Case Study 2 - Benzodiazepines and Combinatorial Chemistry Flashcards
Ring A and drawing
Ring A involved in pie-pie stacking in the receptor protein
Exchanging ring A for a heterocyclic ring leads to lower anxiolytic activity
Ring B drawing
slide 5
Ring C and drawing
• Ring C may contribute to steric or hydrophobic interactions
What Is Combinatorial Chemistry?
- A process where groups of starting materials are reacted together to make every available product
- Mixtures of products are obtained – a library
- Useful for making very diverse mixtures of compounds with drug-like properties for screening
- One set of starting materials may be fixed on a solid support – solid-phase synthesis
- Can be totally automated, increase productivity
- Introduces diversity in lead compound structure
- Rational versus Irrational drug design
What Is A Compound Library?
- A collection of compounds prepared for testing
- A library contains very little of each compound
- Several types of library can be prepared:
- Virtual: in silico
- General: no bias for a target
- Discovery: a library with drug-like features
- Focused: designed to inhibit a target family
- Targeted: designed to inhibit a specific target
Orthodox synthesis:
A+B –> AB
Making a Simple Library
- Parallel synthesis, spatially addressable
* 96- or 384-well plates, ready for HTS
‘Mix And Split’ Library Synthesis
- Substrates are attached to a solid support
- Reactions are driven to completion by using excess reagent
- Screening issues
- Positive results must be deconvoluted
Solid Phase Organic Chemistry 1 - methodology
- The development of automated solid-supported peptide synthesis revolutionised peptide research
- The automated methodology has been adapted to make many peptides in parallel
- All of the chemistry required has been worked out so combinatorial synthesis of peptides is easy
- The methodology can also be applied to the synthesis of any small-molecule library using combinatorial chemistry on a solid support
Solid Phase Organic Chemistry
• Wang linker
- Suitable for a base-labile protecting group strategy
- Requires 1% - 95% TFA for cleavage
- Reveals CO2H
Screening Compound Libraries
- Screen the library in solution
- Must cleave all compounds from the support
- Possibility of false or misleading hits
- Must resynthesize all compounds in an active library
- Screen individual compounds in solution
- Separate the beads manually
- Requires a method for identifying active structures
- Screen compounds still attached to the support
- Colorimetric/ fluorimetric assay
- Support can restrict the conformation of the product
Hit Structure Determination
- The active hit must be prepared on a larger scale once activity has been found
- Structure determination can be the rate-limiting step
- Ideally, the structure of the hit would be determined whilst the compound is still anchored to the solid support
- Nanomolar quantities of the hit can not be characterised easily using spectroscopy so some other methodology, chemical or otherwise, is required to trace the hit structure
Matrix Metalloproteinase (MMP) Enzymes
- A family of zinc-dependant enzymes
- Degradation and remodeling of the extracellular matrix
- Important therapeutic targets with indications in cancer, arthritis, autoimmunity, and cardiovascular disease
- Selectivity is important for therapy
- Diketopiperazine (DKP) enzyme inhibitors chosen as lead compounds following de novo drug design
- Solid-phase combinatorial synthesis chosen as a route to develop a diverse array of analogues for screening
Benzodiazepines
Class A
Class B
Benzodiazepines Class A
Clonazepam Diazepam
Lorazepam
Oxazepam Temazepam
Benzodiazepines Class B
Alprazolam
Estazolam
Midazolam
Triazolam
Clonazepam
Class A R1 - H R3 - H R7 - NO2 X - Cl
Diazepam
Class A R1 - CH3 R3 - H R7 - Cl X - H
Lorazepam
Class A R1 - H R3 - OH R7 - Cl X - Cl
Oxazepam
Class A R1 - H R3 - OH R7 - Cl X - H
Temazepam
Class A R1 - CH3 R3 - OH R7 - Cl X - H
Alprazolam
Class B
R - CH3
X - H
Y - N
Estazolam
Class B
R - H
X - H
Y - N
Midazolam
Class B
R - CH3
X - F
Y - CH
Triazolam
Class B
R - CH3
X - Cl
Y - N
Ring A - position 7
An electronegative group in position 7 markedly increases the functional anxiolytic activity
Ring A - position 6,8,9
Substituents in 6, 8 or 9 positions decrease anxiolytic activity
Ring B - position 1
• Alkylation of the amide nitrogen in position 1 with sterically undemanding groups does not affect activity significantly
Ring B - position 2
- A proton accepting group in position 2 of Ring B is essential, coplanar with Ring A
- Substituting sulfur or oxygen in position 2 does not affect anxiolytic activity significantly
Ring B - position 3
- Substitution on the methylene group at position 3 has no effect on anxiolytic (agonist) activity but decreases antagonist activity
- The stereochemistry of hydroxylation at position 3 does not affect activity
- The stereochemistry at position 3 may be important for steric interactions at the receptor with one enantiomer being favoured
Ring B - position 4,5
• The 4-5 C=N bond is essential for in vivo activity
Ring C - position 2
Substitution at position 2’ is not detrimental to agonist activity
Ring C - position 4
Substitution at position 4’ is unfavourable due to steric interactions
Oxazepam metabolism S
Oxazepam S –> (UGT2B15) S-oxazepam glucuronide
70 - 85%
Oxazepam metabolism R
Oxazepam R –> (UGT1A9, UGT2B7) R-oxazepam glucuronide
15-30%
Metabolism of Alprazolam
CY3A5, CYP3A4 –> Hydroxylation –> Glucuronidation –> Elimination
Metabolism of Triazolam
CY3A5, CYP3A4 –> Hydroxylation –> Glucuronidation –> Elimination
Metabolism of Midazolam
CYP3A4 –> Hydroxylation –> UGT1A4, UGT2B7 UGT2B4 –> Glucuronidation –> Elimination
UGT1A4 –> Glucuronidation –> Elimination
Metabolism of Flurazepam
Hydroxylation/ alkylation –> Glucuronidation –> Elimination
Metabolism of Bromazepam
CYP1A2, CYP2D6 –> Hydroxylation –> Glucuronidation –> Elimination
Metabolism of Lorazepam
UGT2B15 –> Glucuronidation –> Elimination
Metabolism of Clonazepam
NAT2 –> Acetylation –> Elimination
Metabolism of Diazepam
CYP2C19 –> Nordazepam
CYP3A4 –> Tempazepam –>
Metabolism of Nordazepam
CYP3A4 –> S-oxazepam
Metabolism of Temazepam
CYP2C19 –> R-oxazepam
difference in oxazepam and diazepam
oxazepam has an extra OH next C=O
difference in flurazepam and quazepam
quazepam has C=S instead of C=O in flurazepam
quazepam has N-CF3 instead of N-CH2-CH2-N in flurazepam
difference in estazolam, triazolam, temazepam
triazolam has extra CH3 bound to N ring than estazolam
temazepam has an extra C=O and -OH bound to N ring than triazolam
Solid Phase Organic Chemistry 2 - each substrate
- Each substrate is attached to a polymer bead
- All reactions are driven to completion by using an excess of reagent
- The final products are separated easily from the reaction mixture by filtration
- By-products and excess reagents can be washed off the solid support, leaving pure product to be cleaved from the bead
Solid Phase Organic Chemistry 3 - polymeric support
- The polymeric support must be insoluble and inert to the reaction conditions employed
- The means of linking the substrate to the solid support must allow efficient and selective cleavage of some or the entire quantity of product from the solid support
- A protecting group strategy must be used which allows selective protection and deprotection of all reactive groups in the building blocks to be used and the supported substrate
Solid Phase Organic Chemistry 4 - most widely used
- The most widely used solid supports are cross-linked polystyrene beads
- The local environment around the polystyrene bead can limit the range of possible reactions
- Sheppard’s resin is a polyamide and provides a more suitable environment for peptide synthesis
- Tentagel resin has polyethylene glycol residues tethered to polystyrene beads
- The surface environment of Tentagel beads is hydrophilic but also like a polar organic solvent
Solid Phase Organic Chemistry 5 - linker group
- The linker group provides a means by which the substrate is attached to the polymer bead
- The linker dictates what chemistry the growing substrate will withstand, the conditions required to release the library from the solid phase and what functionality will be revealed
- Merrifield resin, developed for peptides: requires HF to cleave the product from the bead, reveals CO2H
Fluoroquinolone Antibiotics
Ciprofloxacin
Levofloxacin
Gatifloxacin
Moxifloxacin
Fluoroquinolone Synthesis
Diagram - slide 27
SPOS Fluoroquinolone Synthesis
Diagram - slide 30
Library DKP-1
L-cysteine gives 19 x 18 = 342 compounds
D-cysteine gives 19 x 18 = 342 compounds
Library DKP-1: Total of 684 compounds in pools of 19 DKPs
Library DKP-2
L-cysteine gives 19 x 18 = 342 compounds
D-cysteine gives 19 x 18 = 342 compounds
Library DKP-2: Total of 684 compounds in pools of 19 DKPs
Most selective inhibitor for Collagenase 1
R1 : PhCH2
R2 : p-CH3OC6H4
Least selective inhibitor
R1 : n-Butyl
R2 : C6H11
Best candidate for optimisation for Gelatinase B Inhibitor
R1 : C6H11CH2
R2 : p-CH3OC6H4
