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OH Ph R1 N H Me H N O R2 O N H O R3 N H O R1 O H N R2 N H O N R3 Me O O HN SO2 Me
1. 2. 3. 4.
Background Primary structure modifications Secondary structure modifications Applications to drug design and development
Gretchen Peterson Evans Group Literature Seminar 04/13/01
01-title 4/11/01 7:03 PM
Peptidomimetic: A molecule bearing identifiable resemblance to a peptide that, as a ligand of a biological receptor, can imitate or inhibit the effect of a natural peptide Isostere: Surrogate functionalities that are isosteric and/or isoelectronic with a peptide amide bond Non-peptidomimetic: A molecule bearing no identifiable resemblance to a peptide or its isosteres, yet which imitates or inhibits the biological effect of a peptide Agonist: A molecule (natural or synthetic) which imitates the function of a ligand of a biological receptor Antagonist: A molecule (natural or synthetic) which inhibits the function of a ligand of a biological receptor Peptide analog: A peptidomimetic where one or more sidechains have been modified Psuedopeptides: A peptidomimetic where one or more peptide bonds have been replaced with an isostere Depsipeptide: A pseudopeptide where the isosteric replacement is an ester bond Retro-inverso peptide: A linear peptide whose amino acid sequence is reversed and the α-center chirality is inverted Exo-peptidase: A non-specific enzyme that degrades peptides by sequential hydrolysis of peptide bonds from either the amino (aminopeptidase) or carboxylic acid (carboxypeptidase) end of an amino acid Endo-peptidase: A peptidase that cleaves an internal amide bond of a specific peptide Pharmacophore: Structural region of a peptide responsible for interaction with its biological target Isostere
R1 N H F R2 O N H O R1 O H N R2 N H R1 N H R2 O H N O
02-definitions 4/11/01 7:10 PM
Why Peptidomimetics are Needed for Drug Potency
R1 N H O
O H N R2 N H
R3 N H O N R2
OH N H
Disadvantages of Peptides as Drugs
Advantages of Peptidomimetics as Drugs
1. Limited stability towards proteolysis by peptidases in the gastroeintesinal tract and in serum (t1/2 on the order of minutes) 2. Poor transport properties from the intestines to the blood and across the blood-brain barrier due to high MW and lack of specific transport systems 3. Rapid excretion through the liver and/or kidneys 4. Inherent flexibility enables interaction with multiple receptors besides the target, and could result in undesired side-effects
1. Conformationally restrained structures can minimize binding to non-target receptors and enhance the activity at the desired receptor. 2. Addition of hydrophobic residues and/or replacement of amide bonds results in better transport properties through cellular membranes. 3. Isosteres, retro-inverso peptides, cyclic peptides and non-peptidomimetics all reduce the rate of degradation by peptidases and other enzymes.
03-why peptides 4/11/01 7:24 PM
Peptidomimetic Drug Design Principles
Receptor scan peptide libraries for binding affinity Potential Peptide Agonist or Antagonists sequencing cloning expression site-directed mutagenesis activity assays Active Peptide Lead Compounds Biologically Active Peptide alanine scan Critical Residues reduce size Define Active Core
acid scan non-coded amino acid scan
Define Local and Global Conformational Parameters cyclization, turn mimetics, isostere replacement
Molecular Modeling of Receptor and/or Ligand
Generate Active Constrained Analogs (Peptidomimetics) conformational analysis physical studies
Identification of Receptor Residues Responsible for Peptide Recognition
Hypothesis of Receptor-Bound Conformation design novel compounds that mimic critical 3-D elements
scan molecule Receptor libraries for affinity
04-drug flowchart 4/11/01 7:24 PM
Potential Agonist/ Antagonist
optimize lead Non-Peptidomimetics for bioactivity
Adapted from Adv. Drug. Res. 1997, 29, 1-78
Ci ψ: Ci . • Conformationally restricted amino acids must retain the crucial side-chain interactions with the receptor. Do these modifications increase the peptide's bioavailability. How can the peptide be modified to rigidify that conformation? 3. Torsional Angles ω: Ni+1 .Determination of a Peptide's Active Conformational Parameters 1. • Constrained amino acids can be categorized by the torsional angles that they restrict in the peptide. What is the conformation of the biologically active core peptide? R1 N H O O H N R2 N H O R3 2. receptor selectivity.C i φ: C α.C i α β α 05-conf constraint 4/11/01 8:52 PM .Ni i χ: C i . and resistance to degradation? Limitation of torsional angles available to active site peptide residues • Sequentially substitute D-amino acids and conformationally constrained amino acids for the natural residues in the target.
1997.C i α β α β-turns I II III φ2 -60 -60 ψ2 -30 -30 φ3 -90 ψ3 0 0 -60 +120 +80 -60 -30 Adapted from Adv. ψ = -47 ° 05a-2 struct 4/11/01 8:28 PM .C i φ: C α. ψ = +135 ° α-helix (3.Peptide Secondary Structure Motifs Torsional Angles ω: Ni+1 . 1-78 β-sheet φ = -139 °.610 helix) φ = -57 °. 29.Ci ψ: Ci . Res.Ni i χ: C i . Drug.
stereochemistry Modification 1. β-alkylation 06-constraint types 4/12/01 10:32 AM .β-dehydrogenation i-1 R H N H2N i i+1 O Cyclization N-alkylation H2N φ ψ ω N H R OH COOH α-alkylation. α. Backbone N-alkylation 2. D-Amino O χ R O Conformational effect φ. ψ are biased towards formation of β-turns or γ-turns. -CH2 S-) ω can be biased to 0 or 180°. Dehydroamino acids 7. may also affect backbone conformation acid/proline substitution 4. φ. ψ are constrained to a helical or extended linear structure Favors formation of β-turn structures ω can be fixed at 0 or 180° (olefins). ψ. facilitates cis-trans amide bond isomerism φ.e.Conformational Constraints β-alkylation R Side chain modification Cyclization. or allowed greater freedom of rotation (i. Cyclic amino acids 6. Backbone Cα-alkylation 3. χ can also be affected Fix χ at 0 or 180° Constrain χ. χ are constrained. Peptide bond isosteres 5.
φ Me Me H 2N ψ COOH α-Aminoisobutyric acid (Aib) R H2N R COOH • Preferred conformation is in an extended structure φ. i-Pr. Toniolo Peptide Res. a peptide responsible for modulating pain response. 6747-6756. resulted in a peptidomimetic with greater in vivo activity. 29. 1989. ψ = 180° R = Et (Deg). 275-281 H2N COOH α-Aminocycloalkane carboxylic acid (Acn+3c) H-Tyr-Gly-Gly-Phe-Leu-OH Enkephalin 07-alpha me aa's 4/11/01 8:33 PM . Ph Dialkylglycine (CH2)n • Preferred conformation is in a β-turn or 310 helix • Substitution of Ac6c into various positions of enkephalin.α-Alkylation of Amino Acids • Most widely studied α-alkylated amino acid • Restricts φ. 2. ψ to angles present in α or 310 helices • Review of its conformational effects in peptides: Biochemistry 1990.
Protected α-Methylcysteine Goodman Pure Appl. 99% y. DCC. RuCl3•H2O. 1303-1308 08-a-me synth 1 4/11/01 8:52 PM . 2. 1996. NaN3. Me SAr CO2Bn BF3•OEt2 CO2Bn HN Me R= OMe H2N RSH 78% y. DMAP. (2 steps) O CO2Bn Me 71% y. Sharpless Asymmetric Epoxidation OH Me OH O Me 1.Synthesis of α-Methylamino Acids Me H 2N Me OH Me OH Me H2N SH COOH COOH H2N COOH α-Methylcysteine α-Methylthreonine D-allo-threonines • When incorporated in a peptidomimetic. 68. 83% y. BnOH 63% y. Chem. NaIO4 2. > 95% ee 1. PPh3. these constrained amino acids have the potential for disulfide bond formation or other cyclizations to furthur rigidify the structure.
63. 82% y. Boc2O OH OBn O Me N BnO O BF3•Et2O Me Me NHCbz N Me Cbz N Me α-Methyl Allo-threonine Goodman J. Pd/C 3. NaIO4. Cbz-OSu. 87% y. NaN3 2.. Chem. 20% H2SO4. Org. Me Me BocHN O OH OBn 1. LiBr. . H2. 5240-5244 Alkylated Tryptophan 09-a-me synth 2 4/12/01 10:38 AM 45-70% y. 94% y. 2. 2. SOCl2 2. Me H OBn O Me BocHN Me 1.Syntheses of α-Methylthreonines Me Me O OBn Sharpless Asymmetric Dihydroxylation AD-Mix-α 91% y. 1998. O O 2S O Me Me OBn O • Use of AD-mix-β allows access to all the 3R threonine and allo-threonine stereoisomers. 1. Me Me N3 O OH OBn 1. 20% H2SO4 87% y. PPh3 2. Pd/C 96% y. RuCl3•H2O. Pyr. DMAP. Boc2O BuOH. 93% y. NaN3 2. 63% y. Me Me Br OH OBn O α-Methyl Threonine 1. >98% ee HO Me OH Me OBn O 1. H2.
LiHMDS CO2t-Bu • no racemization reported in the modified Birch reduction 1. 26. NH3 THF/t-BuOH 76% y. O N Ph O 1. MeI 95% y. LiHMDS 2.α-Methyl Amino Acids and Dipeptides From β-Lactams O O N O Ph N Me O Cl MeO OMe MeO MeO OMe OMe + Et3N 95% y. H3O+ 85% y. 3783 . BnBr 90% y. • β-lactam synthon methodology allows for a wide variety of α-methylated amino acids and peptides to be rapidly synthesized. TFA CO2H O Me Ph 2. Ph CO2t-Bu 1. 44. NH3 THF/t-BuOH 2. 1985. Li. 5307 10-a-me synth 3 4/12/01 4:30 PM Staudinger reaction: Evans Tetrahedron Lett. MeI 92% y. LiHMDS 2. MeO MeO H2N Me COOH 2. >99% ds O Ph H 2N H H N O N Ph O N Me Ph S-α-Methyldopa 1. Li. CO2t-Bu Bn Ojima Tetrahedron 1988. >99% ds Me O N Ph O O Me N Me N O N Ph O O Ph N O O N Ph N O Me 1..
9225-9226 . Soc. Boc-Val-∆Phe-Phe-Ala-Phe-∆Phe-Val-∆Phe-Gly-OMe X-Ray Structure 11-dehydro aa 4/12/01 10:38 AM Chuhan J. Am. • Dehydroamino acid (Z isomer more synthetically accessible than E) rigidifies the conformation of the side chain.Dehydroamino Acids i+2 i+3 Ri+2 O C terminus i+1 χ = 0°C i Ri+1 HN N H O NH HN O Ri+3 Ri O • Favors the formation of β or γ-turns when placed in the (i+2) position of the putative turn sequence. N terminus • Sequential placement of ∆Phe in a peptide gives repeated β-turns. 114. 1992. which form a 310 helix. Chem. χ is fixed at 0°C (Z) or 180 °C (E).
χ -60° 180° +60° descriptor gauche– (g–) trans (t) gauche+(g+) Goodman J Am. 3S Me OC H NH R H H H 2S. Chem. 9390-9401 12-b-me-amino acids 4/12/01 9:50 AM . 114. Preferred conformations are shown. based on the activity changes induced by different configurations at the β center.β-Methylamino Acids Me R Me R Me R Me R H2N COOH H2N COOH H2N COOH H 2N COOH 2R. 1992. 3R Me OC R NH H H R H 2S. 3S NH Me CO R t g– g+ t • Four configurations are accessible from varying the two stereocenters. • Systematic incorporation of β-MePhe into somatostatin peptidomimetics has resulted in a model for the ligand-receptor interaction. 3R NH Me CO H 2R. Soc.
J. preventing α-helix formation.4-Methanoproline (2. ψ angles. Aze. 3036-3043 H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH Angiotensin II N H 5. and Pro residues is largely steric bulk of the side-chain rather than the φ.4-MePro prefers the trans isomer by 6-8 kcal/mol. a key peptide in blood pressure regulation.5-Dimethylthiazolidine4-carboxylic acid (Dtc) 13-proline 4/12/01 10:39 AM . 1991. 34. Med. O (cis isomer) Proline (Pro) 2. • 2. Chem.Conformational Restriction of Proline Analogs H N N R O O (trans isomer) NH H N N O HN φ R • φ is constrained to –65 ± 15°. • Barrier to proline cis-trans isomerism is ~ 2 kcal/mol. and encourages formation of β-turns.4-MePro) (CH2)n HN O OH n=1 Azyline-2-carboxylic acid (Azy) n=2 Azetine-2-carboxylic acid (Aze) n=4 Pipecolic acid (Pip) • Difference between the Azy. whereas 2° amide barrier is 10 kcal/mol. resulted in a peptidomimetic with 39% greater agonist activity than the natural peptide. • Pipecolic acid prefers a chair conformation in which the carboxylic acid is axial. COOH Me S N H Me COOH • Allowed angles for ψ are in the γ-turn region • Substitution of Dtc for Pro in angiotensin II.
1993. 4N HCl/dioxane N CO2Me 2. DEAD 84% y. N Boc O H N CO2Me PPh3. Boc-Pro-OH. O N N O N Boc H 2N O 1.Cyclic Amino Acids Through Lactamization O H N (CH2)n OH O OH Lactamization BocHN ω O ψ N R1 O OR2 LG Displacement n(H2C) O H N O R1 OR2 BocHN BocHN • Lactam favors the ψ and ω angles of a β-turn. N Boc O H N CO2Me OsO4. NaIO4. Et3N 95% y. 58. OH Gly-OMe•HCl N Boc O OH DCC. Chem. (3 steps) N Boc Johnson J. 43% y. NH3/MeOH 48% y. HOBt. O • Spirolactam combines the conformational restraints of both proline and the lactam to fix the tripeptide in a Type II β-turn. NaBH4. 2334-2337 14-lactams 4/12/01 10:39 AM . Org. DCC. HOBt 3.
Parkinson's and Alzheimer's disease. Org. Med. a nervous system modulator and transmitter. 1993. a β-turn in the Phe-Gly-Leu portion of the peptide was believed to be crucial for receptor binding of Substance P. Chem. 1990.7 91. 33. 58. with theraputic potential in treating gastrointestinal inflamation. • By substituting Gly.1 –5. 2334-2337 H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Substance P • Substance P is a tachykinin.9 128. Pro and D-aa in structure-activity relationship (SAR) studies. 1848-1851 . arthritis. O N H H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-N N O O Me Ph Trp-NH2 Me • Incorporation of the spiro-lactam into the optimized peptidomimetic GR71251 resulted in the most potent antagonist of the NK1 Substance P receptor known to date. Chem.4 120 80 0 ideal Type II β-turn –60 Johnson J.Use of Spirolactam to Form an Active Peptidomimetic ψ2 O φ3 φ2 N N O N Boc ψ3 O φ2 X-ray structure ψ2 φ3 ψ3 H 2N –50. GR71251 15-spirolactam applic 4/12/01 10:30 AM Ward J.
Y = OH Methylene isosteres X = S. Ed. 1995. 803-861.Common Amide Bond Isosteres O H N R N H R H N O R S N H R H N O R X R H N O R X R O Peptide Thioamide isostere Trans-olefin isosteres X = H. see: • Rieger. Inc. New York. M. .. Wolff. John Wiley & Sons. S(O). Evans Group Seminar. OH O H N R X Y R H N O R X R O H N O R N H R N O H N R O R N O Ketomethylene isosteres X = Y = H or F X = H. 1991 • Goodman Burger's Medicinal Chemistry and Drug Discovery. F Ethylene isosteres X = H. E. O Azapeptide isostere Peptoid isosteres 16-dipeptide isosteres 4/12/01 10:39 AM For a comprehensive review.
ψi-1 is restricted to 60<ψ<180 in both the cis. Me O N R2 O N H O R3 OH ψi-1 H 2N R1 • The increase in flexibility is tempered by the steric restrictions imposed by the Me group. • Similar or enhanced activity of a tetrazole peptidomimetic indicates a cis-amide bond is favorable for receptor binding. not necessarily that a cis-amide bond is not the bioactive conformation. 17-cis amides 4/12/01 10:39 AM . and is more easily synthesized than a cis-olefin isostere.6 kcal/mol higher in energy than the trans isomer. Tetrazoles N N R1 NH O N N R2 • The tetrazole locks the amide in a cis configuration. • Absence of activity can be due to the increased steric bulk of the tetrazole.Methods for Constraining Peptide Cis-Amides N-Methyl amides • The cis-amide is only 0.and trans-amide.
Pyrrolinone Peptidomimetic Scaffold O R2 H 2N R1 HN O 2 O NH R3 R4 H 1 HN 3 HN O NH • The pyrrolinone backbone fixes φ. 2037-2040 18-pyrrolidines 4/12/01 10:37 AM . Lett. 2000. 2. ω to the angles near that of an anti-parallel β-pleated sheet (ideal is φ = –139°. Rn+2 Rn H N3 Rn+3 O NH Me Me Rn+1 O O OMe N3 Ph O OMe NH2 Me Me OHC OSEM OHC NHBoc OMe O Me Me NH2 ring 3 ring 2 ring 1 stereocenter 4 O i-Pr HN O i-Pr H OSEM H 2N HN Ph O i-Pr HN O i-Pr H OH 2 N3 HN Ph 2 1 O HN 1 O HN 3 3 O N3 NH2 Smith Org. ψ. ψ = 135) • Oligomer is amenable to iterative synthesis for a wide variety of amino acids.
Normal peptide R1 N H O R2 O H N O R3 N H • Reversal of the carboxyl and amino termini reverses the charge structure of the peptide and may be the cause of their low biological activity.Retro-Inverso Peptidomimetics R1 N H O O H N R2 N H O R3 • The reversal of the amide bond direction minimizes peptidase degradation. Verdini J. 1991. A retro-inverso section can also be embedded in a larger normal peptide. • The inversion of each sterocenter from L to D preserves the 3-D orientation of the side chain residues of the original peptide. Chem. OH Retro-inverso peptide R1 H2N N H R2 O H N O R3 H-Thr-Lys-Pro-Arg-OH O HO Me O O N (CH2)4 HN O H N NH2 NH2+ Tuftsin End group modified retro-inverso peptide • Tuftsin is a immune system stimulator which is completely degraded in vivo in 8 min. • End group modifications increased the complementarity of the peptidomimetic with the native peptides and has resulted in several potent peptidomimetics. 3372-3379 H2N N H H2N HOOC Pseudopeptide 19-retro-inverso 4/12/01 10:38 AM . Med. thereby increasing the mimetics' in vivo half-life dramatically. 34. • The retro-inverso peptidomimetic shows less than 2% hydrolysis after 50 min and retention of bioactivity.
β-Turn Non-peptidomimetics R2 HN O R1 NH 7 Å O O O N H HN R4 R3 O O Me S O Me R3 S H2N COOH O O N R1 R2 COOH Representative β-turn mimetics β-turn H2N • β-turns are the most frequently mimicked protein secondary structures. The turn can be stabilized by chelation of a cation. or intramolecular hydrogen bond(s). H 2N H O H CO2H O HN N N O O NHBn COOH NH O NH2 O OH 20-b-turn 1 4/12/01 12:36 PM . • A β-turn is defined as a tetrapeptide sequence where the distance between α-Ci and α-Ci+3 is ≤ 7Å. while conferring better solubility and/or resistance to enzymatic degradation. • Ideal mimic will have a rigid scaffold that orients the sidechain residues in the same direction as the natural peptide. such as Ca2+.
• 1 was the first β-turn mimetic ever synthesized. Tuftsin 1. O O CN EtO P OEt CN O 56% y. (3 steps) O N H 87% y. H2. H2. BF3•OEt2 )2 CuMgBr HO 1. • 1 exhibited some bioactivity in a dose dependent manner. NH2OH•HCl HO N Beckmann 52% y. H2N CN CN H H 1. 1986. m-CPBA 2. Hg(OAc)2 2. Jones oxid. 2. 4841-4844 21-b-turn 2 4/12/01 10:47 AM . Rh/Al2O3 78% y. 27. NaBH4 CN H 1. O3 2. but extensive studies were not undertaken. (4 steps) N 1. N O N 1 NH2 Kahn Tetrahedron Lett. Pd/C 2.Development of β-Turn Mimetics H 2N O N HN Me HO NH2 O O OH HN NH N H NH2 O • A folded structure was thought to be present in the receptor-bound conformation of the immunoregulator tuftsin.
97% y. H2. 93% y. Tentagel-SAC. 5% DBU. 429-441 . Pd/C.has been CO2Me identified as a key target to mimic for developing an effective malaria vaccine. O O NH Ot-Bu H2N N O O OAllyl N Cbz N H 2. HO • The peptide sequence -Asn-Pro-Asn-Ala. 2. PPh3. (2 steps) (mixture) O O t-BuO NH PhthN N O 22-b-turn 3 4/12/01 10:48 AM O O O OAllyl 1. It is present on the surface of N H Plasmodium falciparum parasite which is recognized by the human immune system. TMSCl. CH2Cl2. HOAt 2. (3 steps) PhthN CO2Me O HO CO2Me O 1. Ot-Bu separation. imidazole DMAP.region. 64% y. SOCl2. 84% y. Fmoc-Asp(OAllyl)-OH. Fmoc-Succ. 74% y. Et3N 2. piperidine. • Solid phase amino acid synthesis facilitates the screening of a number of turn mimetics to find the optimal geometry/stability. HO NH FmocN N O O OAllyl Robinson Synlett. (2 steps) 3. Cbz • Solution phase NMR studies indicate a preference for a Type I β-turn in the -Asn-Pro-Asn-Ala. 60% TFA. BrCH2CO2t-Bu 3. 1. 1999. DMF 1. MeNHNH2 2.β-Turn Mimetics for Solid Phase Synthesis HO COOH N H H 1. 58% y. pyridine 2. 10% citric acid 72% y. DCC. 47% y. BnOCOCl Na2CO3. 1. DEAD PhthNH. LDA. MeOH.
peptide coupling: HBTU. DIEA. BOP. NMP 4. TFA (Mtt = methyltrityl) ψ H HN O φ Robinson Synlett. DMF 2. DIEA. Fmoc removal: piperidine.Elaboration of Turn Mimetic O MttHN O O NH H2N N O O OAllyl N O N H O NH O FmocHN Me Me O N HN O O HN O HN Me O NH O 1. Fmoc-aa. Pd(PPh3)4. • NMR studies of the macrocycle indicate the illustrated φ and ψ were in close agreement with a Type I β-turn. 429-441 . piperidine. HOBt. H2N 1. 1999. DMF 2. NMM. repeat sequence O AllylO O N H O NH O Me HN Me O O N HN O O OH 23-b-turn 4 4/12/01 10:54 AM O N O HN Me O NH • Attachment of this peptidomimetic to a carrier protein and injection into mice resulted in production of antisera to malaria sporozoites. DMF 3. AcOH. DMF 3.
Three receptors have been identified: κ. Med. Chem.Morphine: a Non-peptidomimetic For Enkephalin Ph H N O N R N Me H2N O N H O H N O N H O OH Me Me OH Enkephalin HO O O HO O OH HO R = Me: Oxymorphone. and δ. Enkephalin is specific for δ. one of the oldest known analgesics. 1714-1720 24-morphine 4/13/01 10:33 AM . µ agonist R = Allyl: Naloxone. 33. µ. • Exact nature of the binding interaction between either natural or synthetic ligand and a receptor has yet to be elucidated. 1990. µ antagonist Morphine Message: Binds to the receptor Spacer Address: Sequence bound by carrier proteins to direct the peptide to the correct receptor after synthesis N OH HO O N H • Morphine. binds to the opiate receptors. while dynorphin binds to the κ receptor. Naltrindole δ antagonist Portoghese J. An endongenous µ peptide ligand has not been identified yet.
3479-3488 25-g turn 4/12/01 11:35 AM . 49. hydrogen bonding. dipole. a protease involved in blood coagulation. • Does enkephalin then not contain a γ-turn? • Negative results are inconclusive. This turn mimetic concept had successfully been used in developing inhibitors of thrombin. 1 Factors affecting binding: ring conformation.Determination of Active Secondary Structure H-Tyr-Gly-Gly-Phe-Leu-OH Enkephalin R2 O R3 O NH N H O NH R1 N N H-Tyr-Gly-Gly NH O R3 Ph O Me N CO2H Me R2 HN R1 O γ-turn • 1 was designed to probe whether enkepalin's bioactive conformation contained a γ-turn. steric fit. hydrophobicity Huffman Tetrahedron 1993. not proof of the absence of the element in question. • No binding activity was observed at any of the opiate receptors.
Thr(ol) = L-threoninol β-turn 3. D 1995.Somatostatin: Early Investigations • Somatostatin is a cyclic peptide formed by a disulfide bond between two Cys residues. 1133-1140 X-ray: Sheldrick Acta. Cryst. and digestive tract motility and blood supply. regulation of lymphocyte production in the brain. • Biological activity includes the inhibition of growth hormone. 48-59 26-somat 1 4/12/01 11:39 AM X-ray Structure of Octreotide . 31. H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp HO-Cys-Ser-Thr-Phe-Thr-Lys Somatostatin Suspected β-turn in boxed region H-D-Phe-Cys-Phe-D-Trp Thr(ol)-Cys-Thr-Lys Octreotide • Octreotide has similar biological activity to somatostatin and is a drug of first choice for the treatment of several carcinoid syndromes. • Main theraputic use is in the treatment of ulcers and bleeding in the gastrointestinal tract. 1982.2 Å Synthesis: Pless Life Sci. regulation of glucagon/insulin pathway (in pancreas). and found in the brain and digestive organs. but somatostatin has a prohibitively short half life in vivo.
8.301. first synthesized in 1982. 3S)-β-Me-8 (2R. 9 and 11 were important for activity.3R)-β-Me-8 (2S. Am. N OH L-363. 3R)-β-Me-7 (2S.3R)-β-Me-11 (2S. 114.Somatostain Active Site Elucidation O N 7 H O O 11 N H Me H 10 N H N 8 (D) OO NH 9 O NH2 NH • L-363. 3R)-β-Me-11 (2S. 3S)-β-Me-8 (2R.301 IC50 = 1 nM Modification (2S. 9390-9401 . 3S)-β-Me-11 27-somat 2 4/13/01 10:35 AM IC50 (nM) 1 1 >1000 >1000 <1 >1000 10 >1000 50 >1000 >1000 >1000 2D-NMR and Analysis of Conformer Populations Pharmacophore Model Goodman J. was selectively methylated at the 7. 1992. Chem. 3S)-β-Me-11 (2R. 3S)-β-Me-7 (S)-α-Me-7 (R)-α-Me-7 (2R. • NMR and SAR studies of the derivatives led to a model for the somatostatin pharmacophore in which residues 8. Soc. and 11 positions in the putative β-turn region. 3R)-β-Me-8 (2S.
6 10. • Glucose shows promise in development of non-peptidomimetics for several G-protein coupled receptors.5 low conc.3 9.Glucose Scaffold for Non-peptidomimetic of Somatostatin a a O N 7 H O O 11 N H Me H 10 N H 8 (D) N NH OO NH 9 O R2 O O O O O NH b N b d c NH2 d c NHR1 OH L-363.2 14. Chem. but selectivity must be fine-tuned for the desired activity. led to the design of a glucose-based non-peptidomimetic.301 G-Protein Coupled Receptor 1: R1 = R2 = H 2: R1 = CH3CO. 115. 12550-12558 NK1 0.0 6.: antagonist β2-adrenergic 3 antagonist 0. Am. 1993.6 10.2 7. Soc. µM) L-363.301 1 15 2 1.06 antagonist 28-somat 3 4/12/01 12:10 PM .6 8.1 5.1 11.18 agonist • Computer modeling.001 7.3 agonist (all conc. combined with previous biological studies of somatostain peptidomimetics. Hirshmann J.) ___ L-363.: agonist high conc. R2 = OCH2Ph Biological Activity (IC50.301 2 Distance (Å) a-b a-c a-d b-c b-d c-d somatostatin 0.3 9.3 13.
5 pM H2N 29-thrombin 1 4/12/01 12:17 PM . Contraction and dilation of blood vessels are also affected.sequence adjacent to the substrate cleavage site. Antagonists could be theraputic in treating arteriosclerosis and thrombosis. Fibrinogen target sequence NH HN NH2 Ph N O O H N OH B OH Ph O 2S Ph Ph N O O H N H 2N N H A Ki = 3.Peptidomimetic Thrombin Inhibitors Ph N O O H N O N H • Thrombin is a serine protease that promotes blood coagulation by cleavage of fibrinogen to the active protein fibrin.6 pM NH HN NH2 B Ki = 2. • Early antagonist design focused on non-hydrolyzable transition state mimetics of the -Phe-Pro-Arg.
33. Chem. Enzymatic activity is thereby inhibited.Development of Thrombin Peptidomimetics Me O H N O Me HN NH2 N NH O O H N N HO2C S O N H B O S O HN HN NH2 C Ki = 39 nM D Ki = 6. Int.6 nM • Can you assume D is binding in the same pocket as the fibrinogen substrate does? • X-ray structural studies indicate C and D bind to a different site in the enzyme through their hydrophobic residues.which causes collapse of the active site pocket. Me Me O Me O S O N H O H N O H N N Me Me E more potent than D HN NH2 Review summarizing the progression: Gante Angew. Eng. 1699-1720 30-thrombin 2 4/12/01 12:38 PM . Ed. 1994.
4.5 µM • HCl • developed by computer modeling studies of the indole nucleus docked in the thrombin X-ray structure. 287-295 .Non-peptidomimetic Thrombin Inhibitors H N O N N HO HO O HO S O N NH2 NH OBn S O N H O NH Ph N O H N O Fibrinogen target sequence NH HN NH2 LY333545 • developed from a library screening lead Sall Book of Abstracts. A58 Ki = 0. Ed. (racemic) H 2N NH 31-thrombin 3 4/12/01 12:20 PM . 1997. Int.9 µM Selectivity over trypsin: 227:1 O N H N NH NH2 • developed by using a bioactive template (glucose) and altering the periphery to give desired activity. 6. Angew. 36. Wery Protein Sci. Chem. 1412-1417 • developed by computer modeling of lead molecules docked into the X-ray structure. 214th ACS National Meeting 1997. 751-752. 1997. Biol. 1997. O O Me Me H Ki = 13 nM Selectivity over trypsin: >760:1 N N O H Ki = 0. Eng. Diederich Chem.
Ki = 8.) • This drug has been through clincal trials and is currently an established drug for the treatment of AIDS. Am. 358-361 32-hiv protease 1 4/12/01 12:29 PM • Oral bioavailabitlity is good (plasma levels stay above required inhibitory concentration for several hours.Peptidomimetic HIV-1 Protease Inhibitors OH H-Ala-Ala-NH O Val-Val-OMe Ph Ki = 18-180 nM (in vitro against protease) Dreyer Proc. Natl. • The first approach to inhibition was to form transition-state analogues. where the scissile amide bond was replaced with a non-hydroyzable peptide isostere. Inhibition of the protease results in non-infectious virions. 114. meaning that the active site of the enzyme contains the triad -Asp-Thr(Ser)-Gly-. 1992. O N O H N N H CONH2 N OH O NHt-Bu Saquinavir (Ro 31-8959) Roberts Science 1990. • Excellent selectivity for HIV protease over human proteases was observed.2 nM (in vitro against protease) Ile-Val-OMe Janda J. 86. 1989. Soc. Sci. • Most isosteric replacements centered around the Phe-Pro cleavage site in the protease substrate. . 9752-9756 Ph O Ac-Ser-Leu-Asn-NH P OO H N • HIV-1 protease is an aspartyl protease. 7604-7606 Ph H H • Saquinivir has an IC50 = 2 nM in HIV infected cells. Less than 50% inhibition of apartyl proteases was seen at 10 µM. 248. Chem. Acad. The protease is involved in synthesis of the virion's structural proteins.
• Optimization of the core structure to the potent inhibitor PNU-140690 involved computer modeling studies (docking the molecule into the X-ray crystal structure of HIV-1 protease). and pharmacokinetics. 40. 3707-3711 33-hiv 2 4/12/01 7:11 PM . • PNU-140690 is effective against some strains of HIV-1 that have developed resistance to saquinivir.Non-Peptidomimetic HIV-1 Protease Inhibitors Me OH OH S Ph Optimization Ph Me O O HN SO2 N Ph O O Lead Structure IC50 = 3 µM PNU-140690 IC50 = 2. indinavir and other peptide-based protease inhibitors. Hagen J. SAR studies. Chem.7 nM CF3 • Lead found in a high volume library screen at Pharmacia & Upjohn in 1994. Med. The pyrone inhibits HIV-1 protease but has no anti-viral activity. 1997.
1. H2O. (2 steps) 34-pnu synth 1 4/12/01 12:25 PM . Na2CO3.Asymmetric Synthesis of PNU-140690 O O O N Ph Me n-BuLi. THF. TiCl4. CuBr. Hunig's Base. DMS. BnBr. 0 °C N(TMS)2 MgBr 2. THF. HClO4 95% y. -78 °C O NH O Ph Cl Me O 95% y. (2 steps) Me O Me O Xa Me N(Bn)2 1. O MeO O O N O OMe O Ph N(Bn)2 2. Aq. CH2Cl2 78% y.
Soc. dr = 25:1 • Use of Ti(Oi-Pr)Cl3 resulted in an 8:1 dr. F 3C CF3 pyr. 24% overall y. 1997. Chem. Gammill J.Asymmetric Synthesis of PNU-140690 Ti(On-Bu)Cl3. -78 °C. 119. N(Bn)2 O O Xa Ph Me Ph O Xa Me OH O Me N(Bn)2 O Me Me 62% y. Am. CH2Cl2 81% y. 0 °C 67% y. Pd/C Ph O O 2. Me KOt-Bu. CH2Cl2. N SO2Cl Me PNU-140690 9 steps. 3627-3628 35-pnu synth 2 4/10/01 8:05 PM . OH Ph OH O Me O HN SO2 N Me N(Bn)2 1. Hunig's Base. H2. THF..
EtO CO2Et ONa EtO2C R NO2 (separated by chromatography) NO2 86% R = CO2Et R = H (2) 6N HCl 87% y. O Ph Me OLi OEt Me CO2H CO2H 0. >98% ee NO2 HO 1. NaOH.Tapinavir (PNU-140690) Process Route 1.5 eq (1R. MsCl 2. Ph MeCN. crystallization 27% y. (2 crystallized as the cyclohexamine salt) 36-pnu process 1 4/12/01 7:02 PM . 1 Ph Ph (unpurified) (norephedrine removed by extraction and recovered) Et Et Et HO isoprenyl acetate Amano P30 50% y.2S)norephedrine OH Me OH OH Me NH2 2. MeOH 95% y.
quant.H O Me O O Ph Tapranavir (PNU-140690) Process Route Et OH 2. NaHMDS. 1998. pyr. MeOH. NaOH. N SO2Cl Me O O NO2 Tapranavir CF3 F 3C (recrystallized) 13 steps. 84% y. Org. -80 °C Ph 90% y. Ph (precipitation) 2. OH O Me O HN SO2 N Et 1. 2. 63. 4% overall y. H2SO4. Pd/C. 99% y. Gage J. Me CO2Me OPOM (unpurified) NO2 OH Ph Me 1. PCC. CH2Cl2 78% y. 75% y. THF. H2. 3. Chem.. 7356 37-pnu process 2 4/12/01 7:06 PM .
263.12 Å 3. Dock analogs of structure into HIV protease dimer X-ray structure R O HN NH R HO OH Ph Ph HO OH IC50 = 36 nM Lam Science 1994. Prediction of how stereocenters will affect binding. Set of likely inhibitors 2. 380-384 38-hiv imide inhibitor 4/12/01 12:30 PM .5-6.Rational Design of HIV Protease Inhibitor Analysis of HIV protease dimer-ligand X-ray structures Pharmacophore model Ile Ile O H Asp Left enzyme Asp H Right enzyme Results in H-bond d/a 3. and thereby activity.5 Å Aspr 3D-database search R O R OH peptide Lead structure optimization for increased inhibitor binding HO O N N OH Biological assays 1.5-6.5 Å Aspl 8.5 .
However. if a β-turn mimetic shows no activity. decreased rate of excretion. preferential binding to another target (in vivo systems). electronic factors. The benefits include increased bioavailability. better transport through cellular membranes.Summary 1. 2. Control over steric and electronic factors can be exercised through peptide backbone modificatons as well. combinatorial chemistry and screening approaches. the binding site of an antagonist may be different than active site. Compared to native peptides. 4. then optimized to give the best possible pharmacokinetic properties. A tremendous variety of constrained amino acids have been developed to rigidify a particular amino acid or peptide sequence. database searching. where the native peptide is bound. peptidomimetics and non-peptidomimetics offer significant advantages as drug candidates. and analysis of X-ray structure data. Mimetics are usually designed based on analogy to the native peptide structure. 5. The forefront of the field is the rational design of non-peptidomimetics using computer modeling. Failure of a mimetic to give the expected bioactivity is INCONCLUSIVE for determining if that conformational element exists in the receptor-bound conformation of the native peptide. and decreased hydrolysis by peptidases. 39-conclusion 4/12/01 12:30 PM . Biologically active mimetics support the existence of the desired element being present in the receptor-bound conformation of the peptide. etc. For instance. may be the cause. the differences in side chain orientation. 3. 6.
Rubsam. R.-L. Burger's Medicinal Chemistry and Drug Discovery. Kolter. Int. Curr. F. Tetrahedron 1993. John Wiley & Sons. R. A. Pays-Bas 1994. 1991 Ripka. Adv... G. Chem. A. 113. 1-19 Rieger. 1998. M. 441-452 Thorough compliation of constrained amino acid syntheses and secondary structure mimetics Review of peptide bond isosteres Most recent coverage of biologically active peptidomimetics Review of peptidomimetic biological stability Concise explanation of peptidomimetic design process Overview of medicinal chemistry with a section on the beginning of peptidomimetics Fauchere. 1993. E. Recl. 49. T. Opin. 1992. Trav. Int. Chem. Angew. Chim. 1997.. Ro. J. 803-861. Eng.. 30. Drug. R. 1278-1301 40-reviews 4/10/01 8:22 PM .Lead Reviews and References Giannis. Ed. Int. 1244-1267 Goodman. Res. J. Res. D. Adv. Wolff. Thurieau. A. 1699-1720 Giannis.. H. Ed. J. 29. Liskamp. S. Angew. 32. 1991. Drug. Bio. New York. 1994.. D. 1-78 Excellent coverage of design principles and recent examples of the major classes of non-peptidomimetics Overview of peptide isosteres and review of peptidomimetics designed for specific biological receptors Comprehensive coverage of constrained amino acids and peptide isosteres Gante. 3547-3558 Hirshmann. 127-159 Marshall. Eng. Rich. C. M. Chem. Eng. Inc. 33. Evans Group Seminar. 2. M. Ed. Chem. 23. 1995. Ed. S. Angew.
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