Transcript Hydrophobicity of Substituents

Quantitative Structure Activity
Relationships QSAR and 3D-QSAR
Chapter 18
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
•Electronic properties of substituents
•Steric properties of substituents
Most common
properties studied
Hydrophobicity of the Molecule
Partition Coefficient P =
High P
[Drug in octanol]
[Drug in water]
High hydrophobicity
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)
.
.
.
.
.
.
.. .
0.78
•Binding increases as log P increases
•Binding is greater for hydrophobic drugs
3.82
Log 1C  = 0.75 logP + 2.30
Log P
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
Log P o
Log P
Optimum value of log P for anaesthetic activity = log Po
Hydrophobicity of the Molecule
Notes:
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
Hydrophobicity of Substituents
- the substituent hydrophobicity constant (p)
Notes:
•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 values with log P of parent structure
Cl
CONH2
Example:
Benzene
(Log P = 2.13)
Chlorobenzene
(Log P = 2.84)
pCl = 0.71
Benzamide
(Log P = 0.64)
pCONH 2= -1.49
•Positive values imply substituents are more hydrophobic than H
•Negative values imply substituents are less hydrophobic than H
Hydrophobicity of Substituents
- the substituent hydrophobicity constant (p)
Notes:
•The value of p is only valid for parent structures
•It is possible to calculate log P using p values
Example:
Cl
CONH2
Log P(theory)
= log P(benzene) + pCl + pCONH
Log P (observed)
= 2.13 + 0.71 - 1.49
= 1.35
= 1.51
meta-Chlorobenzamide
•A QSAR equation may include both P and p.
•P measures the importance of a molecule’s overall hydrophobicity (relevant to
absorption, binding etc)
• p identifies specific regions of the molecule which might interact
with hydrophobic regions in the binding site
2
Electronic Effects
Hammett Substituent Constant (s)
Notes:
•The constant (s) is a measure of the e-withdrawing or e-donating influence of
substituents
•It can be measured experimentally and tabulated
(e.g. s for aromatic substituents is measured by comparing the
dissociation constants of substituted benzoic acids with benzoic acid)
X
X
CO2H
X=H
KH
CO2
+
[PhCO
2-]
= Dissociation constant =
[PhCO 2H]
H
Hammett Substituent Constant (s)
X= electron withdrawing group (e.g. NO2)
X = electron
withdrawing
group
X
X
CO2H
CO2
+
H
Charge is stabilised by X
Equilibrium shifts to right
KX > KH
s X = log K X = logK X - logK H
KH
Positive value
Hammett Substituent Constant (s)
X= electron donating group (e.g. CH3)
X = electron
withdrawing
group
X
X
CO2H
CO2
+
H
Charge destabilised
Equilibrium shifts to left
KX < KH
s X = log K X = logK X - logK H
KH
Negative value
Hammett Substituent Constant (s)
NOTES:
s value depends on inductive and resonance effects
s value depends on whether the substituent is meta or para
ortho values are invalid due to steric factors
Hammett Substituent Constant (s)
sm (NO2) =0.71
sp (NO2) =0.78
EXAMPLES:
meta-Substitution
O
N
O
e-withdrawing (inductive effect only)
DRUG
para-Substitution
O
O
N
O
O
N
O
O
N
O
O
N
e-withdrawing
(inductive +
resonance effects)
DRUG
DRUG
DRUG
DRUG
Hammett Substituent Constant (s)
sm (OH) =0.12
EXAMPLES:
sp (OH) =-0.37
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
Hammett Substituent Constant (s)
QSAR Equation:
O
O
P
OEt
X
OEt
log1C = 2.282s - 0.348
Diethylphenylphosphates
(Insecticides)
Conclusion: e-withdrawing substituents increase activity
Electronic Factors R & F
•R - Quantifies a substituent’s resonance effects
•F - Quantifies a substituent’s inductive effects
Aliphatic electronic substituents
•Defined by sI
•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
X
CH2
Basic conditions:
+
C
OMe
X
CH2
HOMe
OH
X= electron donating
Rate
sI = -ve
X= electron withdrawing
Rate
sI = +ve
Rate affected by steric + electronic factors
Gives sI after correction for steric effect
Acidic conditions: Rate affected by steric factors only (see Es)
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
kx 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)
Steric Factors
Molar Refractivity (MR) - a measure of a substituent’s volume
MR =
(n 2 - 1)
(n 2 - 2)
Correction factor
for polarisation
(n=index of
refraction)
x
mol. wt.
density
Defines volume
Steric Factors
Verloop Steric Parameter
- calculated by software (STERIMOL)
- gives dimensions of a substituent
- can be used for any substituent
Example - Carboxylic acid
B4
O
B
3
B2
C
H
O
H
L
B3
B
4
O
C
O
B1
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  = - k 1(logP) 2 + k 2 logP + k 3 s + k 4 E s + k 5
Hansch Equation
Example: Adrenergic blocking activity of b-halo-b-arylamines
Y
X
CH CH2
NRR'
1 
Log C  = 1.22 p - 1.59 s + 7.89


Conclusions:
•Activity increases if p is +ve (i.e. hydrophobic substituents)
•Activity increases if s is negative (i.e. e-donating substituents)
Hansch Equation
Example:Antimalarial activity of phenanthrene aminocarbinols
CH2NHR'R"
(HO)HC
X
Y
1
Log  C  = - 0.015 (logP)2 + 0.14 logP + 0.27 SpX + 0.40 SpY + 0.65 SsX + 0.88 SsY + 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)
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
p
0.00 0.56 1.02 1.50 2.13
MR
0.10 0.56 1.03 1.55 1.96
Substituent H Me OMe NHCONH2
p
0.00 0.56 -0.02
-1.30
MR
0.10 0.56 0.79
1.37
Correlated values.
Are any differences
due to p or MR?
I
CN
1.12 -0.57
1.39 0.63
No correlation in values
Valid for analysing effects
of p and MR.
Craig Plot
Craig plot shows values for 2 different physicochemical properties for various substituents
Example:
.
.
. . . ..
. .
.
.
. ..
.
.
.
.
.
.
.
.
.
+
1.0
+s -p
CF3SO 2
.75
CN
CH3SO2
SO 2NH2
NO2
.50
OCF3
.25
CO2H
-2.0
-p
-1.6
-1.2
-.8
-.4
SF5
CF3
CH3CO
CONH2
.4
I
Br
Cl
F
.8
1.2
1.6
CH3CONH
-.25
Me
2.0
+p
Et
t-Butyl
OCH3
OH
+s +p
-.50
NMe 2
NH2
-.75
-s -p
-1.0
-
-s +p
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
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
4-OMe
L
M
E
E
4-CH3
L
M
E
M
3,4-Cl2
L
E
4-But
3-Cl
3-Cl
L
E
M
3-CF3-4-Cl
4-CF3
2,4-Cl2
3-NMe2
See Central
Branch
L
2-Cl
4-NMe2
E
M
3-Me-4-NMe2
4-NH2
3-CH3
4-NO2
4-F
3-CF3
4-NO2
3,5-Cl2
3-NO2
M
3-CF3-4-NO2
Topliss Scheme
Rationale
Replace H with
para-Cl (+p and +s)
Act.
+p and/or +s
advantageous
Add second Cl to
increase p and s
further
Little
change
Favourable p
unfavourable s
Replace with Me
(+p and -s)
Further changes suggested based on arguments of p,s and
steric strain
Act.
+p and/or +s
disadvantageous
Replace with OMe
(-p and -s)
Topliss Scheme
Aliphatic substituents
CH3
L
E
H; CH2OCH3 ; CH2SO2CH3
i-Pr
M
Et
L
E
L
M
Cyclopentyl
E
END
CHCl2 ; CF3 ; CH2CF3 ; CH2SCH3
Ph ; CH2Ph
M
Cyclohexyl
Cyclobutyl; cyclopropyl
t-Bu
CH2Ph
CH2CH2Ph
Topliss Scheme
Example
Order of
Synthesis
SO2NH2
R
1
2
3
4
5
R
H
4-Cl
3,4-Cl2
4-Br
4-NO2
Biological
Activity
High
Potency
M
L
E
M
M= More Activity
L= Less Activity
E = Equal Activity
*
Topliss Scheme
Example
R
N
N
N
N
Order of
Synthesis
CH2CH2CO2H
1
2
3
4
5
6
7
8
R
H
4-Cl
4-MeO
3-Cl
3-CF 3
3-Br
3-I
3,5-Cl 2
Biological
Activity
High
Potency
L
L
M
L
M
L
M
M= More Activity
L= Less Activity
E = Equal Activity
*
*
*
Bio-isosteres
NC
Substituent
p
sp
sm
MR
CN
O
C
C
C
CH3
-0.55
0.50
0.38
11.2
O
CH3
0.40
0.84
0.66
21.5
S
CH3
-1.58
0.49
0.52
13.7
O
O
O
S CH3
S NHCH3
C NMe2
O
O
-1.63
0.72
0.60
13.5
-1.82
0.57
0.46
16.9
-1.51
0.36
0.35
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 sp; SOMe and SO2Me are similar in p)
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
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)
Free-Wilson / Hansch Approach
Advantages
•It is possible to use indicator variables as part of a Hansch equation - see following
Case Study
Case Study
QSAR analysis of pyranenamines (SK & F)
(Anti-allergy compounds)
O
OH
OH
X
3
Y
4
NH
O
O
O
5
Z
O
Case Study
OH
X
3
Y
4
NH
O
Stage 1
OH
O
O
5
19 structures were synthesised to study p and s
1 

Log  C  =
- 0.14Sp - 1.35(Ss )2 - 0.72
Sp and Ss = total values for p and s for all substituents
Conclusions:
•Activity drops as p increases
•Hydrophobic substituents are bad for activity - unusual
•Any value of s results in a drop in activity
•Substituents should not be e-donating or e-withdrawing (activity falls if sis +ve or -ve)
Z
Case Study
O
OH
OH
X
3
Y
4
NH
O
O
O
5
Z
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
Case Study
O
OH
OH
X
3
Y
4
NH
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.
Case Study
O
OH
OH
X
3
Y
4
NH
O
O
O
5
Z
Stage 3 Alter the QSAR equation to take account of new results
1
Log  C  = - 0.30Sp - 1.35(Ss )2 + 2.0(F - 5) + 0.39(345-HBD) - 0.63(NHSO 2)
+ 0.78(M- V) + 0.72(4- OCO) - 0.75
Conclusions
(F-5)
Electron-withdrawing group at position 5 increases activity
(based on only 2 compounds though)
(3,4,5-HBD)
HBD 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
Case Study
O
OH
OH
X
3
Y
4
NH
O
O
O
5
Z
Stage 3 Alter the QSAR equation to take account of new results
1
Log  C  = - 0.30Sp - 1.35(Ss )2 + 2.0(F - 5) + 0.39(345-HBD) - 0.63(NHSO 2)
+ 0.78(M- V) + 0.72(4- OCO) - 0.75
Note
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
Case Study
O
OH
OH
X
3
Y
4
NH
O
O
O
5
Z
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
Case Study
O
OH
X
3
Y
4
NH
O
Stage 5
OH
O
O
5
Z
Revise Equation
1
Log  C  = - 0.034(Sp ) 2 - 0.33Sp + 4.3(F - 5) + 1.3 (R - 5) - 1.7(Ss )2 + 0.73(345- HBD)
- 0.86 (HB - INTRA) - 0.69(NHSO 2) + 0.72(4- OCO) - 0.59
NOTES
a) Increasing the hydrophilicity of substituents allows the identification of an
optimum value for p (Sp = -5). The equation is now parabolic (-0.034 (Sp)2)
b) The optimum value of Sp 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
Case Study
Stage 6
Optimum Structure and binding theory
XH
NH
X
O
OH
C
CH
CH2 OH
C
CH
CH2 OH
O
OH
X
3
RHN
5
NH
NH3
X
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 hydrogen 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 (Ss)2 value
3D-QSAR
Notes
•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
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
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
3D-QSAR
Method
•Place the pharmacophore into a lattice of grid points
.
.
.
.
.
Grid points
•Each grid point defines a point in space
3D-QSAR
Method
•Position molecule to match the pharmacophore
.
.
.
.
.
Grid points
•Each grid point defines a point in space
3D-QSAR
Method
•A probe atom is placed at each grid point in turn
.
.
.
Probe atom
.
.
•Probe atom = a proton or sp3 hybridised carbocation
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
3D-QSAR
Method
•The closer the probe atom to the molecule, the higher the steric energy
•Define the shape of the molecule by identifying grid points of equal steric energy (contour
line)
•Favorable electrostatic interactions with the positively charged probe indicate molecular
regions which are negative in nature
•Unfavorable electrostatic interactions with the positively charged probe indicate
molecular regions which are positive in nature
•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
•Identify steric and electrostatic fields which are favorable or unfavorable for activity
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
3D-QSAR
Method
•Define fields using contour maps round a representative molecule
3D-QSAR CASE STUDY
Tacrine
Anticholinesterase used in the treatment of Alzheimer’s disease
NH2
N
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
NH2
R1
7
R2
6
9
N
Substituents: CH3, Cl, NO2, OCH3, NH2, F
(Spread of values with no correlation)
Log
1 
 C  =
1
1
2
pIC 50 = - 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
3D-QSAR CASE STUDY
CoMFA Study
Analysis includes tetracyclic anticholinesterase inhibitors (II)
NH2
R1
8
R3
1
R2
2
N
7
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
3D-QSAR CASE STUDY
Possible Alignment
Good overlay but assumes similar binding modes
Overlay
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) while still bound to the
binding site (in silico)
The complex was minimized to find the most stable binding mode for structure II
The binding mode for (II) proved to be different from tacrine
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
7
6
Blue areas - addition of steric bulk increases activity
Red areas - addition of steric bulk decreases activity
3D-QSAR CASE STUDY
Prediction
6-Bromo analogue of tacrine predicted to be active (pIC50 = 7.40)
Actual pIC50 = 7.18
NH2
Br
N