Chapter 8

DETERMINATION OF MINIMUM
INHIBITORY CONCENTRATION
(MIC) OF SYNTHESIZED 1,4DIHYROPYRIDINE
DERIVATIVES
INTRODUCTION
Minimum Inhibitory Concentration (MIC) assay consists in the determination of
chemical agent spectrum of action, according to resistance of studied
microorganisms. It was developed the determination of minimum inhibitory
concentration (MIC) for every chemical agent, through the classic method of
successive dilution.
Dilution methods are used to determine the minimum inhibitory concentrations
(MICs) of antimicrobial agents and are the reference methods for antimicrobial
susceptibility testing against which other methods, such as disk diffusion, are
cali-brated.MIC methods are widely used in the comparative testing of new
agents. In dilution tests, microorganisms are tested for their ability to produce
visible growth on a series of agar plates (agar dilution) or in micro plate wells of
broth (broth micro dilution) containing dilutions of the antimicrobial agent. The
lowest concentration of an

antimicrobial agent that will inhibiting the visible

growth of a micro organism known as MIC (1).

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Minimum inhibitory concentrations are important in diagnostic laboratories to

confirm resistance of microorganisms to an antimicrobial agent and also to
monitor the activity of new antimicrobial agents. A lower MIC is an indication
of a better antimicrobial agent.
A series of glass tubes containing different concentrations of the synthesized
compounds (In Dimethyl Sulphoxide) with Mueller Hinton broth was
inoculated with the required amount of the inoculum to obtain a suspension of
microorganism which contains 105 colony forming units per milliliter. Growth
control tube was prepared with the addition of the compound and blank was
prepared without the addition of microorganism. The tubes were incubated at 37
C for 24 h. The turbidity produced in each tube was recorded by using a UV
visible spectrometer (2).
Staphylococcus aureus become resistant to many commonly used antibiotics
due to indiscriminate use of antibiotics. Staphylococcal resistance to penicillin
is mediated by penicillinase(a form of -lactamase) production: an enzyme
which breaks down the -lactam ring of the penicillin molecule. First report of a
penicillin-resistant strain of S.aureus was published in 1945, revealing its
association with penicillinase enzyme produced by the bacteria (Spink and
Ferris,1945).The

-lactamase-resistant

penicillins,

methicillin,

oxacillin,cloxacillin and flucloxacillin) were developed to treat penicillinresistant S. aureus. Penicillinase-resistant penicillins are able to resist
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degradation by staphylococcal penicillinase and are still used as first-line

treatment (3).
The biological activities (MIC) were converted into the corresponding pMIC
values (Eq. 1), where MIC value represents the lowest concentration of drug in
microgram that inhibited the visible growth of a microorganism after overnight
incubation. The MIC values of reference compounds were checked to ensure
that no difference occurred between different groups.

EXPERIMENTAL
1)

MICROBIOLOGY

Minimal inhibitory concentrations (MICs) were determined by broth microdilution. Two Gram-positive (S. aureus ATCC 25923 and E. faecalis ATCC
29212) and two Gram-negative (E. coli ATCC 25922 and P. aeruginosa ATCC
27853) bacteria were used as quality control strains. The MIC values of the
compounds are presented in the table. The stock solutions of the synthesized
compounds were prepared in dimethylsulfoxide (DMSO). MIC was determined
as the lowest concentration of the compound that inhibited visible growth. It
was established that dilution of DMSO lacked antimicrobial activity against any
of the test micro-organisms.

2)

Antibacterial activity assay

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The culture was grown on Mueller-Hinton-Agar (MHA) for all bacteria after
18-24 hour of incubation at 350 C. Before the assay, all of bacteria were grown
in Mueller-Hinton-Agar (MHA) for 2-6 hour. Then the bacterial suspensions
were adjusted to 0.5 McFarland - turbidity. The micro-titer plates were
incubated at 350 C and inspected visually after 18-24 hour for bacteria. The MIC
values were recorded as the lowest concentrations of the substances that had no
visible turbidity.

BIOLOGICAL ACTIVITY
The biological activities (MIC) were converted into the corresponding pMIC
values, where MIC value represents the lowest concentration of drug in
microgram that inhibited the visible growth of a microorganism after overnight
incubation. The MIC values of reference compounds were checked to ensure
that no difference occurred between different groups.

pMIC = -log(MIC) x 10-6

On the basis of QSAR Model -2 mention in chapter 3 we predict some
compounds for synthesis. After the synthesis their predicted value and
experimental values are presented in table-30
The experimental values are presented in table 29-

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Table 1: Minimum Inhibitory Concentration Values

of Synthesized Compound in Microgram/ml

Compound
No.
1.
2.
3.
4.

S.
aureus
g/ml

E. faecalis
g/ml

E. coli
g/ml

P.
aeruginosa
g/ml

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124

128

123

256

251

125

127

128

127

254

253

125

123

251

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The converted experimental values of MIC into p MIC (with the help of
equation mention above) are summarized in table-30

Table 2 : Experimental and Predicted Values in

pMIC50

PREDIC
EXPERIMENTAL
TED
VALUE
VALUE
NO.
1.
2.
3.
4.

S.aur

E.faeca

E.col

P.aerugino

eus
3.59
3.6
3.8
3.9

lius
3.9
3.6
3.8
3.8

i
3.8
4.0
4.1
3.6

sa
4.0
3.8
3.6
3.5

pMIC50
4.3
4.6
6.0
5.6

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The above table shows that for two compounds the predicted value corresponds
with the experimental value while same

is not so in the case of other two

compounds.
The data available do not leads to a hypothesis regarding use of QSAR model as
a predictive tool for identifying new leads. However the same can be part of a
rational approach to provide a direction for synthesized new compounds. new
synthesized compounds were put in appended to data set 2 and combined
dataset was used to develop a new model.

NEW QSAR MODEL

The new QSAR model developed through simulated annealing multiple linear
regression variable selection method, where dissimilarity value set as 3.1.The
chemical structure, substitution and their predicted, experimental values are
given in table-31.

Table 3 : Chemical Structure with Substitution

and their predicted -Experimental Values

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R3

R1O

H3C

OR2

CH3

COMPOU
ND
1
2
3
4

R1,
R2

R3

C2H5
C2H5
C2H5
C2H5

4-NMe2
2-OH
4-Cl
4-OCH3

EXPERIMEN
TAL
VALUES
3.5
3.6
3.8
3.9

PREDICTE
D
ACTIVITY
4.3
4.6
6.0
5.6

New model has correlation coefficient r2=0.9478

Significant cross validation q2=0.7498.
F test 21.16 and degree of freedom 7.
r2se=0.1767, q2se=0.3867, predr2=-5.95, pred_r2se=0.9403.

p MIC = 0.1445(slogp) + 0.0659(3pathcount) + 0.2194(k1alpha)0.9275(SssCH2count)-1.1699(chi2)+0.7631(chi4)

Model is validating by:

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_ran_r2
_ran_q2
best_ran_r2
best_ran_q2
Zscore_ran_r2
Zscore_ran_q2
Zscore pred r2
_ran_predr2
best_ran_predr2

0.000
0.000
0.492
-0.566
-509.013
196.005
-302.96
0.0000
-3.887

INTEPRETATION OF MODEL
It is simple to interpret a 2D QSAR equation where each descriptors
contribution seen by the magnitude and sign of its regression coefficient. A
descriptors coefficient magnitude shows its relative contribution with respect to
other descriptors and sign indicates whether it is directly (+) or inversely (-)
proportional to the activity.
In the developed new QSAR model 6 variables represents the positive and
negative effect of biological activity on compound. SlogP descriptor signifies
log of the octanol/water partition coefficient (including implicit hydrogens).
This property is an atomic contribution model that calculates logP from the
given structure; i.e., the correct protonation state. The direct relationship of this
descriptor suggests that the Octanol/water partition coefficient increases the
activity with 0.939% variance. the 3pathcount is topological parameter which
can signify the total number of fragments of third order (third bond path) in
compound. It is positively correlate and contribute i.e. 11.29% with activity so,
it may be inferred that increase the branching of compound is favorable for
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activity.K1alpha descriptor indicate first alpha modified shape index and

contribute 13.03% .it is directly proportional to the activity.SssCH2count
descriptor defines the total number of CH2 group connected with two single
bonds gave negative effect with -8.33% variance.

Figure 1: Contribution Chart Showing the

Contribution of different Descriptors for new
model
Chi2 descriptor signifies a retention index (second order) derived directly
from gradient retention times and indicates -44.52% with inversely proportional
to the activity.Chi4 descriptor represent Descriptor signifies a retention index

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(second order) derived directly from Gradient retention times positively i.e.
21.8% contribute for the biological activity.
It was observed that the positive coefficient value of slogp, 3pathcount, k1alpha
and chi4 on the biological activity indicated that higher value leads to better
biological activity whereas lower value leads to decrease biological activity.
Negative coefficient values of SssCH2count and chi2 on the biological activity
indicated that lower value leads to good biological activity while higher value
leads to reduced biological activity.
DATA FITNESS PLOT
Data fitness plot for new model is shown in Figure 30.The plot of observed vs
predicted activity provides an idea about how well the model was trained and
how well it predicts the activity of external test
Set.

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Figure 2: Data Fitness Plot of Actual and

Predicted Activity
The graph of observed vs. predicted activity of training and test sets for new
model is shown in Figure 30. the model is able to predict the activity of training
set quite well as well as external test set, providing confidence of model.

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Figure 3: Graph between Actual and Predicted

Biological Activity of Training Set for New Model

Figure 4: Graph between Actual and Predicted

Biological Activity of Test Set for New Model

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