Advanced Medicinal
Chemistry
Barrie Martin
AstraZeneca R&D Charnwood
Lecture 3:
Molecular Interactions and
Drug Potency
Dose-Response Curves
Enzyme Inhibitors (competitive):
Measure inhibition at differing
concentrations of ‘drug’.
10nM 30nM 100nM 300nM 1mM
%
Inhibition
0
50
100
IC50=85nM
[Inhibitor]
IC50 - The inhibitor concentration that
causes a 50% reduction in intrinsic enzyme
activity
pIC50 = - log10(IC50)
IC50 1mM = pIC50 6.0
IC50 1nM = pIC50 9.0
Agonists: Measure % Response vs Agonist concentration
EC50 - The agonist concentration that causes 50% of the maximum response. pEC50 = - log10(EC50)
For a drug, typically target affinity values of pIC50  8 (<10 nM concentration)
[Agonist]
%
Response
EC50=85nM
Antagonists: Situation more complex. Antagonists displace the agonist dose-response curve
rightwards – most accurate measure of potency (pA2) requires measurement of agonist binding at
multiple concentrations of antagonist
iNOS - An AZ Charnwood Discovery Project
Active Site,
Haem & Inhibitor
NH2
NH
NH2
NH
O
OH
NH2
NH
NH2
O
O
OH
iNOS
+ NO
Nitric Oxide Synthases – catalyse production of
NO from arginine in the body – implicated in
inflammatory conditions e.g. rheumatoid arthritis
N
N
N
O
N
N
NH2
F
F
AZ10896372
pIC50 7.5
A potent, selective iNOS inhibitor
How Do Drugs Bind to Enzymes & Receptors?
Drugs bind to particular sites on enzymes and receptors. In the case of an enzyme,
this will often be the active site. Receptors have binding pockets formed between
transmembrane helixes where drugs usually bind (not always the agonist’s binding site).
These sites are comprised of a variety of amino
acid residues which give rise to a specific 3-D
shape and molecular features:
• Charges: CO2
- , NH3
+, =NH-+
• Polar groups: OH, C=O, CONH
• Hydrophobic groups: Ph, Alkyl, SMe
In enzymes, reaction centres are also present:
• Asp-His-Ser in esterases
• SH in some proteases
• Metal ions (CYP-450, iNOS).
N
H
O
N
H
N
H
O
O
O O
O
SER S
GLU E PHE F
N N
NN
Fe
CO2H
HO2C
Haem group – iNOS, CYP-450
Small molecules bind to these pockets by a combination of:
• Shape complementarity
• Energetically favourable interactions
Shape Complementarity
NH
N
S
N
H
N
H
CN
NH
N
NH2
Cimetidine
Histamine
H2 Receptor AntagonistiNOS Enzyme Inhibitor
AZ10896372
Arginine
N
N
+
N
H
NH2
F
F
H
O
N
N
NH2 N
+
NH2
H
NH3+
O
O
The drug must fit into the Binding Site and shape complementarity is an important feature of a
drug molecule. Competitive enzyme inhibitors often bear a resemblance to the substrate, as they
bind to the same Active Site. This is also true for some receptor antagonists, but not all.
The strength of an interaction depends on the complementarity of the physico-chemical properties
of atoms that bind, i.e. protein surface and ligand structure.
The ‘Binding Sites’ are not totally rigid. The side chains of the amino acids that make up the pocket
have some mobility. A variety of related structures can thus be accommodated by movements that
change the shape of the active site. This is known as the ‘Induced Fit Hypothesis’.
Drug-Protein Binding Energies
K = [P:D]
[P] x [D]
Both Enthalpy (DH) and Entropy (DS) changes affect binding strength
DG=-RTlnK and DG=DH-TDS
Gibbs Free Energy Changes
[Protein] + [Drug] [P:D]
K
For a binding Equilibrium between a Protein & a Drug
DG
Drug ProteinDrugProtein
NB. When a drug moves from the aqueous medium into the ‘Binding Site’ it has to
break H-Bonds with water, de-solvate etc. These processes require energy, so the
net energy available for binding is only a fraction of the above bond energies.
Drug-Protein Interactions
Bond Example kJ/mol
Van der Waal Xe…Xe, alkyl groups 2
Hydrophobic Ph…Ph (p-stacking) 5
Dipole - Dipole C=O…HN-R (d+/d-)...(d+/d-) 5
Hydrogen H2O…H2O (X-H) …(Y-R) 35
Ion - Dipole F-…H2O (+/-ve)…(d+/d-) 170
Ion - Ion H+…Cl- (+ve)…(-ve) 450
Covalent C-O 350
Electrostatic Interactions
• These result from the attraction between molecules bearing opposite
electronic charges.
• Strong ionic interactions can contribute very strongly to binding.
• Proteins contain both CO2
- and NH3
+ residues and these may be present
at the binding site to interact with oppositely charged groups on the drug.
Neuraminidase Inhibitor (Antiviral GSK)
O
O
OOH
OH
OH
R
R
N
N
N
H
H
H
H H
ARG
-
+
AZ-10896372 iNOS Inhibitor
N
N
N
H
O
N
N
F
H
+
N
H H
O
O
GLU
F
• The energies involved in a ‘salt bridge’ can be in the order of >30 kJ/mol
• This can lead to increase in observed binding of >106 fold
Hydrogen Bonding Interactions
A hydrogen bond results when a hydrogen is shared between two electronegative atoms
The Donor provides the H, while the Acceptor provides an electron pair
D-X-H….Y-A e.g. R-O-H…..O=C
N
N
N
O
N
N
F
H
+
N
H H
O
H
N
H
O
N
H
H
O
H
H
O
OH
OOH
O
O
R
R
H
HO
O
GLU
AZ10896372 - iNOS complex
Amide to Tyrosine H-Bond
Neuraminidase Inhibitor
Charge re-inforced H-Bond
Hydrophobic Interactions
• Drugs, in general, are hydrophobic molecules
• The ‘Binding Sites’ of proteins are also hydrophobic in character
• Thus a mutual attraction can result (like attracts like).
• What drives this attraction?
• Each -(CH2)- group can contribute >1 kJ/mol towards binding
• Each -Ph ring can contribute >2 kJ/mol towards binding
• These effects are additive and hence Hydrophobic Bonding
can make a very high contribution to binding
• Entropy gains are achieved when water molecules are displaced
from ‘active site’, and return to a more random (high S) state.
• Enthalpy gains may result from van der Waals bonding:
• Between Alkyl, Aryl, Halogen groups
• p-p Stacking is an important type of this
Hydrophobic Bonding : D Entropy
Water molecules are
in a highly disordered
state. Each molecule
maximises H-Bonds
to other molecules
of water.
When a hydrophobic drug
is placed into water, the
structure of the water around
the drug is more ordered.
This allows the H2O-H2O
H-bonds to be maintained.
This leads to lower entropy
and is not favoured.
Hydrophobic Bonding : D Entropy
D E
• Hydrophobic interaction between protein and drug is favoured by entropy gains:
• Bulk water returns to less ordered state
• Water molecules may be expelled from being bound in active site.
• In addition enthalpy gains due to new bonds may also be favourable
(e.g. van der Waals interactions)
DE
Probing Hydrophobicity
in Drug Discovery
NH
N
H
NH2
R
F
F
New iNOS lead identified: R =Me, small lipophilic substituent iNOS pIC50 7.8
Aim: Probe lipophilic pocket – what else could we put there?
How would we make it?
NH2
NH2
NHF
F
R
O
Effect of Hydrophobicity on Activity
Binding into Lipophilic pocket of iNOS
NH
N
H
NH2
R
F
F R cLogP IC50 mM
Me 1.13 0.016
Et 1.66 0.009
CF3 1.75 0.008
Thiophene 2.02 0.003
Phenyl 2.34 0.015
2-Me-Thiophene 2.48 0.026
cLogP
7.6
7.8
8
8.2
8.4
8.6
1
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6
iNOS_pIC50
Too big to fit in pocket optimally
(Shape complementarity)
Bioisosteres
Isostere:
Similarities in physicochemical props. of atoms/groups/molecules with similar electronic structures
(no. and arrangement of electrons in outermost shell). Often observed with groups in the same
periodic table column (Cl  Br, C  Si).
Grimm – Hydride Displacement Law (1925) - Replacement of
chemical groups by shifting one column to the right & adding H.
Bioisostere:
Simplest definition - any group replacement which improves the molecule in some way
Two different interchangeable functionalities which retain biological activity.
Bioisosteric replacements can offer improvements both in potency and other properties (e.g.
metabolic stability, absorption)
O
O N
N
NN N
O
S O
O
--
Carboxylic acid & bioisosteres
O S
O
N
H
N
H
N
N
S
N
H
O O
-CH2 & bioisosteres
amide & bioisosteres
C N O F Ne Na+
CH NH OH FH
CH2 NH2 OH2 FH2
+
CH3 NH3 NH4+
Invisible Bioisosteres
N
N
MeO
MeO
NH Br
N
MeO
MeO
NH Br
N
EGF-R 2.2 nM EGF-R 7.5 nM
N
MeO
MeO
NH
Br
N
H
O
Me
NH
O
N
N
HMeO
MeO
NH
Br
H O
H
H O
Me NH
O
H-bonds can be directly to protein or via water molecules
Optimising Potency
N
N
N
NH2
F
F
O
N
N
Develop understanding of which molecular features are
important for activity – remove substituents.
Look at incorporating new groups for additional potency e.g.
through lipophilic interactions, hydrogen bonds etc.
Functional group bioisosteres.
Use available structural information – e.g. crystal structures of
compound bound to enzyme.
Use of modelling to design/evaluate new targets.
Develop and test hypotheses.
Identify good disconnections/robust chemistry to allow rapid
synthesis of multiple analogues – build up information.
pIC50 7.5
N.B. Potency is one of many properties that needs to be optimised in drug
discovery - need to consider absorption, metabolism, selectivity etc.
NH
N
N
NH2
F
F
OH
O
N
R
OH
O
Ar
+ or
How might we improve potency further from this compound?
NH2
F
F NH2
NH
N
O
O
OEt
NN
H
F
F NH2
NH
OH
OEt
O
N
+
F
F NH
NH2
N
OEt
O
N
N
F
F NH
NH
OEt
O
N
N
F
F NH2
N
OEt
O
Tautomerism
Forward Synthesis - 1
NH2
F
F
N
NH2
F
F NH
NH
OH
NH2
F
F NH2
NH
N
O
O
OEt N
N
H
F
F NH2
N
O
OEt
i, NH2OH, NaOMe,
methanol, reflux
ii, H2, Raney Ni,
ethanol, 60C
iii, ethanol,
reflux
Forward Synthesis - 2
N
N
H
F
F NH2
N
O
OEt
NH
N
H
F
F NH2
N
N
N
H
F
F NH2
N
O
N
N
OH
O
N
N
iv, NaOH, H2O,
EtOH, D
v, (COCl)2, CH2Cl2,
then amine, NEt3, CH2Cl2