Patrick

An Introduction to Medicinal Chemistry 3/e

Chapter 13

QUANTITATIVE STRUCTURE-
ACTIVITY RELATIONSHIPS (QSAR)

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Contents

1. Introduction
2. Hydrophobicity of the Molecule (4 slides)
3. Hydrophobicity of Substituents (2 slides)
4. Electronic Effects
4.1. Hammett Substituent Constant (s) (7 slides)
4.2. Electronic Factors R & F
4.3. Aliphatic electronic substituents
5. Steric Factors (3 slides)
6. Hansch Equation (4 slides)
7. Craig Plot (2 slides)
8. Topliss Scheme (5 slides)
9. Bio-isosteres
10. Free-Wilson Approach (3 slides)
11. Case Study (10 slides)
12. 3D-QSAR (10 slides)
13. 3D-QSAR Case Study(7 slides)

[62 slides]
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 1. Introduction
• Aims
• To relate the biological activity of a series of compounds to
their physicochemical parameters in a quantitative fashion
using a mathematical formula
• Requirements
• Quantitative measurements for biological and
physicochemical properties

• Physicochemical Properties
• Hydrophobicity of the molecule
• Hydrophobicity of substituents Most common
• Electronic properties of substituents properties studied
• Steric properties of substituents

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2. Hydrophobicity of the Molecule

Partition Coefficient P = [Drug in octanol]

[Drug in water]

High P High hydrophobicity

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 2. Hydrophobicity of the Molecule
• Activity of drugs is often related to P
e.g. binding of drugs to serum albumin
(straight line - limited range of log P)

Log (1/C)

. . .
.
.. . . . Log 1C 0.75 logP + 2.30

0.78 3.82 Log P

• Binding increases as log P increases

• Binding is greater for hydrophobic drugs

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 2. Hydrophobicity of the Molecule
Example 2 General anaesthetic activity of ethers
(parabolic curve - larger range of log P values)

Log (1/C)
1
Log  C - 0.22(logP)2 + 1.04 logP + 2.16

o
Log P Log P

Optimum value of log P for anaesthetic activity = log Po

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 2. Hydrophobicity of the Molecule

• QSAR equations are only applicable to compounds in the

same structural class (e.g. ethers)

• However, log Po is similar for anaesthetics of different

structural classes (ca. 2.3)

• Structures with log P ca. 2.3 enter the CNS easily

(e.g. potent barbiturates have a log P of approximately 2.0)

• Can alter log P value of drugs away from 2.0 to avoid CNS
side effects

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 3. Hydrophobicity of Substituents
- the substituent hydrophobicity constant (p)
• A measure of a substituent’s hydrophobicity relative to
hydrogen
• Tabulated values exist for aliphatic and aromatic substituents
• Measured experimentally by comparison of log P’s with parent
structure
Cl CONH2

Example :

Benzene Chlorobenzene Benzamide

(Log P = 2.13) (Log P = 2.84) (Log P = 0.64)

Cl = 0.71 CONH = -1.49

2

• Positive values imply substituents are more hydrophobic than H

• Negative values imply substituents are less
hydrophobic than H ©1
 3. Hydrophobicity of Substituents
- the substituent hydrophobicity constant (p)
• The value of is only valid for parent structures
• It is possible to calculate log P using  values

Cl
Example :
Log P(theory) = log P(benzene) + Cl + CONH
2
= 2.13 + 0.71 - 1.49
= 1.35
CONH2
Log P (observed) = 1.51
meta-Chlorobenzamide

• A QSAR equation may include both P and .

• P measures the importance of a molecule’s overall
hydrophobicity (relevant to absorption, binding etc)
•  identifies specific regions of the molecule which might
interact with hydrophobic regions in the binding site
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 4. Electronic Effects
4.1 Hammett Substituent Constant (s)
• The constant () a measure of the e-withdrawing or e-
donating influence of substituents
• It can be measured experimentally and tabulated
(e.g.  for aromatic substituents is measured by comparing the
dissociation constants of substituted benzoic acids with benzoic acid)

X X
CO2H CO2 + H

-
X=H KH = Dissociation constant= [PhCO2 ]
[PhCO2H]

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 4.1 Hammett Substituent Constant (s)

X= electron withdrawing group (e.g. NO2)

X = electron
withdrawing X X
group CO2H CO2 + H

Charge is stabilised by X
Equilibrium shifts to right
KX > KH

K
X = log X = logKX - logKH
KH

Positive value

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 4.1 Hammett Substituent Constant (s)
X= electron donating group (e.g. CH3)

X = electron
withdrawing X X
group CO2H CO2 + H

Charge destabilised
Equilibrium shifts to left
KX < K H

K
 X = log X = logKX - logKH
KH

Negative value

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 4.1 Hammett Substituent Constant (s)

 value depends on inductive and resonance effects

 value depends on whether the substituent is meta or para

ortho values are invalid due to steric factors

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 4.1 Hammett Substituent Constant (s)

EXAMPLES: p (NO2) m (NO2)

meta-Substitution
O

N
O e-withdrawing (inductive effect only)

DRUG

para-Substitution

O O O O O O O O
N N N N
e-withdrawing
(inductive +
resonance effects)
DRUG DRUG DRUG DRUG

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 4.1 Hammett Substituent Constant (s)

EXAMPLES: m (OH) p (OH)

meta-Substitution

OH

e-withdrawing (inductive effect only)

DRUG

para-Substitution

OH OH OH OH

e-donating by resonance
more important than
inductive effect
DRUG DRUG DRUG DRUG

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 4.1 Hammett Substituent Constant (s)

QSAR Equation:

X
O P OEt
log 1C 2.282 - 0.348
OEt

Diethylphenylphosphates
(Insecticides)

Conclusion : e-withdrawing substituents increase activity

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4.2 Electronic Factors R & F

• R - Quantifies a substituent’s resonance effects

• F - Quantifies a substituent’s inductive effects

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 4.3 Aliphatic electronic substituents
• Defined by I
• Purely inductive effects
• Obtained experimentally by measuring the rates of hydrolyses
of aliphatic esters
• Hydrolysis rates measured under basic and acidic conditions
O O
Hydrolysis
C C + HOMe
X CH2 OMe X CH2 OH

X= electron donating Rate I = -ve

X= electron withdrawing Rate I = +ve

Basic conditions: Rate affected by steric + electronic factors

Gives I after correction for steric effect
Acidic conditions: Rate affected by steric factors only (see Es)
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5. Steric Factors
Taft’s Steric Factor (Es)
• Measured by comparing the rates of hydrolysis of substituted
aliphatic esters against a standard ester under acidic
conditions

Es = log kx - log ko k
x represents the rate of hydrolysis of a substituted ester

ko represents the rate of hydrolysis of the parent ester

• Limited to substituents which interact sterically with the

tetrahedral transition state for the reaction
• Cannot be used for substituents which interact with the
transition state by resonance or hydrogen bonding
• May undervalue the steric effect of groups in an
intermolecular process (i.e. a drug binding to a receptor)
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5. Steric Factors
Molar Refractivity (MR) - a measure of a substituent’s volume

(n 2 - 1) mol. wt.
MR = x
(n 2 - 2) density
Correction factor Defines volume
for polarisation
(n=index of
refraction)

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5. Steric Factors
Verloop Steric Parameter
- calculated by software (STERIMOL)
- gives dimensions of a substituent
- can be used for any substituent

Example - Carboxylic acid

B4 B3

O B
3

B2
C
H O C O B1
O B
4

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 6. Hansch Equation
• A QSAR equation relating various physicochemical properties
to the biological activity of a series of compounds

• Usually includes log P, electronic and steric factors

• Start with simple equations and elaborate as more structures

are synthesised

• Typical equation for a wide range of log P is parabolic

Log 1C  - k1(logP)2 + k 2 logP + k 3  + k 4 Es + k 5

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6. Hansch Equation
Example: Adrenergic blocking activity of -halo--arylamines
Y X

CH CH2 NRR'

1 

Log  C  1.22  - 1.59  + 7.89

Conclusions:
• Activity increases if  is +ve (i.e. hydrophobic substituents)
• Activity increases if  is negative (i.e. e-donating substituents)

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 6. Hansch Equation
Example: Antimalarial activity of phenanthrene aminocarbinols
CH2NHR'R"
(HO)HC

1
Log C - 0.015 (logP)2 + 0.14 logP + 0.27 X + 0.40 Y + 0.65 X + 0.88 Y + 2.34

Conclusions:
• Activity increases slightly as log P (hydrophobicity) increases
(note that the constant is only 0.14)
• Parabolic equation implies an optimum log Po value for activity
• Activity increases for hydrophobic substituents (esp. ring Y)
• Activity increases for e-withdrawing substituents (esp. ring Y)
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6. Hansch Equation
Choosing suitable substituents
Substituents must be chosen to satisfy the following criteria:

• A range of values for each physicochemical property studied

• values must not be correlated for different properties (i.e. they
must be orthogonal in value)
• at least 5 structures are required for each parameter studied
Substituent H Me Et n-Pr n-Bu Correlated values.
 0.00 0.56 1.02 1.50 2.13 Are any differences
MR 0.10 0.56 1.03 1.55 1.96
due to  or MR?

Substituent H Me OMe NHCONH2 I CN No correlation in values

 0.00 0.56 -0.02 -1.30 1.12 -0.57 Valid for analysing effects
MR 0.10 0.56 0.79 1.37 1.39 0.63 of  and MR.

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7. Craig Plot
Craig plot shows values for 2 different physicochemical properties
for various substituents
Example: + -
. . + 1.0

+ +

. . . .. . .
CF3SO 2
.75
NO2

.
CN SF5
CH3SO2 .50

. ..
SO 2NH2 CF3

.
CH3CO
CONH2
OCF3

.
.25
I

.
CO2H Cl Br

.
-.8 -.4 .4

.
-2.0 -1.6 -1.2 F .8 1.2 1.6 2.0

-
. .
CH3CONH +

. OCH3
-.25 Me Et
t-Butyl

. .
OH
-.50

NH2 NMe 2
-.75

- - -1.0 - +
-
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 7. Craig Plot

• Allows an easy identification of suitable substituents for a

QSAR analysis which includes both relevant properties

• Choose a substituent from each quadrant to ensure

orthogonality

• Choose substituents with a range of values for each property

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8. Topliss Scheme
Used to decide which substituents to use if optimising compounds
one by one (where synthesis is complex and slow)
Example: Aromatic substituents
H

4-Cl
L E M
4-OMe 4-CH3 3,4-Cl2
L E M L E M L E M
4-But 3-CF3-4-Cl

3-Cl 3-Cl 4-CF3 3-CF3-4-NO2

L E M
2,4-Cl2
See Central 3-NMe2 3-CH3 3-CF3
Branch 4-NO2
2-Cl 3,5-Cl2
4-NMe2
M 4-NO2 3-NO2
L E
3-Me-4-NMe2 4-F
4-NH2

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8. Topliss Scheme
Rationale
Replace H with
para-Cl (+ and +)
Act. Little Act.
change

+ and/or + favourable  + and/or +

advantageous unfavourable  disadvantageous

add second Cl to
replace with OMe
increase  and  replace with Me
(- and -)
further (+ and -)

Further changes suggested based on arguments of  and

steric strain
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8. Topliss Scheme
Aliphatic substituents

CH3

i-Pr
L E M

H; CH2OCH3 ; CH2SO2CH3 Et Cyclopentyl

L E M L E M
END Cyclohexyl

CHCl2 ; CF3 ; CH2CF3 ; CH2SCH3 Cyclobutyl; cyclopropyl CH2Ph

Ph ; CH2Ph t-Bu CH2CH2Ph

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8. Topliss Scheme
Example

Order of R Biological High

Synthesis Activity Potency

SO2NH2 1 H -
R 2 4-Cl M
3 3,4-Cl2 L
4 4-Br E
5 4-NO2 M *

M= More Activity
L= Less Activity
E = Equal Activity

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8. Topliss Scheme
Example

N N Order of R Biological High

Synthesis Activity Potency

R 1 H -
N N L
2 4-Cl
CH2CH2CO2H 3 4-MeO L
4 3-Cl M *
5 3-CF3 L
6 3-Br M *
7 3-I L
8 3,5-Cl2 M *

M= More Activity
L= Less Activity
E = Equal Activity

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9. Bio-isosteres
NC CN
O C O O O O

Substituent C C S S CH3 S NHCH3 C NMe2

CH3 CH3 CH3
O O
 -0.55 0.40 -1.58 -1.63 -1.82 -1.51
p 0.50 0.84 0.49 0.72 0.57 0.36
m 0.38 0.66 0.52 0.60 0.46 0.35
MR 11.2 21.5 13.7 13.5 16.9 19.2

• Choose substituents with similar physicochemical properties

(e.g. CN, NO2 and COMe could be bio-isosteres)
• Choose bio-isosteres based on most important
physicochemical property
(e.g. COMe & SOMe are similar in p; SOMe and SO2Me are similar in
)
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 10. Free-Wilson Approach
Method
• The biological activity of the parent structure is measured
and compared with the activity of analogues bearing
different substituents
• An equation is derived relating biological activity to the
presence or absence of particular substituents
Activity = k1X1 + k2X2 +.…knXn + Z

• Xn is an indicator variable which is given the value 0 or 1

depending on whether the substituent (n) is present or not
• The contribution of each substituent (n) to activity is
determined by the value of kn
• Z is a constant representing the overall activity of the
structures studied
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 10. Free-Wilson Approach
Advantages
• No need for physicochemical constants or tables
• Useful for structures with unusual substituents
• Useful for quantifying the biological effects of molecular
features that cannot be quantified or tabulated by the
Hansch method
Disadvantages
• A large number of analogues need to be synthesised to
represent each different substituent and each different
position of a substituent
• It is difficult to rationalise why specific substituents are
good or bad for activity
• The effects of different substituents may not be additive
(e.g. intramolecular interactions)
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 10. Free-Wilson / Hansch Approach
Advantages
• It is possible to use indicator variables as part of a Hansch
equation - see following Case Study

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11. Case Study
QSAR analysis of pyranenamines (SK & F)
(Anti-allergy compounds)

O OH OH X
3
Y
NH 4
Z
5
O O O

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 O OH OH X
3
11. Case Study NH
Y
4
Z
5
O O O

Stage 1 19 structures were synthesised to study  and 

1 

Log C - 0.14 - 1.35( )2  0.72
 and  = total values for  and  for all substituents

Conclusions:
• Activity drops as  increases
• Hydrophobic substituents are bad for activity - unusual
• Any value of  results in a drop in activity
• Substituents should not be e-donating or e-withdrawing
(activity falls if is +ve or -ve)
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 O OH OH X
3
11. Case Study NH
Y
4
Z
5
O O O

Stage 2 61 structures were synthesised, concentrating on

hydrophilic substituents to test the first equation
Anomalies
a) 3-NHCOMe, 3-NHCOEt, 3-NHCOPr.
Activity should drop as alkyl group becomes bigger and more
hydrophobic, but the activity is similar for all three substituents

b) OH, SH, NH2 and NHCOR at position 5 : Activity is greater than expected

c) NHSO2R : Activity is worse than expected

d) 3,5-(CF3)2 and 3,5(NHMe)2 : Activity is greater than expected

e) 4-Acyloxy : Activity is 5 x greater than expected

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 O OH OH X
3
11. Case Study NH
Y
4

Theories O O O
5
Z

a) 3-NHCOMe, 3-NHCOEt, 3-NHCOPr.

Possible steric factor at work. Increasing the size of R may be good for activity
and balances out the detrimental effect of increasing hydrophobicity

b) OH, SH, NH2, and NHCOR at position 5

Possibly involved in H-bonding

c) NHSO2R
Exception to H-bonding theory - perhaps bad for steric or electronic reasons

d) 3,5-(CF3)2 and 3,5-(NHMe)2

The only disubstituted structures where a substituent at position 5 was electron
withdrawing

e) 4-Acyloxy
Presumably acts as a prodrug allowing easier crossing of cell membranes.
The group is hydrolysed once across the membrane.
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 O OH OH X
3
11. Case Study NH
Y
4
Z
5
O O O

Stage 3 Alter the QSAR equation to take account of new results

1
Log C - 0.30 - 1.35( )2 + 2.0(F- 5) + 0.39(345 -HBD) - 0.63(NHSO2 )
+ 0.78(M - V) + 0.72(4- OCO) - 0.75
Conclusions
(F-5) e-withdrawing group at position 5 increases activity
(based on only 2 compounds though)
(3,4,5-HBD) H-bond donor group at positions 3, 4,or 5 is good for activity
Term = 1 if a HBD group is at any of these positions
Term = 2 if HBD groups are at two of these positions
Term = 0 if no HBD group is present at these positions
Each HBD group increases activity by 0.39
(NHSO2) Equals 1 if NHSO2 is present (bad for activity by -0.63).
Equals zero if group is absent.
(M-V) Volume of any meta substituent. Large substituents at meta
position increase activity
4-O-CO Equals 1 if acyloxy group is present (activity increases by 0.72).
Equals 0 if group absent
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 O OH OH X
3
11. Case Study NH
Y
4
Z
5
O O O

Stage 3 Alter the QSAR equation to take account of new results

1
Log C - 0.30 - 1.35( )2 + 2.0(F- 5) + 0.39(345 -HBD) - 0.63(NHSO2 )
+ 0.78(M - V) + 0.72(4- OCO) - 0.75
The terms (3,4,5-HBD), (NHSO2), and 4-O-CO are examples of indicator
variables used in the free-Wilson approach and included in a Hansch equation

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 O OH OH X
3
11. Case Study NH
Y
4
Z
5
O O O

Stage 4
37 Structures were synthesised to test steric and F-5 parameters,
as well as the effects of hydrophilic, H-bonding groups

Anomalies
Two H-bonding groups are bad if they are ortho to each other
Explanation
Possibly groups at the ortho position bond with each other rather
than with the receptor - an intramolecular interaction

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 O OH OH X
3
11. Case Study NH
Y
4
Z
5
O O O

Stage 5 Revise Equation

1
Log C  - 0.034( ) 2 - 0.33 + 4.3(F- 5) + 1.3 (R- 5) - 1.7( )2 + 0.73(345- HBD)
- 0.86 (HB- INTRA) - 0.69(NHSO2) + 0.72(4- OCO) - 0.59

a) Increasing the hydrophilicity of substituents allows the identification of an

optimum value for  ( = -5). The equation is now parabolic (-0.034 ()2)

b) The optimum value of  is very low and implies a hydrophilic binding site

c) R-5 implies that resonance effects are important at position 5

d) HB-INTRA equals 1 for H-bonding groups ortho to each other (act. drops -086)
equals 0 if H-bonding groups are not ortho to each other

e) The steric parameter is no longer significant and is not present

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11. Case Study
Stage 6 Optimum Structure and binding theory

XH X
O OH
X
NH C CH CH2 OH

3
RHN
5

NH C CH CH2 OH X

O OH

NH3

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 11. Case Study

NOTES on the optimum structure

• It has unusual NHCOCH(OH)CH2OH groups at positions 3 and
5

• It is 1000 times more active than the lead compound

• The substituents at positions 3 and 5

• are highly polar,
• are capable of H-bonding,
• are at the meta positions and are not ortho to each other
• allow a favourable F-5 parameter for the substituent at
position 5

• The structure has a negligible (2 value ©1

12. 3D-QSAR
• Physical properties are measured for the molecule as a whole
• Properties are calculated using computer software
• No experimental constants or measurements are involved
• Properties are known as ‘Fields’
• Steric field - defines the size and shape of the molecule
• Electrostatic field - defines electron rich/poor regions of
molecule
• Hydrophobic properties are relatively unimportant
Advantages over QSAR
• No reliance on experimental values
• Can be applied to molecules with unusual substituents
• Not restricted to molecules of the same structural class
• Predictive capability

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12. 3D-QSAR
Method
• Comparative molecular field analysis (CoMFA) - Tripos
• Build each molecule using modelling software
• Identify the active conformation for each molecule
• Identify the pharmacophore

OH

HO NHCH3

HO
Build 3D
model
Active conformation Define pharmacophore

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12. 3D-QSAR
Method
• Comparative molecular field analysis (CoMFA) - Tripos
• Build each molecule using modelling software
• Identify the active conformation for each molecule
• Identify the pharmacophore

OH

HO NHCH3

HO
Build 3D
model
Active conformation Define pharmacophore

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12. 3D-QSAR
Method
• Place the pharmacophore into a lattice of grid points

.
.
. .
.

Grid points

• Each grid point defines a point in space

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12. 3D-QSAR
Method
• Position molecule to match the pharmacophore

.
.
. .
.

Grid points

• Each grid point defines a point in space

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12. 3D-QSAR
Method
• A probe atom is placed at each grid point in turn

.
. Probe atom
. .
.

• Probe atom = a proton or sp3 hybridised carbocation

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12. 3D-QSAR
Method
• A probe atom is placed at each grid point in turn

.
. Probe atom
. .
.

• Measure the steric or electrostatic interaction of the probe

atom with the molecule at each grid point ©1
12. 3D-QSAR
Method
• The closer the probe atom to the molecule, the higher the steric
energy
• Can define the shape of the molecule by identifying grid points of
equal steric energy (contour line)
• Favourable electrostatic interactions with the positively charged
probe indicate molecular regions which are negative in nature
• Unfavourable electrostatic interactions with the positively charged
probe indicate molecular regions which are positive in nature
• Can define electrostatic fields by identifying grid points of equal
energy (contour line)
• Repeat the procedure for each molecule in turn
• Compare the fields of each molecule with their biological activity
• Can then identify steric and electrostatic fields which are favourable
or unfavourable for activity

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12. 3D-QSAR
Method
.
. . ..

Tabulate fields for each

compound at each grid point

Compound Biological Steric fields (S) Electrostatic fields (E)

activity at grid points (001-998) at grid points (001-098)
S001 S002 S003 S004 S005 etc E001 E002 E003 E004 E005 etc
1 5.1
2 6.8
3 5.3
4 6.4
5 6.1

Partial least squares

analysis (PLS)

QSAR equation Activity = aS001 + bS002 +……..mS998 + nE001 +…….+yE998 + z

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12. 3D-QSAR
Method
• Define fields using contour maps round a representative
molecule

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13. 3D-QSAR - CASE STUDY

Tacrine
Anticholinesterase used in the treatment of Alzheimer’s disease

NH2

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13. 3D-QSAR - CASE STUDY
Conventional QSAR Study
12 analogues were synthesised to relate their activity with the
hydrophobic, steric and electronic properties of substituents at
positions 6 and 7 9
NH 2

R1 7

R2 6 N

Substituents: CH3, Cl, NO2, OCH3, NH2, F

(Spread of values with no correlation)
1 1 1 2
Log  C pIC50 = - 3.09 MR(R ) + 1.43F(R , R ) + 7.00

Conclusions
• Large groups at position 7 are detrimental
• Groups at positions 6 & 7 should be electron withdrawing
• No hydrophobic effect ©1
13. 3D-QSAR - CASE STUDY
CoMFA Study
Analysis includes tetracyclic anticholinesterase inhibitors (II)
NH2

R1 8 R3
1

2
R2 7 N R4
3

II R5

• Not possible to include above structures in a conventional

QSAR analysis since they are a different structural class
• Molecules belonging to different structural classes must be
aligned properly according to a shared pharmacophore

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13. 3D-QSAR - CASE STUDY
Possible Alignment
Good overlay but assumes similar binding modes

Overlay

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13. 3D-QSAR - CASE STUDY
X-Ray Crystallography
• A tacrine / enzyme complex was crystallised and analysed
• Results revealed the mode of binding for tacrine
• Molecular modelling was used to modify tacrine to structure
(II) whilst still bound to the binding site (in silico)
• The complex was minimised to find the most stable binding
mode for structure II
• The binding mode for (II) proved to be different from tacrine

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13. 3D-QSAR - CASE STUDY
Alignment
• Analogues of each type of structure were aligned according to
the parent structure
• Analysis shows the steric factor is solely responsible for
activity

• Blue areas - addition of steric bulk increases activity

• Red areas - addition of steric bulk decreases activity
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13. 3D-QSAR - CASE STUDY
Prediction
6-Bromo analogue of tacrine predicted to be active (pIC50 = 7.40)
Actual pIC50 = 7.18

NH2

Br N

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