Transcript Lecture 10, molecular diversity - Cal State LA

Molecular diversity and Drug Design
Molecular diversity: differences in physical properties that exist in
different molecules. (shape, size, polarity, charge, lipophilicity,
polarizability, flexibility).
Access to a diverse set of molecules increases the likelihood
of finding an effective drug.
Sources of Diversity:
Natural products
•Organisms, manipulation of biosynthetic machinery of microorganisms
•Rate of discovery of truly novel natural product drugs has decreased.
Synthetic compounds
•Pharmaceutical companies optimizing their own drugs
•Wide variety of starting materials and reaction types (unlike biosynthesis)
All 10 largest selling drugs in 1994 were small organic molecules
(<700g/mole) 8 were heterocycles and 1 was a natural product.
Challenges for molecular diversity by organic synthesis:
1. Develop a variety of reactions with good yields for a broad
set of starting materials
2. Efficiently identify the synthetic products
Synthetic approaches: traditional (“target-oriented”) method is to
make a desired compound (natural product or drug) using
retrosynthetic analysis to plan the route:
Synthetic approaches:
A. Traditional- (solution phase) - linear or convergent. 25-50
compounds/person/year
Linear:
A
A
B
+ B
D
+
C
A
D
C
B
A
B
C
D
B
Convergent:
C +
A
D
C
D
Traditional- (solid phase). Ex. - Peptide synthesis=optimized amide
bond formation. Plan of synthetic route is simple…Linear polymer!
amino acid
R2
H2N
R1
O
C
H
dipeptide
amino acid
OH
+
H2N
electrophile
O
R2
poor yields
H2N
C
OH
H
Nucleophile
O
R1
H
N
C
H
O
C
H
OH
(and other combinations)
How to solve problem of low yield of desired dipeptide?
•Use protecting groups (“P” below) -so the correct groups react
•Improve reactivity of CO2H (make a better electrophile)
•Use solid phase resin beads which allow
the use of excess reagents (high yield)
improved purification because you can physically separate product
R2
P
H
N
C
H
R2
P
H
N
O
"activation"
Resin bead (insoluble solid)
OH
electrophile
O
C
H
Better electrophile
OR
R1
+ H2N
R2
O
C
H
Good Nucleophile
"coupling"
P
H
N
C
H
O
R1
H
N
C
H
O
Traditional- (solid phase, continued)
Solid phase peptide synthesis (SPPS) details
1. N-protected amino acid (Boc or Fmoc)
is attached to bead by the C-terminus
2. Deprotect N-terminus, rinse with
solvent.
3. Couple: Add solution of activated next
amino acid. Let react, rinse with solvent.
4. Repeat deprotection and coupling for
each subsequent monomer
5. Cleave peptide from resin
Note: synthesize peptide from C to N
terminus
“Linker” attached
to polystyrene
resin bead R
Synthetic approaches (continued):
Parallel synthesis - simultaneous synthesis of multiple products,
each in different reaction vessels with aid of automation. (Increased
productivity 100-fold or more; more efficient).
Each well has one compound, and can be
identified by its position on the grid.
Ex.: Parallel synthesis of a 96-member library of
dipeptides in a microtiter plate with 8 rows and 12
columns. (partially shown)
Step 1: each row starts with a different amino acid
attached to a bead
Step 2: each column adds a different second amino
acid. (results in 96 different dipeptides)
Step 3: Remove dipeptide from the bead
Note: Each well contains one compound
Step 4: Test each for biological activity.
Note: pin and well grid technique also
Synthetic approaches (continued):
Example. Hydantoins can help control epilepsy. Parke-Davis made a
library of 39 hydantoins using parallel synthesis
BOC = protecting group for amines
Synthetic approaches (continued):
B. Combinatorial Synthesis: Multiple reactions in one reaction vessel,
quickly generating a very large set of somewhat diverse products
known as a combinatorial library. Usually the reaction is the same, but
reactants are different. (more than one product in each reaction
vessel/well)
Number of compounds possible: bx (x = # steps)
Design of combinatorial synthesis: sequential (a) or template (b):
Combinatorial Synthesis
Statistical: several reagents react at same time to get all possible
combinations of reactants. (works if reaction rates are equal; hard
to identify products).
Mix and Split synthesis:
1. Synthesize a “mix and split” library
on beads. Each bead has one
compound on it.
2. Identify active compound by
Step 1
combination of analytical techniques
of synthesis
and reaction history (need encoding
molecules or deconvolution).
To create 10,000 compounds: for a 3-step
synthesis, this technique requires 22
different reaction vessels (not 30,000
vessels for standard synthesis!).
Step 2 of
synthesis
Mix and split: Each bead has one compound on it, but each
vessel/well contains a mixture of different beads!
Identification of structure of compound on a bead: Deconvolution
1. ID most active mixture. Recall: in each of the final mixtures (pots) of beads,
the residue coupled last is the same. Here, the pot (B) with “Y” as the last residue
is active
2. Take all saved resin beads from the prior synthetic step (beads attached
to dimers) and couple the appropriate (active) last residue. (Here, Y).
3. ID most active
mixture. Here it is the
one with X as the
second residue.
4. Resynthesize all compounds
with variations in the first residue.
ID active compound.
Mix and split: Disadvantages of Deconvolution
•Deconvolution takes a lot of time and reagents
•Most active mixture identified in round 1 may be active due
to the cumulative action of multiple compounds (not the very
strong action of one very active compound).
Identification of structure of compound on a bead:
Chemical encoding or tagging
For very large libraries in which deconvolution is too difficult…
Building Coding
Block
compound
If you isolate one bead with activity from
that vessel/well, how do you know which
compound it is? Chemical encoding!
X
Y
Z
A
B
C
XYZ
XYZ
CBA
ABC
1. Isolate one bead
XYZ
2. Cleave test compound
frombead
XYZ = test compound
ABC = encoding tag molecule
CBA
ABC
3. Assay
4. If active, analyze bead for
encoding molecule
Example: Chemical encoding or tagging
Coding compound: must be determined easily in small amounts.
(oligonucleotides).
Identification of structure of compound on a bead:
Chemical encoding or tagging
To screen the beads for activity:
The encoded tag is removed, amplified by PCR, and sequenced
to determine the structure of the active compound.
Other methods of encoding:
Peptides (coding for non-peptide and peptide libraries)
Haloaromatic tags, separated by CE
Still’s Binary code; isotopic labeling
Computerized tagging, radiofrequency tags (microchips)
Identification of structure of compound on a bead:
Chemical encoding or tagging
Example of binary encoding: Library of dipeptides from three
amino acids (9 possible) using 4 tags. The tag (or tags) that
uniquely represent the library monomer are reacted with the resin
just before the library monomer is attached.
Ala Phe Leu
Tag for positi on 1 1
2
1+2
Tag for positi on 2 3
4
3+4
Dipeptide
Ala-Ala
Phe-Phe
Leu- Leu
Tag
1,3
2,4
1,2,3,4
Dipeptide
Ala-PHe
Phe-Ala
Leu-Ala
Tag
1,4
2,3
1,2,3
Dipeptide
Ala-Leu
Phe-Leu
Leu- Phe
Tag
1,3,4
2,3,4
1,2,4
n tags will code for 2n-1 library compounds
How does the synthetic approach fit in with drug design?
The traditional (target-oriented) approach : trial and error.
Chemists develop a hypothesis about the structure of a potential drug, synthesize this
substance, and have biological tests conducted. The hypothesis is confirmed or falsified.
In the latter case, the chemist then proposes a new structural hypothesis and synthesizes
a new molecule. Statistically, this cycle has to be repeated an average of 10 000 times
before a new drug is found. A characteristic feature of this approach is that the structure
is known before the test.
The combinatorial principle: trial and selection.
By means of combination (permutation) of the individual components (scaffolds and
building blocks), all possible molecules in a substance family (chemotypes) are
synthesized simultaneously/in parallel. The active representatives are subsequently
selected from this library of compounds by using an assay and their structure is
determined. It follows the principle of evolution, in which the most active
representative is selected from a number of compounds (survival of the fittest).
Role of combinatorial chemistry in drug design:
•Early combichem: peptides (HIV protease inhibitors,
antimicrobial agents, opiate receptor ligands)
•Now: want diverse structures to find a lead - start with scaffolds
that are small and will allow a wide variety of substituents for
determining favorable binding interactions.
Features of orally active drugs:
MW <500g/mole
logP<5
<6 H-bond donating groups
<11 H-bond accepting groups
Potential scaffolds for drug design
Example: Benzodiazepine synthesis
Example: Protease inhibitor libraries:
Target = thermolysin
Pharmacophore:
O
P
R
OR
OH
(incorporated in a peptide)
Trimer peptidyl phosphonates:
540 member library by split pool:
Three side-chains: P1, P1’, P2’
Cbz-X(6)-Y(5)-Z(18)-NH-resin
(“Prot” = protecting group)
Library was assayed for thermolysin inhibition:
Assays revealed:
1. An active compound that
matched the most potent inhibitor
found in literature (KI = 49nM)
(P1= phe; P1’= Leu; P2’ = ala)
2. New active sequences
P2’ = arg, his, and gln (polar,
charged) - unlike any reported in
literature (hydrophobic)
Examining only related analogs
may lead to biases in design. But,
combinatorial chemistry can
reveal new potent structures.
How biologically relevant is the diversity created in chemistry?
Limited “chemical structure space”…
Even the most beautiful molecules synthesized using the most
elegant methods are useless if they do not affect a biological
target.
Theoretically, one could make every possible drug structure, and
you will find at least ONE that works. However, the universe
does not contain enough atoms to synthesize even one copy of
every conceivable molecule!
Possible solution to better match of biological and chemical
structure space: Natural Product-Guided Combichem. Analogs of
known individual natural products.
Example: active metabolite of vitamin D3
Variations:
Synthetic approaches: C. Diversity-oriented synthesis (DOS)
method is to make a large collection of structurally complex and
diverse compounds using forward analysis to plan the route:
Creates natural product-like
molecules that are
stereochemically and skeletally
diverse. (Not available by
conventional combichem).
Synthetic approaches: Diversity-oriented synthesis (cont).
Diversity-Oriented Synthesis-Based Combinatorial Chemistry:
Libraries of Natural Product-like Compounds
Evans asymmetric aldol
Sharpless asymmetric dihydroxylation
O
Furan
Note: furan can
•Ring open (7.6)
•Ring expand (7.7, 7.8)
Note: addition of Br to
furan allows more chemical
additions to the molecule
DOS libraries
•10-100 compounds (not 10,000 as in combichem)
•Mostly cyclic, complex, resemble natural products, lots of stereochem
•few synthetic steps
Potential role of diversity-oriented synthesis and combinatorial
chemistry in more efficient drug discovery:
Small molecules obtained by DOS may find use in “chemical
genetics” (later) as well as drug design.
Alternatives: Other ways to generate potent drugs…
“Click chemistry”
Biological receptor selects the best fitting partial ligands that don’t
fill the binding site from a range of modules that can react with
one another. When the modules bind, they can react and form a
compound to block the entire binding site. Target selects its own
ligand! Angew. Chem. Int. Ed. 2001, 40, 2004 p. 2021
Dynamic combinatorial chemistry
Biological receptor is exposed to a library of potential ligands,
each of which is formed by reversible combinations of small
building block compounds in equilibrium. More of the more
strongly bound ligands will form. Target selects its own ligand!
Dozens of azides and acetylenes were added to the enzyme
(acetylcholinesterase), but only one pair binds and reacts…
Azide = blue
Acetylene = yellow triangle
Proc. Natl. Acad. Sci. USA, 101, 1449 (2004)].
References for Diversity in Drug Design.
Patrick, G. L. An Introduction to Medicinal Chemistry; Oxford University Press: New York, NY, 2001
Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action ; Academic Press: San Diego,
CA, 1992.
Thomas, G. Medicinal Chemistry An Introduction; John Wiley and Sons, Ltd.: New York, NY, 2000.
Combinatorial Chemistry and Molecular Diversity in Drug Discovery; Gordon, E. M.; Kerwin, J. F., Eds.;
Wiley-Liss: New York, NY, 1998
Terrett, N. K. Combinatorial Chemistry; Oxford Chemistry Masters; Oxford University Press: New York,
NY, 1998.
Arya, P.; Joseph R.; Gan,Z.; Rakic, B. “Exploring New Chemical Space by Stereocontrolled DiversityOriented Synthesis” Chemistry & Biology, 2005, 12, 163–180.
Burke, M. D.; Schreiber, S. L. “A planning strategy for diversity-oriented synthesis” Angew. Chem. Int.
Ed. 2004, 43, 46–58.
Schreiber, S. L. “Target-oriented and diversity-oriented organic synthesis in drug discovery” Science, 2000,
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Chem. 198 7, 30, 7A± 8A; d) G. deStevens, J. Med. Chem. 19 91, 34,
26 65 ± 267 0.