

Sulphamethazine (D)

2.0

2.0

Preparation and separation of the partition layers

Safety glasses and gloves were worn and a Gilson p1000 pipette was used to pipette 2.0 ml of 1-octanol into each tube. The caps were securely replaced and the rack with the tubes was taken back to the bench after wearing fresh gloves. Each tube was vortexed in turn for about half a minute using the maximum setting on the mixer taking care not to allow any leakage. Vigorous vortexing allowed the 1-octanol and aqueous liquid to emulsify. The vortexing step was repeated for a period of 5 minutes and half a minute intervals was allowed between each 30 second vortexing. The eight tubes were placed in the centrifuge ensuring the rotor was balanced and centrifuged at 3,000 rpm for 5 minutes to completely separate the two phases.

Preparing the 1-octanol samples for measurement in the spectrophotometer

Nine cuvettes were labeled, 1 as "O" (the 1-octanol blank) and A1-D1 and A2-D2 making sure not to write on the clear sides and placed on a rack. The rack was taken to the fume cupboard and 1.0ml of 1-octanol was pipette from the stock bottle into the "O" cuvette. Gloves and safety glasses was worn. After 5 minutes the 8 tubes were removed from the centrifuge, placed in a rack and taken to the fume cupboard. 1.0ml of the upper 1-octanol phase was carefully pipette from each of the 8 tubes and placed into the corresponding cuvette using a disposable bulb pipette. A fresh bulb pipette was used for each tube. The screw caps were replaced on the tubes and they were put on the used rack to be disposed of. The used pipettes were also placed in the disposable bag in the fume cupboard. The top of the 9 cuvettes were sealed carefully with parafilm and care was taken not to allow the film to go too far down the sides of the cuvette. New gloves were worn to take the cuvettes to the spectrometer.

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Measuring the absorbance of the samples using the spectrophotometer- wavelength scan

The O cuvette was used as a blank to zero the spectrophotometer to determine the λ max of each of the four sulphonamide drugs in the 1-octanol phase. The wavelength was initially set to 250nm using the instructions in part 1. After the machine was zeroed the absorbance of each drug (A1-D1) was measured at 250nm and then at 5nm intervals up to 280nm. The machine was re-zeroed after the change in each wavelength. The results were tabulated. After the λ max was determined, another wavelength scan at 1.0 nm intervals over a range of 10nm was performed to determine the λ max to the nearest nm. This was done using the replicas A2-D2. The results were once more recorded and a graph was plotted. The absorbance at the λ max for A1-D1 and A2-D2 were recorded in Table 2 along with the calculated averages of each drug.

The cuvettes were taken back to the fume cupboard to be disposed of safely.

Results

Table 1b: Composition of dilution series of sulphonamide drugs to be used in calibration. The table shows the calculated final concentration of drug (mM) in each dilution with corresponding recorded absorbance readings (nm) for each drug series.

Tube number

H2O (ml)

0.1mM drug (ml)

Final [drug] (mM)

A 258nm (sulphanilamide) (Drug A)

A 258nm (sulphacetamide) (Drug B)

A 261nm (sulphapyridine) (Drug C)

A 261 nm (sulphamethazine) (Drug D)

1

1.0

­

0

0.00

000

0.00

0.00

2

0.9

0.1

0.01

0.195

0.312

0.346

0.163

3

0.8

0.2

0.02

0.385

0.439

0.371

0.314

4

0.7

0.3

0.03

0.567

0.585

0.606

0.456

5

0.6

0.4

0.04

0.752

0.678

0.697

0.601

6

0.5

0.5

0.05

0.938

0.838

0.896

0.749

7

0.4

0.6

0.06

1.100

0.991

1.056

0.928

8

0.3

0.7

0.07

1.316

1.182

1.182

1.032

9

0.2

0.8

0.08

1.468

1.244

1.340

1.164

10

0.1

0.9

0.09

1.636

1.442

1.508

1.306

11

­

1.0

0.10

1.838

1.666

1.768

1.476

The four sulphonamide drugs all showed an increase in absorbance at their respective wavelengths as their concentration increased. Sulphanilamide had the highest absorbance reading at 1.838nm followed by sulphapyridine sulpacetamide and sulphamethazine at 1.768nm, 1.666 nm, and 1.476nm respectively.

Fig 2 above shows a linear relationship between the absorbance against the concentration of the drugs. The average λmax of the drugs with 1-octanol were plotted on the graph to determine their concentration. Drug A is 0.075mM at 1.38nm, Drug B: 0.012mM at 0.319nm, Drug C: 0.062mM at 1.088 and Drug D: 0.046mM at 0.699nm.

Table 2b: Composition of screw-capped tubes for the determination of partition coefficient of sulphonamide drugs.

Tube name

Sulphonamide Drug (0.1mM)

Drug (ml)

1­octanol (ml)

λ max (nm)

Absorbance at λmax

Absorbance at λmax of replicate

Ave. value of Absorbance at λmax

A1/A2

Sulphanilamide (A)

2.0

2.0

269

1.370

1.390

1.38

B1/B2

Sulphacetamide (B)

2.0

2.0

261

0.318

0.320

0.319

C1/C2

Sulphapyridine (C)

2.0

2.0

271

1.130

1.046

1.088

D1/D2

Sulphamethazine (D)

2.0

2.0

271

0.693

0.704

0.699

Table 3: Wavelength scan of the four drugs between 250-280nm at 5 nm intervals. Maximum absorbance is indicated by highlight.

Wavelength (nm)

Absorbance readings (nm)

A1

B1

C1

D1

250

0.596

0.221

0.52

0.236

255

0.813

0.286

0.652

0.359

260

1.084

0.323

0.835

0.504

265

1.306

0.308

0.995

0.632

270

1.378

0.243

1.048

0.694

275

1.276

0.166

0.988

0.667

280

1.086

0.113

0.872

0.584

Table 4: Second wavelength scan for drugs A1, C1 and D1 at 1nm intervals to determine which wavelength has the maximum absorbance.

Wavelength (nm)

Absorbance readings (nm)

A1

C1

D1

265

1.302

0.993

0.63

266

1.332

1.016

0.651

267

1.354

1.024

0.668

268

1.364

1.068

0.675

269

1.37

1.086

0.685

270

1.368

1.122

0.689

271

1.364

1.13

0.693

272

1.35

1.12

0.69

273

1.326

1.106

0.684

274

1.304

1.094

0.675

275

1.272

1.076

0.662

Table 5: Second wavelength scan for Drug B1 to determine which wavelength has the maximum absorbance.

Wavelength (nm)

Aborbance (nm) of Drug B1

255

0.279

256

0.289

257

0.297

258

0.308

259

0.314

260

0.316

261

0.318

262

0.317

263

0.315

264

0.309

265

0.307

Table 6: Analysis of data for the four sulphonamide drugs.

Drug

Concentration in 1-octanol phase (mM)

Partition coefficient (P)

log P

Lipophilicity constant (Ï€)

Minimum concentration (Cr) (um)

1/Cr

log (1/Cr)

A

0.075

3

0.477

0

200

0.005

-2.301

B

0.012

0.136

-0.866

1.343

7

0.143

-0.845

C

0.062

1.632

0.213

0.264

6

0.167

-0.778

D

0.046

0.852

-0.07

0.547

3

0.333

-0.477

Discussion

The low logP values of all the sulphonamide drugs indicates that the drugs are not capable of partitioning the lipophilic phase which causes them to be localized in the aqueous phase. This may be caused by the branching and crowding of the lipophilic groups which decreases the lipophilicity constant and logP.

Sulphamethazine should be a more lipophilic molecule because the large size of the molecule due to the steric effect. This makes it more hydrophobic and it does not react with water while passing through the membrane. It is more soluble in the octanol phase due to lipophilicity effect and is more localize in the lipophilic phase.

Sulphapyridine is a lipophilic molecule as its substituent groups causes an increase in size. The increase in size increases the hydrophobicity of the compound and the electronic distribution can be polarized. This makes the drug more soluble in 1-octanol phase and not to have any reaction with water.

Sulphacetamide tends to stay in the lipophobic (water phase) rather than in the octanol phase. This s because a ketone structure replaces the R substituent group which has a reaction with water to produce other molecules and end up staying in this aqueous phase.

Sulphonamides are antibacterial drugs that kill the bacteria in vitro. The activity of the drug has a relationship with lipophilicity as drugs that are nearer to the optimum value of the logP which is zero are more likely to pass from a lipophobic to lipophilic through the membrane. Drugs that are more far away from the optimal value of the logP are less likely to go through a lipophobic to a lipophilic site within the membrane. From Table 6, it shows that Drug A and B are further away from the optimal value and therefore has a lower chance of passing to the lipophilic site in the membrane. Drug C and D as indicated from Table 6 is more closer to the optimal value of logP and are more likely to pass through to the lipophilic site in the membrane. They are referred to as bacteriostasis agents.

E.coli are gram negative bacteria which affects different parts of the body so the drug needed for each case has to be different in their mechanism of action. The membranes which they need to pass through can be different at different sites in the body. Some drugs may need to pass through a lipophobic membrane while others need to pass through a lipophilic membrane. So they are needed to be specially adapted for the membrane they go through.

This relationship is not necessarily to free cell assay of bacterial dihydropteroate synthase enzyme activity in vitro to measure drug activity. Activity of the drug increases as the there is an increase in the bacterial enzyme activity. The increase in bacterial activity produces more enzymes for the drugs to bind to and inhibit the growth of the bacteria. This produce shorter reaction time for the drug to bind to the enzymes and in turn increases the drug activity over this timescale.