Advanced Medicinal Chemistry Lecture 3:

Molecular Interactions and Drug Potency

Barrie Martin AstraZeneca R&D Charnwood

100 50 0

Dose-Response Curves

Enzyme Inhibitors (competitive): Measure inhibition at differing concentrations of ‘drug’.

IC 50 - The inhibitor concentration that causes a 50% reduction in intrinsic enzyme activity IC 50 =85nM 10nM 30nM 100nM 300nM 1

m

M pIC 50 = - log 10 (IC 50 ) IC 50 1

m

M = pIC 50 6.0

IC 50 1nM = pIC 50 9.0

Agonists: Measure EC 50 % Response vs Agonist concentration - The agonist concentration that causes 50% of the maximum response. pEC 50 = - log 10 (EC 50 ) Antagonists: Situation more complex. Antagonists displace the agonist dose-response curve rightwards – most accurate measure of potency (pA 2 ) requires measurement of agonist binding at multiple concentrations of antagonist For a drug, typically target affinity values of pIC 50



8 (<10 nM concentration)

iNOS - An AZ Charnwood Discovery Project

Active Site, Haem & Inhibitor H 2 N H N NH NH 2 O OH O NH NH 2 iNOS H 2 N O OH + NO Nitric Oxide Synthases – catalyse production of NO from arginine in the body – implicated in inflammatory conditions e.g. rheumatoid arthritis AZ10896372 pIC 50 7.5

A potent, selective iNOS inhibitor F F N N NH 2 N O N N

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: CO 2 -

•

Polar groups: , NH 3 + , =NH OH, C=O, CONH +

•

Hydrophobic groups: Ph, Alkyl, SMe

N H

GLU E

O O O O H N N H O

SER S PHE F

O

In enzymes, reaction centres are also present:

•

Asp His Ser in esterases

• •

SH in some proteases Metal ions (CYP-450, iNOS).

N N N Fe N

Small molecules bind to these pockets by a combination of:

•

Shape complementarity

•

Energetically favourable interactions

CO 2 H HO 2 C

Haem group – iNOS, CYP-450

Shape Complementarity

iNOS Enzyme Inhibitor AZ10896372 Arginine O F F H N N + H NH 2 N NH 3 + O N H 2 N N + H NH 2 O N H 2 Receptor Antagonist H N H N S H N N CN H N N NH 2 Cimetidine Histamine 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

For a binding Equilibrium between a P rotein & a D rug K [ P rotein] + [ D rug] [ P : D ]

D

G Protein Drug Drug Protein K = [ [ P P : D ] x [ ] D ] Gibbs Free Energy Changes

D

G=-RTlnK and

D

G=

D

H -T

D

S Both Enthalpy (

D

H) and Entropy (

D

S) changes affect binding strength

Bond

Van der Waal Hydrophobic Dipole - Dipole Hydrogen Ion - Dipole Ion - Ion Covalent

Drug-Protein Interactions

Example kJ/mol

Xe…Xe, alkyl groups Ph…Ph (

p

-stacking) 2 5 C=O…HN-R (

d

+/

d

-)...(

d

+/

d

-) 5 H 2 O…H 2 O ( X H) …(Y-R) F …H 2 O (+/ ve)…(

d

+/

d

-) H + …Cl (+ve)…(-ve) 35 170 450 C-O 350 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.

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 CO 2 and NH 3 + residues and these may be present at the binding site to interact with oppositely charged groups on the drug.

AZ-10896372 iNOS Inhibitor O F H N N F N H + H N H O O GLU N N Neuraminidase Inhibitor (Antiviral GSK) H O OH O OH R R O O H H N + N H H N H ARG

• •

The energies involved in a ‘salt bridge’ can be in the order of This can lead to increase in observed binding of >10 6 fold >30 kJ/mol

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

O N H H N OH O H O O GLU H O R O R O OH H H O H N O H O N N N H + F H N H AZ10896372 - iNOS complex Amide to Tyrosine H-Bond N 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?

•

Enthalpy gains may result from van der Waals bonding:

• •

Between Alkyl, Aryl, Halogen groups

p-p

Stacking is an important type of this

•

Entropy gains are achieved when water molecules are displaced from ‘active site’, and return to a more random (high S) state.

• • •

Each -(CH 2 )- 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

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 H 2 O-H 2 O H-bonds to be maintained. This leads to lower entropy and is not favoured.

Hydrophobic Bonding :

D

Entropy

D E E D •

Hydrophobic interaction between protein and drug is favoured by

•

Bulk water returns to less ordered state

•

entropy gains 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)

Probing Hydrophobicity in Drug Discovery

F H N NH R F NH 2 New iNOS lead identified: R =Me, small lipophilic substituent iNOS pIC 50 7.8

Aim: Probe lipophilic pocket – what else could we put there? How would we make it? F F NH 2 NH 2 NH O R

Effect of Hydrophobicity on Activity Binding into Lipophilic pocket of iNOS

F F H N NH NH 2 R R Me Et CF 3 Thiophene Phenyl 2-Me-Thiophene cLogP 1.13

1.66

1.75

2.02

2.34

2.48

IC 50

m

M 0.016

0.009

0.008

0.003

0.015

0.026

8.6

8.4

iNOS_pIC 50 8.2

8 7.8

7.6

1

1.2

1.4

1.6

1.8

cLogP 2 2.2

2.4

2.6

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.

C N O CH NH F OH Ne FH CH 2 NH 2 OH 2 Na + FH 2 + CH 3 NH 3 NH 4 +

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 N N N S O O O Carboxylic acid & bioisosteres O -CH 2 & bioisosteres O N H O O S N H amide & bioisosteres S N N H N

Invisible Bioisosteres

H N MeO N MeO N EGF-R 2.2 nM Br H N MeO MeO N EGF-R 7.5 nM N Br MeO MeO Br Me NH H N H O H O H N NH O MeO MeO Br NH Me N H O H N O N H-bonds can be directly to protein or via water molecules

Optimising Potency

F F N NH 2 N

How might we improve potency further from this compound?

N O

pIC 50 7.5

N N F

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.

O NH O N N + H O R N or H O Ar F NH 2

N.B. Potency is one of many properties that needs to be optimised in drug discovery - need to consider absorption, metabolism, selectivity etc.

F F F F NH 2 N i, NH 2 OH, NaOMe, methanol, reflux

Forward Synthesis - 1

F NH 2 NH F H N OH ii, H 2 , Raney Ni, ethanol, 60C F F O N NH 2 NH O NH 2 iii, ethanol, reflux OEt O N OEt NH 2 NH O NH 2 F F OH H N NH NH 2 N O OEt F F F F H N N NH 2 N O N + NH NH 2 N OEt O OEt F F N N NH 2 O N OEt Tautomerism F F O N NH NH N OEt

Forward Synthesis - 2

F F O H N NH 2 N N OEt iv, NaOH, H 2 O, EtOH, D F F H N N NH 2 O H O N NH N v, (COCl) 2 , CH 2 Cl 2 , then amine, NEt 3 , CH 2 Cl 2 F F H N N NH 2 N O N N