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Combinatorial chemistry
1.
2.
3. 1) In 1960-combinatorial chemistry was
introduced.
2) The drug discovery process become highly
parallel.
3) Even thousands of structures could be
synthesised one at a time.
4) Interestingly biologist introduced HTS to
perform invitro assays.
In 1963,combinatorial synthesis of peptide on resin bead was done by Bruce
Merrifield.
It is a tool which allows large no of compounds to be synthesized simultaneously in a
time taken to prepare only handful of compounds by traditional synthesis.
A chosen set of building blocks are reacted together to make a collection of products
known as “library” or “an array”.
4. A1 B1
A2 B2
An Bn
• The structure of the compounds in the mixture are not known with certainty
• They are not separated instead each mixture is tested for biological activity as a
whole & if active the mixture is analyzed for active compound.
8. 1. In this method ,the reaction is carried out on a solid
support such as resin beads.
2. The solid phase synthesis was pioneered by
Merrifield –synthesis of peptides.
The bead is treated with different starting materials
which bound together.
Then it is mixed with another reagent to get the
product which is bound to solid support.
The excess reagent or by product can be easily
removed by washing with appropriate solvent.
9. A cross linked insoluble polymeric
support-resin.
An anchor or linker covalently linked
to resin.
A bond linking the substrate to linker
which will be stable to reaction
condition.
Chemical protecting groups for
protecting the functional group not
involved in the synthesis.
12. The linker is the molecule that site between the
compound and the support.
The linker moves the point of attachment of
substrate away from the surface of the bead.
Different linkers are used depending on the
functional group(which is present on the
substrate)
functional group(which is desired on the final
product once it is released)
13. WANG RESIN: linker suitable
RESIN LINKER F for attachment & release of
carboxylic acids.
OH
O
RESIN LINKER F MERRIFIELD RESIN : linker
suitable for peptide products.
O
O
RINK RESIN : Linker suitable
RESIN LINKER F for attachment & release
of carboxamide.
NH2
O
OMe
MeO
14. Protecting groups are important for blocking
& regenerating certain functional groups in a
reaction sequence.
Some example of protecting groups are
FMOC(fluoro methoxy carbonyl)
TBOC(tertiary butyloxy carbonyl)
15. R2
HO [BOC]
N
O H O R2
O H
NH 2 N [BOC]
N
H
R1 R1 O
peptide
F3C-COOH
N-Et3
O R2
H
N
[REPEAT] NH 2
R1 O
16. Here the compounds are synthesised in separate vessels but at
the same time parallel.
The array of reactions are taken either in
grid well in a plastic plate(in bead method)
or
pins(grid of plastic rods) called crowns.
The building blocks are attached to these beads or crowns.
The structure of product is identified from the grid code.
17. A bath of resin is divided in to equal portion in reaction
vessel.
Each portion of resin is treated with treated with
different derivative of first block(A,B & C).
After washing the beads are pooled together in one pot
& mixed thoroughly.
Then split in to equal portion again for coupling to the
next portion.
This process is continued until the required library is
synthesised.
18. A B C
A A A
B B B
C C C
A A B A A
B B B B
C B C C
23. The reaction vessel consists of brush like
array of pins, at the end of it consists of bead
(lollypop) with suitable linker. Here the
synthesis takes place.
It is inserted in to the plates where the
reagents & the solvents kept ,and continously
changed.
24.
25.
26. Spatial array of microchips , which is
embedded with resin beads
27.
28.
29.
30. Unlike one bead - one compound synthesis
,solution phase synthesis often lead to mixture of
products in one pool.
Most of the org reaction occurs in solution phase .
For this reason there has been much interest in
solution phase synthesis.
The main problem here is the difficulty of removing
unwanted impurities at each step in synthesis.
31.
32.
33. R-CO-Cl + RI-NH2 R-CO-NH-RI + HCl
Acid chloride based set:-
A1 + (B1,B2,B3…………B10) mixture1 containing all possible A1-B compounds
A2 + (B1,B2,B3…………B10) mixture2 containing all possible A2-B compounds
A10+(B1,B2,B3…….B10) mixture10 containing all possible A10-B compounds
Amine based set:-
B1 + (A1,A2,A3…………A10) mixture1 containing all possible B1-A compounds
B2 + (A1,A2,A3…………A10) mixture2 containing all possible B2-A compounds
B10+(A1,A2,A3…….A10) mixture10 containing all possible B10-Acompounds
34. R1 COOH R2 NH2 R3 CHO NC
O R3 H
N
R1 N
R2 O
OH O R3 R5 R4 R2
OR OH N R3
O R1 N N R3
OR R1
R2 N
R1 R2 O R2 O
35. POOL
A subset of a given combinatorial library.
The process of combining & mixing library
component.
BUILDING BLOCKS: One of a set of
interchangable reagents that can be used in
synthesis of library.
DECONVOLUTION
Isolation & identification of the most active
compound in a mixture is known as
deconvolution.
37. It is the process as a result of which the combinatorial
exp becomes less complex.
It is usually done by backtracking & reanalysing or
resynthesising a subset of structures in the library.
The goal of deconvolution is to determine which of
the mixture of compounds is actually responsible for
the activity.
Eg: micromanipulation & sequential release.
38. The specific quantity of beads are allocated
for each possible structure in library.ie beads
contains only molecules of the library
member. They may be tagged.
The advantage of this is simplicity of
analysis & screening.
Advances in robotics & automation reduces
problems in this strategy.
39. This is same as mix and split technique.
At each gp of beads bearing a variety of
compounds,but a given structure only appears in one
group.
In this a small library of structures selected &
screened.
We find activity in the middle gp of beads ie
BAC,BBC,BCC….This tells us that in position ‘1’,B is
essential for the activity.
The final step is to synthesise this beaded
compounds,keeping then separate & screened each &
find out ‘BAC’ IS THE ACTIVE ONE
40. This is similar to iterative deconvolution but
uses negative logic ie eliminate a functional
group if activity is absent.
Thus functional group that is missing must be
needed for activity.
It is useful for “QSAR” studies.
41. Example:chlorine gp is placed in several position of
phenyl ring.
1) The entire library is screened to get baseline
activity level.
2) If activity is detected,a set of sub library is
prepared,each with subtraction of functional
group & screened to identify most important
functional group.
3) The reduced library contains only these
functional gp,these are screened to get the
active compound.
42. The term “orthogonal” means perpendicular
In this type of pooling ,we distribute the functional
groups to be considered in to a set of sub
libraries, A,B,C…etc. which can contain mixture of
same compounds also.
But the functional groups distributed such that any
subset in A & B shares only one functional gp.
eg: if we have a very small library of structures-
aa,ab,ac……
43. A B C
(aa,ab) (aa,ac) (ab,ac)
Shows the pharmacological activity
‘ab’ is the active compound
44. This is a non iterative screening in which a sub set
library is created with a single building block fixed at one
position & all other building blocks in other position.
Here by selecting the functional group from the most
active subset at each position ,the most active
compound is over all discovered.
45. Encoding/tagging used as a code to indicate what
happened at each step in the synthesis ,thus identify
the structure of most active library member.
TAGGING
LASER
CHEMICAL ISOTOPIC DYE
R.F.TAGGING OPTICAL
TAGGING TAGGING
ENCODING
46. Here specific compounds (tags) are used as a code for
the individual step in synthesis.
Eg :ss DNA(oligonucleotide) 6 bases are used for DNA
generic code .For decoding, DNA tag is amplified by
PCR.
--A—B—C—B—C--etc Library compound
--R—S—T—S—T--etc Code compound
49. A tiny micro chip is added to resin/to solution.
As the various reactions are conducted to
generate the product,at each step a rf signal is
stored in microchip.
This signal can be recalled to identify the
sequence of reaction that generated the product.
50. The tags are encapsulated in glass casing which is stable to chemical &
synthetic conditions.
Each tag code is associated with the identity of its library member &
detected by data base computer.
51. Here the solid support acts as an eye to view the reaction
52. The solid support consists of a ceramic chip
covered with a polypropylene-poly styrene
polymer in which barcode pattern is burned at
each step & is decoded visually with the use of
a microscope.
55. 1) Chemically cleave the compounds from the support & filter
off the beads
2) The filter the solution to get the product.
3) If the solution contains just a single compound we use
• IR
• UV
• MASS
SPECTROPHOTOMETER
• FLUORESCENCE
• NMR
If it contains a mixture we use HPLC
56. HPLC is highly effective method for separation & detection of components
57.
58.
59.
60. •IR light is transparent to resin beads ,hence we can analyse
the resin bead directly without cleaving the product from it.
•FTIR will amplify the very small spectral signal from one or
more beads.
•The shape of the beads affect the IR spectra,flattned beads
gives strong signal than spherical beads.
61.
62.
63. NMR gives more structural informations
than IR/UV.
The solid support broadens the peak &
hence low resolution.
Magic angle spinning NMR: Here the
sample is inserted in to the mag.field at the
angle of 550 ,this will reduce the peak
broadening,and has been used to analyse
swollen polymer beads directly.
64.
65. MASS spectroscopy analysis is highly automated.
The measurement is made on resin beads directly.
It is the most widely used technique in combinatorials.
66.
67. Solution containing compounds is passed
through electrically charged capillary & they
“explore” in to smaller droplets.
68.
69. The sample is embedded in the solid matrix(2,5 dihydroxy
benzoic acid)
Bombarded with laser
The sample molecule are vaporized &ionised
The analysis is done with the use of time of flight
analyser(TOF)
In TOF ions of different mass travels different distance in
a specified of time.
75. In recent years the peptide-protien & peptide-antibody
interactions have gained importance in the area of autoimmune
diseases.
By using combinatorials a library of hexa peptides was generated
& screened for B7 antibodies binding with CD-2 T-cell proliferation.
Peptides screened for inhibition of T cell for type 1-DM & RA.
Certain polypeptide screened for inhibition of kinase & protease
for AIDS &cancer.
76. Peptide suffer from disadvantages like poor bioavailability &
unfavorable ph.kinetic profile.
Thus the focus has shifted to synthetic peptido-mimetics like
peptoids.
Peptoids are molecules in which the variation
occurs in the attachment of amide nitrogen.
Similarity: Difference:
Molecular weight <500 Lack peptide H-bond
More rational flexibility
Side chain,functional gps More stability
in same position. Less double bond
77. O R1 O R3
H
N
N N
H H
O R2
O R1 O R3
N N
N
O R2
Peptoids are N substituted glycine with increased biological
half life.
80. oIn contrast to biopolymers,synthetic oligomers are stable to
proteases & nucleases.
oThey may be linear chain molecules like
oligo nucleotide(ssDNA & ssRNA)
oligo ureases.
oA library of 5000 oligo peptoids was generated & screened for
7-transmembrane G protien coupled receptor inhibition.certain
peptoids were active at nano mol conc.
81.
82. Solid phase chemistry can also synthesise oligo
saccharides.
Carbohydrate antibiotics including vancomycin
& aminoglycosides has been target in
combinatorial chemistry.
Examples
Bauhinia purpurea “lectin” analogues.
Erythromycin analogues.
Neocarzinostatin analogues.
83. R2 NH-FMOC
NH2
O R2
O
HO
O
R1 R1
2 amino benzophenone
Solid support:tenta gel
R2 NH2
H
O O
N O
R2
R3
O R1
R1
Benzodiazepine
84. CO CONH2
NH CO NH
RCOCl RSO2Cl
CH2Cl2 OH OSO2R1
H2N OH HN RCONH
CO
R
Uses:leukotriene antagonist ,carbapenam
antibiotics etc.
85.
86. It involves identification &
validation of target & docking to
identify the hit drug.
Virtual libraries are created in
combinatorial manner to screen
the molecules.
This helps to find out novel
drugs against sp.diseases.
87.
88. Virtual library: a combinatorial library that has no
physical existance,it exists in computer & can be
generated automatically.
They are screened againt “rule of 5” or to be docked by
using molecular docking.
89. CADD is a combination of
computational chemistry and
information technology tools that help
us to discover new therapeutic
solutions.
ADVANTAGES
target specific & structure based
fast and automatic
very low cost
high success rate
Two types:
Ligand based drug design
Structure/receptor based drug
design
90.
91. Combinatorial synthesis is now being used in lead
optimsation program of many potent drugs of natural
origin.
Eg:Rhodacyanin:
It is a natural dye with anti malarial activity. it is
considered as a lead to develop new anti malarials
effective againt quinine resistant plasmodium.
In lead optimisation process,Takasu et al,reported a
simple one pot method for synthesis of no of
rhodacyanin analogue with variation in “N” containing
ring.
94. Nucleoside analogue are useful as
antimetabolites & treatment of cancer.
By using combinatorial approach,the
synthesis of several nucleoside has been
synthesised.
These are effective againts various cancers.
Green Burg et al developed a method of
synthesis of nucleosides in solid state which
are the building blocks for antisense
polynucleotide.
95. R1
N R1
N
R N
O G N N
N
N
N
HO OH
R O
R1
N HO OH
N O
H
R1
N
N O
R O
PYRAMIDINE NUCLEOTIDE
HO OH
97. HIGH THROUHGPUT SCREENING
Automated microprocessor controlled robotic process
HTS : 50000-100000 compounds can be screened per week
against biological target
u HTS : 10000-100000 compounds within 24hrs
HTS assay system consist of sample wells for handling samples
98. CHEMICAL LIBRARY
HTS ASSAYS
INFORMATICS AND ANALYSIS
PROCESS ENGINEERING
99. MICROPLATE TECHNOLOGIES
a.96 well microtiter plate
8 rows (A-H)
12 columns
88 test samples
8 controls
b. 384 well microtiter plate
16 rows (A-P)
24 columns
352 test samples
32 controls
100. c. 1536 well microtiter plate
32 rows ( A-AF)
48 columns
1,408 test samples
128 controls
101. IN VITRO MARTIX –
PHARMACODYNAMIC LIGAND
STUDIES INTERACTION
STUDIES
PHARMACOKINETIC NATURAL PRODUCT
STUDIES ISOLATION
METABOLISM
DRUG SYNTHESIS
STUDIES
102. Here an effective target is identified & validated
for its function.
HIT: it is a molecule with confirmed activity
from primary hts assay,with good profile in 20
assays & with confirmed structure.
LEAD: Lead is explained as a hit series for which
SAR is studied.
103. Fl.correlation
spectroscopy
Scintillation
proximity Fl.anisotropy
assay(SPA)
Surface Fl.resonance
sensitive energy
fl.detection transfer
Fl.life time
imaging
microscopy
104. This procedure is suitable to detect receptor
ligand binding reactions.
107. When two different chromophores(drug & receptor) interact via
dipole-dipole mechanism, transfers excitation energy non
radially to acceptor chromophore.
108.
109. The sample is illuminated with pulsed laser
& the life time of fl.probe is determined
from the phase shift between the
modulation of excitation light & emission of
flourescence.
Eg: measurement of tyrosine kinase activity.
110.
111. The membrane receptor can be immobilised using
affinity tags
Thus flourescence labelled ligand binds with it then
the receptor emits fluorescence.
Eg:flourescence antagonist on immobilized 5-HT3a
receptor.
112. An antibody or a receptor
molecule, which is bound to a bead
emits light when beta emission from an
isotope occurs in close proximity; ie
when a radiolabelled ligand binds to
bead with receptor or antibody .
113.
114.
115. Absorption studies: caco(colon
cancer)cell line
grow confluently & form a monolayer on
polycarbonate support or collagen coated
polycarbonate support. These are used
for permeation studies.
116.
117. Due to development of 2-D multi parallel HPLC
(SEPBOX,SEPIAtec,Germany) it is now possible
to load up to 5g of NP extract & to isolate all
compounds in 70-80% purity with in 24hrs.
118. SEPBOX system works by using gradient elution & polarity based
trapping in solid phase extraction(SPE).
SEPBOX is coupled with photodiode array detector & light
scattering detector in series enables the identification &
quantification of the significant compounds.
NP database containing about 10,000 structurally characterized
natural compounds ,is being commercialized using MS & 2-D
NMR.
119. 1. Modern methods of drug discovery by
A.Hillisch and R.Hilgenfeld page no: 72-98
2. Foye’s Medicinal chemistry by Thomas and
David page no: 56-43
3. Medicinal chemistry by K.Illango page no: 389-407
4. http://www.sciencedirect.com/science
5. http://www.liebertonline.com/loi/adt
6. http://www.ingentaconnect.com/content
7. http://jbx.sagepub.com
8. http://www.nature.com/nmeth/index.html
9. http://mli.nih.gov/
10. http://pubchem.ncbi.nlm.nih.gov/