Combinatorial Chemistry by Shraddha (uploaded by Dr ANTHONY)1)


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Combinatorial Chemistry by Shraddha Ghodke (uploaded by Dr ANTHONY MELVIN CRASTO) helping millions with websites , million hits on google


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CONTENT 2 Definition Principle Basic characteristics Conventional Vs combinatorial synthesis Combinatorial libraries Method of combinatorial synthesis Solid phase Vs solution phase Strategy of peptide synthesis Encoding combinatorial libraries Analytical method conclusion 2

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What is Combinatorial Chemistry? Combinatorial Chemistry is a synthetic strategy which , utilising different techniques, aims at the rapid synthesis of large collections (libraries) of compounds DEFINATION:- PRINCIPLE:- The goal of the combinatorial chemistry is to synthesize, purify, chemically analyze & biologically test the all the structures in the library using few synthetic experiments 3

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4 Basic characteristics of Combichem: Combinatorial Chemistry prepares a large number of different compounds simultaneously under identical reaction in a systematic manner 4

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5 Conventional Synthesis: A + B AB one educt (A) reacts with one educt (B) to yield one product AB Conventional Vs combinatorial 5

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6 Combinatorial Synthesis: A 1-n + B 1-n A 1-n B 1-n different building blocks of type A react combinatorially with different building blocks of type B to yield a combinatorial library 6

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7 Combinatorial Synthesis: A 1 A 2 A 3 B 1 B 2 B 3 A 1 B 1 A 1 B 2 A 1 B 3 A 2 B 1 A 2 B 2 A 2 B 3 A 3 B 1 A 3 B 2 A 3 B 3 7

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Why Combinatorial Chemistry? large numbers of compounds promise to increase the chance of finding hits/leads Faster lead generation Low risk of failure systematic variations in parent structure increase the chance to find Structure-Activity Relationships (SAR) hits can be followed up more rapidly 8

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9 Combinatorial Libraries: Library refers to a collection of molecules. two common type s of chemical libraries- 1) A generic library (scaffold based):- - based on a single parent or scaffold sructure & multiple substituents or residues 2) A mixture based (Backbone-based) libraries :- -Containg a variety of structure types 9

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10 Scaffold-based libraries: (Example: benzodiazepines ) Building block A (2-Aminobenzophenones) Building block B (Amino acids) Building block C (Alkylating agents) C B A 10

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11 Scaffold-based libraries: (Example: benzodiazepines ) 11

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12 Building block A (Amino acid) Building block B (Amino acid) B A Backbone-based(mixture) libraries: (Example: peptides) 12



Two Major Approaches:

14 Two Major Approaches Split & Mix “Real Combinatorial Chemistry” Array Synthesis “Parallel Synthesis‘ 14

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15 Resin Split v 1. Coupling (3 products) Mix v Split v v 2. Coupling (9 products) The Split-mix Synthesis Strategy: 15

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16 Mix, Split, Couple G G G R R R D D D GG G R G D R G RR R D D G D R DD GGG GG R GG D G D G G D R G DD G R G G RR G R D R GG R G R R G D R D G R D R R DD RR G RRR RR D D GG D G R D G D DD G DD R DDD D R G D RR D R D G R D Polymer Bead n n/3 n/9 n/27 Mix, Split, Couple The Split-mix Synthesis Strategy 16

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17 A more rigorous approach to generating libraries. Resin is split into n equal portions. • To create library with 3 possible amino acids at a position, split resin into 3 equal portions. • Each aliquot of resin is coupled separately with 1 of the 3 different amino acids. •Aliquots are then recombined after coupling reaction. Resin is split again into 3 equal aliquots. Each aliquot containing an equimolar mixture of the 3 different amino-acyl resins. • A second round of coupling reactions similar to the first. • The process can be repeated until the peptides in the library have reached the desired size. The Split-mix Synthesis Strategy: 17

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18 Array-based Synthesis Stratergy: Parallel synthesis Resin in Array 1. Coupling v 2. Coupling Use array of plastic pins (8 x 12 array) →20 reagents →within 1 or 2 reactions →gives 96 different products . 18


19 Synthesis is carried out on polystyrene pins(act as solid-phase support) Pins are compatible with 96 well micro titer plates. Each individual well could contain a different amino acid and coupling reagent. Syntheses on pins are carried out in parallel (by immersing these pins into individual reaction vessel) Washing & protecting group removal can be performed in a reaction bath Adv :- Useful for epitope mapping and overlapping peptide sequences. parallel synthesis 19

Reaction Path:

20 Reaction Path 20

Automated parallel synthesis: Pin Method:

21 Automated parallel synthesis: Pin Method Polypropylene pins derivatized with acrylic acid Reagents or wash solvents in a 96 deep-well plate Drop it in to run reactions 21

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22 Solid Phase Chemistry Reactor, Beckman 96 deep-well titer plate Solid Phase Chemistry Reactor Plate in a Plate Clamp 22


23 METHODS OF COMBINATORIAL SYNTHESIS 1) Methods of parallel synthesis Houghton's Tea Bag procedure Automated parallel synthesis 2)Methods in mixed combinatorial synthesis The mix and split method 23

Houghton's Tea Bag procedure :

24 Houghton's Tea Bag procedure Alternative approach to parallel synthesis. Initially, the resin (~100 mg) is distributed into individual polypropylene meshed bags & each bag is sealed & labeled The tea bag are then distributed in to individual reaction vessel & resin is acylated with sp. Amino acid . The tea bag can then redistributed into fresh reaction vessel for the addition of next amino acid The cycle repeated until the desire peptide length is achieved 24

Solid Phase Vs solution phase:

25 Solid Phase Vs solution phase Solid-Phase Organic Synthesis The compound library have been synthesized on solid phase such as resin bead, pins, or chips Solution-Phase Organic Synthesis The compound library have been synthesized in solvent in the reaction flask 25

Planning of solid phase synthesis:

26 Planning of solid phase synthesis The resin The linker The protective group Activating group Coupling agent Purification Encoding techniques Analytical techniques 26

Types of Resins:-:

27 Types of Resins:- Polystyrene resin Polyamide resin PEG based resin Tenta Gel resin Cellulose & other inorganic support material 27

The resin(solid support) :

28 The resin(solid support) Can be functionalised ; Chemical stab ility ( it must be inert to all applied chemicals ); Mechanical stab ility ( it shouldn’t break under stirring) ; It must swell extensively in the solvents used for the synthesis; Peptide-resin bond should be stable during the synthesis ; Peptide-resin bond can be cleaved effectively at the end of the synthesis ; ideal properties of resin

Polystyrene resin:

29 Polystyrene resin This is versatile resin Quite useful in multi-well, automated peptide synthesis, due to its minimal swelling in dichloromethane. It swells in non polar solvent Eg-Lightly cross linked gel type of polystyrene (1-2% divinylbenzene) and -poly(styrene-oxyethylene). 29

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Polyamide resin:

31 Polyamide resin Useful and versatile resin - closely mimic the properties of the peptide chain themselves and have greatly improved solvation properties in polar and aprotic solvants. 31

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PEG Based Resins:

33 PEG Based Resins These are swells in polar and non polar solvents. ChemMatrix® is a new type of resin which is based on PEG that is crosslinked. ChemMatrix® has claimed a high chemical and thermal stability and has shown higher degree of swellings in acetonitrile, dichloromethane, DMF. Eg- 33

TentaGel Resins :

34 TentaGel Resins polyethylene glycol attached to cross-linked polystyrene through an ether link, and combines the benefits of the soluble polyethylene glycol support with the insolubility and handling characteristics of the polystyrene bead. PEG-Polystyrene graft polymers 34

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Peptides Peptoids Oligo- carbamates Oligo- ureas vinyl. Sulfonyl- peptides vinyl. Peptides Oligosulfones Oligosulfoxides Biopolymers used in Combinatorial Chemistry

Linkers :

37 Linkers A linker covalently connects molecules to the solid support, and should provide a means for their chemical attachment and cleavage Linker has many similarity to protecting groups in solid phase synthesis. 37

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38 Properties of a linker - Stable to the reaction conditions -Cleaved selectively at the end of synthesis -Re-useable -Facilitate reaction monitoring -Sequential / Partial release - allow some room for the rotational freedom of the molecule attached to the polymer 38


39 Linkers Carboxylic acid 2. Carboxamide 3. Alcohol 39


40 Linkers 4. Carbamates and Amines 5. Traceless 40

Protective group:

41 Protective group - A species conjugated to a functional group that block the reactivity of that group. - Good protective group are easily attached and removed using reaction. 41

Solid phase synthesis: protecting groups:

42 Solid phase synthesis: protecting groups A few protecting groups used in solid phase synthesis. For amines. Boc (t-butoxycarbonyl) Fmoc (9-fluorenylmetoxy carbonyl) Tmsec (2 [ trimethylsilyl ] ethoxycarbonyl) For carboxylic acids. Tert Bu ester(t-butyl ester) Fm ester(9-fluronyl methyl ester) Tmse ester(2 [trimethylsilyl] ethyl) 42

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Activating Group :

46 Activating Group For coupling the peptides the carboxyl group is usually activated. This is important for speeding up the reaction. There is two main type of activating group:- Carbodiimides Triazoles. HoBt HoAt 46

Peptide Coupling:

47 Peptide Coupling Amides are formed by treating a mixture of an acid and amine with dicyclohexylcarbodiimide (DCC) 47



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49 N N N OH 1-hydroxybenzotriazole (HOBt) N N N OH N 1-hydroxy-7-aza-benzotriazole (HOAt) N N N O P + (CH 3 ) 2 N N(CH 3 ) 2 N(CH 3 ) 2 PF 6 - benzotriazol-1-yl-oxy-tris(dimethylamino)- phosphonium hexafluoro phosphate (BOP) N N N O P + PF 6 - N N N benzotriazol-1-yl-oxy- tris(pyrrolidino)phosphonium hexafluoro phosphate (PyBOP) Hexamethylphosphoramide (carcinogen)! 49

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50 2-(1H-benzotriazol-1-yl)-1,1,3,3,- tetramethyluronium hexafluorophosphate HBTU N N N O C + (CH 3 ) 2 N N(CH 3 ) 2 PF 6 - N N N O C + (CH 3 ) 2 N N(CH 3 ) 2 BF 4 - 2-(1H-benzotriazol-1-yl)-1,1,3,3,- tetramethyluronium tetrafluoroborate TBTU N N N O C + N(CH 3 ) 2 PF 6 - N(CH 3 ) 2 + - N -[(1H-benzotriazol-1-yl)(dimethylamino)- methylene]- N -methanaminium hexafluorophosphate N -oxide 50



General Considerations:

52 General Considerations Making peptide bonds between amino acids is not difficult. The challenge is connecting amino acids in the correct sequence. for example, Random peptide bond formation in a mixture of phenylalanine and glycine, will give four dipeptides. Phe—Phe Gly—Gly Phe—Gly Gly—Phe 52

General Strategy:

53 General Strategy 1. Limit the number of possibilities by "protecting" the nitrogen of one amino acid and the carboxyl group of the other. N-Protected phenylalanine C-Protected glycine N HCHCOH CH 2 C 6 H 5 O X H 2 N CH 2 C O Y 53

General Strategy:

54 General Strategy 2. Couple the two protected amino acids. N HCH 2 C O Y N HCHC CH 2 C 6 H 5 O X N HCHCOH CH 2 C 6 H 5 O X H 2 N CH 2 C O Y 54

General Strategy:

55 General Strategy 3. Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus . N HCH 2 CO O H 3 N CHC CH 2 C 6 H 5 O + – Phe-Gly N HCH 2 C O Y N HCHC CH 2 C 6 H 5 O X 55



Protect Amino Groups as Amides:

57 Amino groups are normally protected by converting them to amides. Benzyloxycarbonyl (C 6 H 5 CH 2 O—) is a common protecting group. It is abbreviated as Z . Z -protection is carried out by treating an amino acid with benzyloxycarbonyl chloride. Protect Amino Groups as Amides 57

Protect Amino Groups as Amides:

58 Protect Amino Groups as Amides CH 2 OCCl O + H 3 N CHCO CH 2 C 6 H 5 O – + 1. NaOH, H 2 O 2. H + N H CHCOH CH 2 C 6 H 5 O CH 2 OC O (82-87%) 58

Protect Amino Groups as Amides:

59 Protect Amino Groups as Amides N H CHCOH CH 2 C 6 H 5 O CH 2 OC O is abbreviated as: Z N H CHCOH CH 2 C 6 H 5 O or Z-Phe 59

Removing Z-Protection:

60 An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by: a) Hydrogenolysis b) Cleavage with HBr in acetic acid Removing Z-Protection 60

Hydrogenolysis of Z-Protecting Group:

61 Hydrogenolysis of Z-Protecting Group N H CHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 O CH 2 OC O H 2 , Pd H 2 N CHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 O CH 3 CO 2 (100%) 61

HBr Cleavage of Z-Protecting Group:

62 HBr Cleavage of Z-Protecting Group N H CHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 O CH 2 OC O HBr H 3 N CHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 O CH 2 Br CO 2 (82%) + Br – 62

The tert- Butoxycarbonyl Protecting Group:

63 The tert- Butoxycarbonyl Protecting Group N H CHCOH CH 2 C 6 H 5 O (CH 3 ) 3 COC O is abbreviated as: Boc N H CHCOH CH 2 C 6 H 5 O or Boc-Phe 63

HBr Cleavage of Boc-Protecting Group:

64 HBr Cleavage of Boc-Protecting Group N H CHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 O (CH 3 ) 3 C OC O HBr H 3 N CHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 O CO 2 (86%) + Br – CH 2 C H 3 C H 3 C 64



Protect Carboxyl Groups as Esters:

66 Carboxyl groups are normally protected as esters. Deprotection of methyl and ethyl esters is by hydrolysis in base. Benzyl esters can be cleaved by hydrogenolysis. Protect Carboxyl Groups as Esters 66

Hydrogenolysis of Benzyl Esters:

67 Hydrogenolysis of Benzyl Esters H 2 , Pd H 3 N CHC N HCH 2 CO CH 2 C 6 H 5 O C 6 H 5 CH 3 CO 2 (87%) N H CHC N HCH 2 CO CH 2 C 6 H 5 CH 2 C 6 H 5 O C 6 H 5 CH 2 OC O O + – CH 3 C 6 H 5 67



Forming Peptide Bonds:

69 The two major methods are: 1. coupling of suitably protected amino acids using N , N' -dicyclohexylcarbodiimide (DCCI) 2. via an active ester of the N-terminal amino acid. Forming Peptide Bonds 69

DCCI-Promoted Coupling:

70 DCCI-Promoted Coupling Z N H CHCOH CH 2 C 6 H 5 O + H 2 N CH 2 COCH 2 CH 3 O DCCI, chloroform Z N H CHC CH 2 C 6 H 5 O N HCH 2 COCH 2 CH 3 O (83%) 70

The Active Ester Method:

71 A p -nitrophenyl ester is an example of an "active ester." p -Nitrophenyl is a better leaving group than methyl or ethyl, and p -nitrophenyl esters are more reactive in nucleophilic acyl substitution. The Active Ester Method 71

The Active Ester Method:

72 The Active Ester Method Z N H CHC O CH 2 C 6 H 5 O + H 2 N CH 2 COCH 2 CH 3 O NO 2 72

The Active Ester Method:

73 The Active Ester Method Z N H CHC O CH 2 C 6 H 5 O + H 2 N CH 2 COCH 2 CH 3 O chloroform Z N H CHC CH 2 C 6 H 5 O N HCH 2 COCH 2 CH 3 O (78%) + H O NO 2 NO 2 73

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74 Merrifield peptide synthesis :- 74

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78 Encoding combinatorial libraries A) Microsequencing -determination of -C terminal amino acid unit Carboxypeptidase method Akabori method -determination of -N terminal amino acid unit 1)Sanger`s method 2)Edman degradation 3)Dansyl method B)Tag-based encoding methods 78

Microsequencing of peptides by C-Terminal Residue Determination:

Microsequencing of peptides by C-Terminal Residue Determination Carboxypeptidase enzymes cleave the C-terminal amide bond Analysis determines the appearance of the first free amino acid, which must be at the carboxy terminus of the peptide 79

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80 Microsequencing of peptides by Edman degradation 80 The Edman degradation cleaves amino acids one at a time from the N-terminus and forms a detectable, separable derivative for each amino acid

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81 Encoding principle: each individual bead contains an encoding tag each tag uniquely encodes the building blocks on the bead Encoding tags in use: DNA (analysis by PCR) Peptides (analysis by Edman degradation) Haloaromatic groups (suitable for "binary code") Secondary amides (suitable for "binary code") Radiofrequency transponders Tag-based encoding methods 81

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82 Encoding by Radiofrequency chips Polymer beads are located in a microreactor vessel Each microreactor vessel contains one semi-conductor chip which acts as like a bar code ,identifying each lilrary member Unique radiofrequency signals can be recorded and detected Inert and porous mantle Radiofrequency semiconductor chip Polymer bead Ref.: K.C. Nicolaou, Angew. Chem . 107 (1995) 2476-79 82

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.. Extraction Techniques for Purification Liquid-Liquid extraction Extensivley used for solution-phase combinatorial synthesis. Automated by freezing liquid phase. Fails when ; Emulsions formed The impurities have the same solubility properties Fluorous phase technique Attach a insoluble perfluorinated moiety to the compound. Retain the molecules from fluorous solvent. Solid-phase extraction Based on adsorption to a suitable surface. Impurities are washed away with a solvent in which the compound are insoluble.


POOLING STRATEGIES Used during course of the reaction to generate new libraries Approaches:- One bead one compound strategy Iterative deconvolution Subtractive deconvolution Bogus-coin detection Positional scanning 96

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1) One bed one compound strategy X Template Y Linker P reaction block with reaction vessels for synthesis, washing and cleavage of products (only one compound/vessel - no mixtures!)

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2) Iterative Deconvolution: X 1-n Y 1-n Z 1-n a b C Y 1-n Z 1-n Y 1-n Z 1-n Y 1-n Z 1-n Z 1-n Z 1-n Z 1-n

3) Subtractive deconvolution :

3) Subtractive deconvolution This is similar to iterative deconvolution but uses Negative logic ,namely leave out the functional group that is missing must be needed for the activity This is particularly important for QSAR type studies. 99

4)Bogus-coin detection:

4)Bogus-coin detection This begins generating and screening the entire library as a single mixture If activity is detected the building blocks are divided in to three groups ( α , β , γ )and additional sub libraries are prepared . In these subset the number of building blocks of α group is decreased and the number from the β group is increased and the number of γ group is unchanged the resulting effect on activity (up, down, unchanged)suggest which group of the building blocks was contributing most to activity 100

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5)Parallel Deconvolution(Positional Scanning) Y 1-n Z 1-n Y 1-n Z 1-n Y 1-n Z 1-n Z 1-n Z 1-n Z 1-n X 1-n X 1-n X 1-n Y 1-n Y 1-n Y 1-n X 1-n X 1-n X 1-n

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Solution-phase combinatorial chemistry Template R1 R2 R1 R2 102

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103 103 Applications of synthetic peptides Immune peptides: synthetic antigens; vaccines diagnostic tools immunostimulator peptides; muramyl dipeptide tuftsin derivatives Hormones: oxytocin vasopressin insulin somatostatin GnRH etc. Neuropeptides: substance P cholecystokinin neurotensin Antibiotics: tachikinin gramicidine S Toxins: conotoxins spider toxins snake toxins ionchanel blockers Enzymes and enzyme inhibitors: Ribonuclease A Carriers: templates miniproteins Peptides for structural studies: turn mimicking cyclic peptides Transporter peptides: penetratin o ligoarginine HIV-Tat protein

Trends over the Last Decade:

104 Trends over the Last Decade 0 1000+ no of Compounds time Dev. times for solid phase Sld P Array Sld P S & M Solu P Array 2004 Classical Organic Synthesis Solu P Array 1996 10,000+ Solution Phase Array or Parallel Synthesis now dominates CONCLUSION 104


105 BIBLIOGRAPHY Wilson and Gisvold`s Textbook of organic medicinal and Pharmaceutical chemistry, John H. Block John M. Beale,Jr. Eleventh edition ,43-64 Smith and Williams' Introduction to the principles of Drug design and action , 4 th Edition ,355-375 Abraham D. J.; Burger’s Medicinal Chemistry Drug Design edited by. Vol. 2 nd Drug Discovery and Development; Ed.6 th , 56-67 Ghose A. K., Vellarkad N. V.; Combinatorial Library Design and Evaluation; Ed.5 th , 30-31 105

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106 A Textbook Of Organic Chemistry By Raj K. Bansal,3 rd Edition, New Age International Publishers, New Delhi,1997. Lebl, M. 1999. Parallel personal comments on “classical” papers in combinatorial chemistry. J. Comb. Chem., , 1(1): 3-24 Baum, R.M. 1994. Combinatorial approaches provide fresh leads for medicinal chemistry. Chemical & Eng. News, 72(6): 20-26. www 106

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