Recent Advances in the Design and Synthesis of Peptidomimetics

Me
OH

Ph

R1 O R3 R1 O R3 O O
Me H H
N N HN
N N N N Me
H H H H SO2
O R2 O O R2 O
N

CF3

Overview

1. Background
2. Primary structure modifications
3. Secondary structure modifications
4. Applications to drug design and development

Gretchen Peterson

Evans Group Literature Seminar

04/13/01
01-title 4/11/01 7:03 PM
 Definitions
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 O
H R1 O
R1 O H
N
N N
H N N
N R2 H H
H O
R2 O
F R2

Pseudopeptide Peptide analog Retro-inverso peptide

02-definitions 4/11/01 7:10 PM
 Why Peptidomimetics are Needed for Drug Potency

R1 O R3 OH R3
H
N N
N N N N
H H H H
O R2 O O R2 O

Disadvantages of Peptides as Drugs Advantages of Peptidomimetics as Drugs

1. Limited stability towards proteolysis 1. Conformationally restrained structures can

by peptidases in the gastroeintesinal tract minimize binding to non-target receptors and
and in serum (t1/2 on the order of minutes) enhance the activity at the desired receptor.

2. Poor transport properties from the intestines 2. Addition of hydrophobic residues and/or
to the blood and across the blood-brain barrier replacement of amide bonds results in better
due to high MW and lack of specific transport systems transport properties through cellular membranes.

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
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 Biologically Active Peptide

scan peptide libraries alanine scan

for binding affinity
Critical Residues
Potential Peptide
Agonist or Antagonists reduce size
sequencing Define Active Core
cloning activity assays
expression D-aminoacid scan
site-directed non-coded amino acid scan
mutagenesis Active Peptide
Lead Compounds Define Local and Global Conformational Parameters
cyclization, turn mimetics,
isostere replacement

Molecular Modeling of Generate Active Constrained Analogs

Receptor and/or Ligand (Peptidomimetics)

conformational analysis
physical studies

Identification of Receptor Residues

Hypothesis of Receptor-Bound Conformation
Responsible for Peptide Recognition
design novel compounds that
mimic critical 3-D elements
scan molecule Potential optimize lead
Receptor Agonist/ Non-Peptidomimetics
libraries for affinity Antagonist for bioactivity

Adapted from Adv. Drug. Res. 1997, 29, 1-78

04-drug flowchart 4/11/01 7:24 PM
 Determination of a Peptide's Active Conformational Parameters

1. What is the conformation of the biologically active core peptide?

R1 O R3
H 2. How can the peptide be modified to rigidify that conformation?
N
N N
H H 3. Do these modifications increase the peptide's bioavailability, receptor
O R2 O
selectivity, and resistance to degradation?

Limitation of torsional angles

available to active site peptide residues Torsional Angles

ω: Ni+1 - Ci
• Sequentially substitute D-amino acids and conformationally
α
constrained amino acids for the natural residues in the target. ψ: Ci - C i
• Conformationally restricted amino acids must retain the crucial φ: C αi - Ni
side-chain interactions with the receptor.
α β
χ: C i - C i
• Constrained amino acids can be categorized by the
torsional angles that they restrict in the peptide.

05-conf constraint 4/11/01 8:52 PM

 Peptide Secondary Structure Motifs Torsional Angles

ω: Ni+1 - Ci
α
ψ: Ci - C i

φ: C αi - Ni
α β
χ: C i - C i

β-turns φ2 ψ2 φ3 ψ3

I -60 -30 -90 0

II -60 +120 +80 0
III -60 -30 -60 -30

Adapted from Adv. Drug. Res. 1997, 29, 1-78

β-sheet
φ = -139 °, ψ = +135 °

α-helix (3.610 helix)

φ = -57 °, ψ = -47 °

05a-2 struct 4/11/01 8:28 PM

 Conformational Constraints

β-alkylation Side chain modification

i-1 i i+1
R
φ ψ
Cyclization Cyclization, α,β-dehydrogenation R O
ω R
H
H2N COOH N OH
N-alkylation H2N N
H
α-alkylation,
stereochemistry
O χ R O

Modification Conformational effect

1. Backbone N-alkylation φ, ψ, χ are constrained, facilitates cis-trans

amide bond isomerism

2. Backbone Cα-alkylation φ, ψ are constrained to a helical or extended

linear structure

3. D-Amino acid/proline substitution Favors formation of β-turn structures

4. Peptide bond isosteres ω can be fixed at 0 or 180° (olefins), or allowed

greater freedom of rotation (i.e. -CH2 S-)

5. Cyclic amino acids ω can be biased to 0 or 180°, φ, ψ are biased

towards formation of β-turns or γ-turns, χ can
also be affected
6. Dehydroamino acids Fix χ at 0 or 180°

7. β-alkylation Constrain χ, may also affect backbone

conformation

06-constraint types 4/12/01 10:32 AM

 α-Alkylation of Amino Acids

Me Me • Most widely studied α-alkylated amino acid

φ
H 2N COOH • Restricts φ, ψ to angles present in α or 310 helices
ψ
• Review of its conformational effects in peptides:
α-Aminoisobutyric acid (Aib) Biochemistry 1990, 29, 6747-6756.

R R

H2N COOH • Preferred conformation is in an extended structure

φ, ψ = 180°
R = Et (Deg), i-Pr, Ph
Dialkylglycine

(CH2)n • Preferred conformation is in a β-turn or 310 helix

• Substitution of Ac6c into various positions of enkephalin, a

H2N COOH
peptide responsible for modulating pain response, resulted in
α-Aminocycloalkane a peptidomimetic with greater in vivo activity.
carboxylic acid (Acn+3c) Toniolo Peptide Res. 1989, 2, 275-281

H-Tyr-Gly-Gly-Phe-Leu-OH
Enkephalin

07-alpha me aa's 4/11/01 8:33 PM

 Synthesis of α-Methylamino Acids

Me OH Me OH • When incorporated in a
Me SH
peptidomimetic, these constrained
H 2N amino acids have the potential for
H2N COOH COOH H2N COOH
Me disulfide bond formation or other
cyclizations to furthur rigidify the
α-Methylcysteine α-Methylthreonine D-allo-threonines structure.

Sharpless
Asymmetric OH 1. RuCl3•H2O,
Epoxidation O NaIO4 O CO2Bn
OH
Me 2. DCC, DMAP, BnOH Me
Me 71% y.
> 95% ee 63% y. (2 steps)
1. NaN3, 99% y.
2. PPh3, 83% y.

BF3•OEt2
SAr CO2Bn
Me
HN Me
R= H2N CO2Bn RSH
78% y.
Protected
OMe α-Methylcysteine

Goodman Pure Appl. Chem. 1996, 68, 1303-1308

08-a-me synth 1 4/11/01 8:52 PM

 Syntheses of α-Methylthreonines

Sharpless
Me Asymmetric O Me
HO Me
Dihydroxylation 1. SOCl2
Me OBn Me OBn O 2S
OBn
O
AD-Mix-α OH O 2. RuCl3•H2O, Me
O O
91% y., >98% ee NaIO4, 94% y.
1. NaN3 1. LiBr, 93% y.
• Use of AD-mix-β allows access to all 2. 20% H2SO4 2. 20% H2SO4, 87% y.
the 3R threonine and allo-threonine 87% y.
stereoisomers.

Me OH 1. H2, Pd/C Me OH Me OH
96% y.
Me OBn Me OBn Me OBn
BocHN 2. Boc2O N3 Br
O BuOH, 63% y. O O
α-Methyl Threonine
1. PPh3 1. NaN3
2. Cbz-OSu, 2. H2, Pd/C
DMAP, Pyr. 3. Boc2O
82% y.

Me Me OH
H
BF3•Et2O
Me N Me Cbz N BocHN OBn
OBn
Me Me
BnO Me O
O
NHCbz
O N α-Methyl Allo-threonine
Me
Alkylated Tryptophan
45-70% y. Goodman J. Org. Chem. 1998, 63, 5240-5244
09-a-me synth 2 4/12/01 10:38 AM
 α-Methyl Amino Acids and Dipeptides From β-Lactams

OMe MeO MeO

O OMe OMe
MeO O O
Cl Et3N O 1. LiHMDS O
O N +
N 2. MeI N Me
O 95% y.
Ph 95% y.
N Ph >99% ds Ph
Me N N
O Me O Me

• no racemization 1. Li, NH3

O O reported in the THF/t-BuOH
O O
modified Birch 2. H3O+
N Ph N Ph reduction 85% y.
1. LiHMDS
Ph Ph
N 2. MeI N
CO2t-Bu 92% y. CO2t-Bu MeO Me
O O
Me H2N COOH
MeO

1. LiHMDS S-α-Methyldopa
2. BnBr
90% y., >99% ds

O O • β-lactam synthon methodology

Ph H 1. TFA allows for a wide variety of
H
N CO2H
N Ph α-methylated amino acids and
H 2N
2. Li, NH3 peptides to be rapidly synthesized.
Ph
O Me THF/t-BuOH N
O CO2t-Bu
Ph 76% y.
Me
Bn Ojima Tetrahedron 1988, 44, 5307
Staudinger reaction: Evans Tetrahedron Lett. 1985, 26, 3783
10-a-me synth 3 4/12/01 4:30 PM
 Dehydroamino Acids

i+2 i+3

Ri+2
O
C terminus Ri+1 O
N
i+1 H
HN O HN Ri+3

χ = 0°C
Ri NH O
i

• Favors the formation of β or γ-turns when placed in

the (i+2) position of the putative turn sequence.

• Dehydroamino acid (Z isomer more synthetically

accessible than E) rigidifies the conformation of the
side chain. χ is fixed at 0°C (Z) or 180 °C (E).

N terminus • Sequential placement of ∆Phe in a peptide gives

repeated β-turns, which form a 310 helix.

Boc-Val-∆Phe-Phe-Ala-Phe-∆Phe-Val-∆Phe-Gly-OMe
X-Ray Structure Chuhan J. Am. Chem. Soc. 1992, 114, 9225-9226
11-dehydro aa 4/12/01 10:38 AM
 β-Methylamino Acids

Me R Me R Me R Me R

H2N COOH H2N COOH H2N COOH H 2N COOH

2R, 3R 2S, 3R 2R, 3S 2S, 3S

NH NH NH NH
Me H R Me Me R H Me

OC H H CO OC H H CO
R H H R

t g– g+ t

• Four configurations are accessible from varying the

two stereocenters. Preferred conformations are shown. χ descriptor

• Systematic incorporation of β-MePhe into somatostatin -60° gauche– (g–)

peptidomimetics has resulted in a model for the 180° trans (t)
ligand-receptor interaction, based on the activity changes +60° gauche+(g+)
induced by different configurations at the β center.

Goodman J Am. Chem. Soc. 1992, 114, 9390-9401

12-b-me-amino acids 4/12/01 9:50 AM
 Conformational Restriction of Proline Analogs

H
H
N N • φ is constrained to –65 ± 15°, preventing α-helix
N N formation, and encourages formation of β-turns.
φ R O
O O
O (trans isomer) • Barrier to proline cis-trans isomerism is ~ 2
R (cis isomer) kcal/mol, whereas 2° amide barrier is 10 kcal/mol.
NH
HN
• 2,4-MePro prefers the trans isomer by 6-8 kcal/mol.
2,4-Methanoproline
Proline (Pro) (2,4-MePro)

• Difference between the Azy, Aze, and Pro

(CH2)n residues is largely steric bulk of the side-chain
HN OH
n=1 Azyline-2-carboxylic acid (Azy) rather than the φ, ψ angles.
n=2 Azetine-2-carboxylic acid (Aze)
n=4 Pipecolic acid (Pip) • Pipecolic acid prefers a chair conformation in
O
which the carboxylic acid is axial.
COOH
Me
Me
S • Allowed angles for ψ are in the γ-turn region N
H
N COOH
• Substitution of Dtc for Pro in angiotensin II, a
H
key peptide in blood pressure regulation,
resulted in a peptidomimetic with 39% greater
5,5-Dimethylthiazolidine- agonist activity than the natural peptide.
4-carboxylic acid (Dtc) J. Med. Chem. 1991, 34, 3036-3043

H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH

Angiotensin II

13-proline 4/12/01 10:39 AM

 Cyclic Amino Acids Through Lactamization
O
H
N LG
OH ψ O
Lactamization ω Displacement n(H2C) O
(CH2)n N H
BocHN OR2 N
BocHN OR2
OH O R1
BocHN O R1
• Lactam favors the ψ and ω
O
angles of a β-turn.

OH

Gly-OMe•HCl OsO4, NaIO4;

H H
N OH DCC, HOBt, Et3N N N NaBH4, 43% y. N N
95% y. CO2Me CO2Me
Boc O Boc O Boc O

PPh3, DEAD
84% y.
O
• Spirolactam combines O
the conformational N O
1. 4N HCl/dioxane
restraints of both proline N N CO2Me
H 2N
and the lactam to fix the 2. Boc-Pro-OH, N
tripeptide in a Type II O DCC, HOBt
β-turn. Boc
N 3. NH3/MeOH
Boc 48% y. (3 steps)

Johnson J. Org. Chem. 1993, 58, 2334-2337

14-lactams 4/12/01 10:39 AM

 Use of Spirolactam to Form an Active Peptidomimetic

ψ2 O φ3 ψ3
φ2 ψ2 φ3 ψ3
φ2 N O
N
H 2N X-ray structure –50.9 128.7 91.1 –5.4
O ideal Type II β-turn –60 120 80 0
N
Boc

Johnson J. Org. Chem. 1993, 58, 2334-2337

• Substance P is a tachykinin, a nervous system

modulator and transmitter, with theraputic potential in
treating gastrointestinal inflamation, arthritis,
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Parkinson's and Alzheimer's disease.
Substance P
• By substituting Gly, Pro and D-aa in structure-activity
relationship (SAR) studies, a β-turn in the Phe-Gly-Leu
portion of the peptide was believed to be crucial for
O
receptor binding of Substance P.

N • Incorporation of the spiro-lactam into the optimized

Trp-NH2 peptidomimetic GR71251 resulted in the most potent
H N
O Me antagonist of the NK1 Substance P receptor known to
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-N
O date.
Me
Ph

GR71251 Ward J. Med. Chem. 1990, 33, 1848-1851

15-spirolactam applic 4/12/01 10:30 AM
 Common Amide Bond Isosteres

O R S R X R X R
H H H H
N N N N
N N
H H
R O R O R O R O

Peptide Thioamide isostere Trans-olefin isosteres Ethylene isosteres

X = H, F X = H, OH

O R R O R O R
H H H H
N N N N N N
X N
H
R X Y O R O R O R O

Ketomethylene isosteres Methylene isosteres Azapeptide isostere Peptoid isosteres

X = Y = H or F X = S, S(O), O
X = H, Y = OH

For a comprehensive review, see:

• Rieger, Evans Group Seminar, 1991
• Goodman Burger's Medicinal Chemistry and
Drug Discovery. Ed. M. E. Wolff. New York, John
Wiley & Sons, Inc., 1995, 803-861.
16-dipeptide isosteres 4/12/01 10:39 AM
 Methods for Constraining Peptide Cis-Amides

N-Methyl amides
• The cis-amide is only 0.6 kcal/mol higher in energy
than the trans isomer.
Me O R3
O N OH
• The increase in flexibility is tempered by the steric
N restrictions imposed by the Me group. ψi-1 is restricted
ψi-1 H to 60<ψ<180 in both the cis- and trans-amide.
R2 O
H 2N R1

Tetrazoles
• The tetrazole locks the amide in a cis configuration,
and is more easily synthesized than a cis-olefin isostere.
N
N N
• Similar or enhanced activity of a tetrazole
R1 N R2 peptidomimetic indicates a cis-amide bond is favorable
for receptor binding.
NH
O • Absence of activity can be due to the increased steric
bulk of the tetrazole, not necessarily that a cis-amide
bond is not the bioactive conformation.

17-cis amides 4/12/01 10:39 AM

 Pyrrolinone Peptidomimetic Scaffold
O HN O
R2 R4 H
2 • The pyrrolinone backbone fixes φ, ψ, ω to
H 2N 1 the angles near that of an anti-parallel
R1 3
HN O
R3
HN β-pleated sheet (ideal is φ = –139°, ψ = 135)

• Oligomer is amenable to iterative synthesis

NH O NH
Rn+2 Rn for a wide variety of amino acids.

H Rn+3 Rn+1
O NH O
N3
Me O
O

Me OMe OHC OSEM

N3 OMe
NHBoc NH2
Me Ph NH2
OHC OMe
Me Me Me

O
ring 3 ring 2 ring 1 stereocenter 4

O i-Pr HN O i-Pr H O i-Pr HN O i-Pr H

2 OSEM 2 OH
N3 1 H 2N 1
3 3
HN O HN HN O HN O
Ph Ph

N3 NH2

Smith Org. Lett. 2000, 2, 2037-2040

18-pyrrolidines 4/12/01 10:37 AM
 Retro-Inverso Peptidomimetics
R1 O R3
H
N • The reversal of the amide bond direction minimizes peptidase
N N degradation, thereby increasing the mimetics' in vivo half-life
H H dramatically.
O R2 O
• The inversion of each sterocenter from L to D preserves the 3-D
Normal peptide orientation of the side chain residues of the original peptide.

R1 O R3 • Reversal of the carboxyl and amino termini reverses the charge

H structure of the peptide and may be the cause of their low biological
N
N N activity.
H H
O R2 O • End group modifications increased the complementarity of the
peptidomimetic with the native peptides and has resulted in several
Retro-inverso peptide potent peptidomimetics. A retro-inverso section can also be
embedded in a larger normal peptide.
R1 O R3
H
N OH
H2N N
H H-Thr-Lys-Pro-Arg-OH
R2 O O
HO Me
Tuftsin
End group modified O O
retro-inverso peptide
• Tuftsin is a immune system H2N N N
H
stimulator which is completely (CH2)4
degraded in vivo in 8 min. H2N
HN O
• The retro-inverso peptidomimetic H
N NH2
shows less than 2% hydrolysis after HOOC
50 min and retention of bioactivity.
NH2+
Verdini J. Med. Chem. 1991, 34, 3372-3379 Pseudopeptide

19-retro-inverso 4/12/01 10:38 AM

 β-Turn Non-peptidomimetics

R2 O R3 Representative β-turn mimetics

N
HN H O
HN O O
O Me S Me R3
R1 R4 S
R1
NH 7 Å O
O N R2
H2N
β-turn H2N COOH O COOH

• β-turns are the most frequently mimicked protein

secondary structures. O
H 2N

H N
• A β-turn is defined as a tetrapeptide sequence where the O
H
CO2H
distance between α-Ci and α-Ci+3 is ≤ 7Å. The turn can be N

stabilized by chelation of a cation, such as Ca2+, or

O O
intramolecular hydrogen bond(s). COOH NHBn
HN
• Ideal mimic will have a rigid scaffold that orients the NH
sidechain residues in the same direction as the natural
O O OH
peptide, while conferring better solubility and/or resistance
NH2
to enzymatic degradation.

20-b-turn 1 4/12/01 12:36 PM

 Development of β-Turn Mimetics
H 2N

O • A folded structure was thought to be present in the

receptor-bound conformation of the immunoregulator
N tuftsin.
HN O

Me O HN • 1 was the first β-turn mimetic ever synthesized.

NH • 1 exhibited some bioactivity in a dose dependent
HO NH2 O manner, but extensive studies were not undertaken.
N
OH H NH2
Tuftsin

1. m-CPBA 1. Jones oxid. Beckmann

2. BF3•OEt2 2. NH2OH•HCl 52% y.

(3 steps) N
CuMgBr HO N O H
)2 HO
87% y.

H2N CN 1. Hg(OAc)2
CN 2. NaBH4
H CN
H
1. H2, Pd/C 1. O3 H 56% y.
N
N 2. H2, Rh/Al2O3
2. O N
78% y. (4 steps)
O P CN
O
EtO O
1 CN OEt

NH2
Kahn Tetrahedron Lett. 1986, 27, 4841-4844
21-b-turn 2 4/12/01 10:47 AM
 β-Turn Mimetics for Solid Phase Synthesis
1. SOCl2, HO
HO • The peptide sequence -Asn-Pro-Asn-Ala- has been
MeOH, 93% y. CO2Me identified as a key target to mimic for developing an
COOH effective malaria vaccine. It is present on the surface of
2. BnOCOCl N H Plasmodium falciparum parasite which is recognized
N H
H Na2CO3, 84% y. Cbz by the human immune system.

• Solution phase NMR studies indicate a preference

1. TMSCl, Et3N for a Type I β-turn in the -Asn-Pro-Asn-Ala- region.
2. LDA,
BrCH2CO2t-Bu • Solid phase amino acid synthesis facilitates the
3. 10% citric acid screening of a number of turn mimetics to find the
72% y. (3 steps) optimal geometry/stability.

PhthN HO
1. PPh3, DEAD O
CO2Me PhthNH, 74% y. CO2Me O
O O
N N
2. H2, Pd/C; NH O
H Ot-Bu separation, 47% y. Cbz Ot-Bu H2N
N
(mixture) OAllyl

1. Fmoc-Asp(OAllyl)-OH, O
DCC, HOAt
2. 5% DBU, 64% y. (2 steps) 1. Tentagel-SAC,
pyridine
O O 2. piperidine, DMF
O O
t-BuO HO
1. MeNHNH2
NH O NH O
PhthN FmocN
N 2. Fmoc-Succ, imidazole N
OAllyl OAllyl
DMAP, 58% y. (2 steps)
O 3. 60% TFA, CH2Cl2, 97% y. O

Robinson Synlett. 1999, 429-441

22-b-turn 3 4/12/01 10:48 AM
 Elaboration of Turn Mimetic
O

N O
N H
O
MttHN O HN
O 1. peptide coupling: Me
O HBTU, HOBt, Fmoc-aa, NH O
DIEA, DMF O NH
NH O Me Me
H2N FmocHN
N 2. Fmoc removal:
OAllyl O O
piperidine, DMF O
HN
O 3. repeat sequence N
AllylO HN
O
O

N O
H 1. Pd(PPh3)4, NMM, AcOH, DMF
O N
O 2. piperidine, DMF
H2N HN 3. BOP, DIEA, NMP
• Attachment of this peptidomimetic
to a carrier protein and injection into Me 4. TFA
NH
mice resulted in production of anti- O (Mtt = methyltrityl)
sera to malaria sporozoites. O
Me NH
HN Me
• NMR studies of the macrocycle
indicate the illustrated φ and ψ were ψ
O
O O
in close agreement with a Type I β-turn. HN
H N φ
HN
O
O
OH Robinson Synlett. 1999, 429-441
23-b-turn 4 4/12/01 10:54 AM
 Morphine: a Non-peptidomimetic For Enkephalin

Me Ph
R N O O O
N H H
H2N N N
N N OH
OH H H
O O Me

Enkephalin Me
O O HO
HO HO OH
O

R = Me: Oxymorphone, µ agonist Morphine

R = Allyl: Naloxone, µ antagonist
Message: Spacer Address:
Binds to the Sequence bound by
receptor carrier proteins to
direct the peptide to
the correct receptor
N after synthesis

OH
• Morphine, one of the oldest known analgesics, binds to the opiate
receptors. Three receptors have been identified: κ, µ, and δ.
Enkephalin is specific for δ, while dynorphin binds to the κ receptor.
O N An endongenous µ peptide ligand has not been identified yet.
HO
H
• Exact nature of the binding interaction between either natural or
Naltrindole synthetic ligand and a receptor has yet to be elucidated.

δ antagonist

Portoghese J. Med. Chem. 1990, 33, 1714-1720

24-morphine 4/13/01 10:33 AM

 Determination of Active Secondary Structure

H-Tyr-Gly-Gly-Phe-Leu-OH
Enkephalin

R2 R2 Ph
O O O
HN R3 O Me
R1 R3 Me
N R1
O N N
H N
NH NH CO2H
O H-Tyr-Gly-Gly NH
γ-turn 1

• 1 was designed to probe whether enkepalin's bioactive conformation

contained a γ-turn. This turn mimetic concept had successfully been
used in developing inhibitors of thrombin, a protease involved in blood
coagulation.

• No binding activity was observed at any of the opiate receptors.

• Does enkephalin then not contain a γ-turn?

• Negative results are inconclusive, not proof of the absence of the

element in question.

Factors affecting binding: ring conformation, dipole, steric fit, hydrogen bonding, hydrophobicity

Huffman Tetrahedron 1993, 49, 3479-3488

25-g turn 4/12/01 11:35 AM
 Somatostatin: Early Investigations

• Somatostatin is a cyclic peptide formed by a disulfide

bond between two Cys residues, and found in the brain
and digestive organs.
H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp
• Biological activity includes the inhibition of growth
hormone, regulation of lymphocyte production in the
HO-Cys-Ser-Thr-Phe-Thr-Lys brain, regulation of glucagon/insulin pathway (in
pancreas), and digestive tract motility and blood supply.
Somatostatin
• Main theraputic use is in the treatment of ulcers and
bleeding in the gastrointestinal tract, but somatostatin
Suspected β-turn has a prohibitively short half life in vivo.
in boxed region

H-D-Phe-Cys-Phe-D-Trp

Thr(ol)-Cys-Thr-Lys

Octreotide
β-turn
• Octreotide has similar biological activity to
3.2 Å
somatostatin and is a drug of first choice for the
treatment of several carcinoid syndromes.
Thr(ol) = L-threoninol

X-ray Structure
of Octreotide
Synthesis: Pless Life Sci. 1982, 31, 1133-1140
X-ray: Sheldrick Acta. Cryst. D 1995, 48-59

26-somat 1 4/12/01 11:39 AM

 Somatostain Active Site Elucidation

O • L-363,301, first synthesized in 1982, was selectively

H
N 8 methylated at the 7, 8, and 11 positions in the putative β-turn
N 7 region.
H (D) NH
N OO
O • NMR and SAR studies of the derivatives led to a model for
H NH the somatostatin pharmacophore in which residues 8, 9 and
O 11 10 N 9 11 were important for activity.
N
H
O
Me OH
NH2
L-363,301
IC50 = 1 nM

Modification IC50 (nM)

(2S, 3R)-β-Me-7 1
(2S, 3S)-β-Me-7 1
(S)-α-Me-7 >1000
2D-NMR and
(R)-α-Me-7 >1000
(2R, 3S)-β-Me-8 <1
(2R,3R)-β-Me-8 >1000
Analysis of Conformer
(2S, 3R)-β-Me-8 10 Populations
(2S, 3S)-β-Me-8 >1000
(2R, 3S)-β-Me-11 50
(2R,3R)-β-Me-11 >1000
(2S, 3R)-β-Me-11 >1000
Pharmacophore Model
(2S, 3S)-β-Me-11 >1000

Goodman J. Am. Chem. Soc. 1992, 114, 9390-9401

27-somat 2 4/13/01 10:35 AM
 Glucose Scaffold for Non-peptidomimetic of Somatostatin

a
a NH
O
H 8 (D) O O
N
N 7
NH
b
H
N OO R2 O
O
H NH b
O 11 10 N 9 O O
N NHR1
d H
c
O d
Me OH
c 1: R1 = R2 = H
NH2
2: R1 = CH3CO, R2 = OCH2Ph
L-363,301

G-Protein
Coupled
Receptor Distance (Å)
Biological Activity (IC50, µM)
a-b a-c a-d b-c b-d c-d
L-363,301 1 2
L-363,301 7.1 11.3 9.2 7.3 9.2 14.1
somatostatin 0.001 15 1.3 2 5.6 10.6 8.0 6.6 10.3 13.5
low conc.: agonist agonist
high conc.: antagonist (all conc.) • Computer modeling, combined with previous
___ biological studies of somatostain peptidomimetics, led
β2-adrenergic 3 to the design of a glucose-based non-peptidomimetic.
antagonist
• Glucose shows promise in development of
NK1 0.18 0.06 non-peptidomimetics for several G-protein coupled
agonist antagonist receptors, but selectivity must be fine-tuned for the
desired activity.

Hirshmann J. Am. Chem. Soc. 1993, 115, 12550-12558

28-somat 3 4/12/01 12:10 PM
 Peptidomimetic Thrombin Inhibitors

Ph
• Thrombin is a serine protease that promotes blood
N H O coagulation by cleavage of fibrinogen to the active protein fibrin.
N N Contraction and dilation of blood vessels are also affected.
H
O Antagonists could be theraputic in treating arteriosclerosis and
O
thrombosis.
Fibrinogen • Early antagonist design focused on non-hydrolyzable
target sequence
transition state mimetics of the -Phe-Pro-Arg- sequence
NH adjacent to the substrate cleavage site.
HN
NH2

Ph Ph Ph

N H OH Ph O 2S N H
H 2N N N N
B H
O O OH O O

A Ki = 3.6 pM B Ki = 2.5 pM
NH
H2N
HN
NH2

29-thrombin 1 4/12/01 12:17 PM

 Development of Thrombin Peptidomimetics
Me

O
NH O O H
O S N
B H N N
N H
S N O
O O
HO2C Me

HN
C Ki = 39 nM HN D Ki = 6.6 nM
HN NH2
NH2

Me O O
O H H
• Can you assume D is binding in the same
S N N
pocket as the fibrinogen substrate does? N N
H
Me O
• X-ray structural studies indicate C and D
bind to a different site in the enzyme through O Me
Me
their hydrophobic residues,which causes Me
collapse of the active site pocket. Enzymatic
activity is thereby inhibited.
E HN
NH2
more potent than D

Review summarizing the progression:

Gante Angew. Chem. Int. Ed. Eng. 1994, 33, 1699-1720
30-thrombin 2 4/12/01 12:38 PM
 Non-peptidomimetic Thrombin Inhibitors
H NH
N Ph
O
HO NH2
N N HO N H O
N N
O NH H
O O
S OBn
HO S O O
N Fibrinogen
target sequence
LY333545 Ki = 0.9 µM NH
Selectivity
• developed from a library screening lead HN
over trypsin: NH2
Sall Book of Abstracts, 214th ACS 227:1
National Meeting 1997, A58

• developed by using a
H bioactive template (glucose)
N
O and altering the periphery to Ki = 13 nM
NH give desired activity. Angew. Selectivity
N Chem. Int. Ed. Eng. 1997, 36, Me Me over trypsin:
NH2 751-752. >760:1
H
O
N
O
N
Ki = 0.5 µM
O H
• HCl

• developed by computer modeling

studies of the indole nucleus docked • developed by computer modeling of lead
in the thrombin X-ray structure. molecules docked into the X-ray structure. (racemic)
Wery Protein Sci. 1997, 6, 1412-1417 Diederich Chem. Biol. 1997, 4, 287-295 H 2N NH
.

31-thrombin 3 4/12/01 12:20 PM

 Peptidomimetic HIV-1 Protease Inhibitors

OH
H-Ala-Ala-NH Val-Val-OMe

O Ki = 18-180 nM • HIV-1 protease is an aspartyl protease, meaning that

Ph the active site of the enzyme contains the triad
(in vitro against
protease) -Asp-Thr(Ser)-Gly-. The protease is involved in
synthesis of the virion's structural proteins. Inhibition of
the protease results in non-infectious virions.
Dreyer Proc. Natl. Acad. Sci. 1989, 86, 9752-9756
• The first approach to inhibition was to form
Ph transition-state analogues, where the scissile amide
O H bond was replaced with a non-hydroyzable peptide
isostere.
Ac-Ser-Leu-Asn-NH P N Ki = 8.2 nM
(in vitro against • Most isosteric replacements centered around the
O-
protease) Phe-Pro cleavage site in the protease substrate.
O
Ile-Val-OMe

Janda J. Am. Chem. Soc. 1992, 114, 7604-7606

• Saquinivir has an IC50 = 2 nM in HIV infected cells.

Ph H
O
H • Excellent selectivity for HIV protease over human
N H proteases was observed. Less than 50% inhibition of apartyl
N N N
H proteases was seen at 10 µM.
O OH
CONH2 • Oral bioavailabitlity is good (plasma levels stay above
O NHt-Bu
required inhibitory concentration for several hours.)
Saquinavir (Ro 31-8959)
• This drug has been through clincal trials and is currently
Roberts Science 1990, 248, 358-361 an established drug for the treatment of AIDS.

32-hiv protease 1 4/12/01 12:29 PM

 Non-Peptidomimetic HIV-1 Protease Inhibitors

Me
OH

OH Ph

S Optimization
Ph O O
Me HN
Ph O O SO2
Lead Structure
PNU-140690 N
IC50 = 3 µM IC50 = 2.7 nM

CF3
• Lead found in a high volume library screen
at Pharmacia & Upjohn in 1994. The pyrone
inhibits HIV-1 protease but has no anti-viral activity.

• 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), SAR studies,
and pharmacokinetics.

• PNU-140690 is effective against some strains

of HIV-1 that have developed resistance to saquinivir,
indinavir and other peptide-based protease inhibitors.

Hagen J. Med. Chem. 1997, 40, 3707-3711

33-hiv 2 4/12/01 7:11 PM

 Asymmetric Synthesis of PNU-140690

O O O
n-BuLi, THF, -78 °C Me
O NH O N
O
Ph Me Ph
Cl

95% y. 1. CuBr, DMS,

THF, 0 °C
N(TMS)2

MgBr

2. Na2CO3, BnBr,
H2O, CH2Cl2
78% y. (2 steps)

Me
Me 1. TiCl4; O O
O
Hunig's Base;
N(Bn)2 O N
Me
MeO Ph
O Xa OMe
O N(Bn)2
O

2. Aq. HClO4
95% y. (2 steps)

34-pnu synth 1 4/12/01 12:25 PM

 Asymmetric Synthesis of PNU-140690

Ti(On-Bu)Cl3,
Me Me
CH2Cl2, -78 °C; Me
O Hunig's Base; OH O
N(Bn)2
Me N(Bn)2
Ph
O
O Xa
O Xa
Ph
Me 62% y.
dr = 25:1

• Use of Ti(Oi-Pr)Cl3 resulted in an 8:1 dr.

KOt-Bu,
THF, 0 °C
Me
67% y.
OH

Ph Me
OH
O O 1. H2, Pd/C Ph N(Bn)2
Me HN
SO2
O O
2. SO2Cl
N
PNU-140690 Me
N
9 steps, 24% overall y. F 3C
CF3
pyr., CH2Cl2
81% y.
Gammill J. Am. Chem. Soc. 1997, 119, 3627-3628

35-pnu synth 2 4/10/01 8:05 PM

 Tapinavir (PNU-140690) Process Route

OLi CO2H CO2H

1.
0.5 eq (1R,2S)-
O OEt norephedrine
Me OH Me OH
OH
Ph Me MeCN,
2. NaOH, MeOH crystallization Me
Ph 1 Ph Ph
95% y. 27% y.
NH2
(unpurified)
(norephedrine
removed by extraction
and recovered)

Et Et Et
isoprenyl
acetate
Amano P30 1. MsCl EtO2C
HO HO
50% y. 2. EtO R
>98% ee CO2Et
NO2 NO2 ONa NO2
(separated
86% R = CO2Et
by chromatography) 6N HCl
R = H (2) 87% y.

(2 crystallized as the
cyclohexamine salt)

36-pnu process 1 4/12/01 7:02 PM

 H Tapranavir (PNU-140690) Process Route
O

Me O Et
OH

O
2, NaHMDS, THF, -80 °C
Ph Ph
CO2Me
90% y. OPOM
Me NO2
(unpurified)

1. PCC, 99% y.
2. H2SO4, 84% y.
Me 3. NaOH, MeOH, 75% y.
OH

Ph
Et
OH
O O 1. H2, Pd/C, quant.
Me HN Ph
(precipitation) SO2
2. SO2Cl O O
N NO2
Me
N
F 3C (recrystallized)
Tapranavir
CF3 pyr., CH2Cl2
13 steps, 4% overall y. 78% y.

Gage J. Org. Chem. 1998, 63, 7356

37-pnu process 2 4/12/01 7:06 PM

 Rational Design of HIV Protease Inhibitor

Analysis of HIV protease

dimer-ligand X-ray structures Pharmacophore model

Ile Ile H-bond O

d/a 3D-database
Left O
Right Results in 3.5-6.5 Å 3.5-6.5 Å search
H H
enzyme enzyme
Asp Asp Aspl Aspr R R
8.5 - 12 Å OH
peptide
Lead structure
optimization
for increased
inhibitor binding

HO OH 1. Set of likely O
O Biological Dock analogs
assays inhibitors
of structure
HN NH
N N
2. Prediction of how
stereocenters will R R
affect binding, and into HIV protease
Ph Ph thereby activity. dimer X-ray structure OH
HO OH HO

IC50 = 36 nM

Lam Science 1994, 263, 380-384

38-hiv imide inhibitor 4/12/01 12:30 PM

 Summary

1. Compared to native peptides, peptidomimetics and non-peptidomimetics offer

significant advantages as drug candidates. The benefits include increased
bioavailability, better transport through cellular membranes, decreased rate of
excretion, and decreased hydrolysis by peptidases.

2. A tremendous variety of constrained amino acids have been developed to rigidify

a particular amino acid or peptide sequence. Control over steric and electronic
factors can be exercised through peptide backbone modificatons as well.

3. 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. For instance, if a β-turn mimetic shows no
activity, the differences in side chain orientation, electronic factors, preferential
binding to another target (in vivo systems), etc. may be the cause.

4. Biologically active mimetics support the existence of the desired element being
present in the receptor-bound conformation of the peptide. However, the binding
site of an antagonist may be different than active site, where the native peptide is
bound.

5. Mimetics are usually designed based on analogy to the native peptide structure,
then optimized to give the best possible pharmacokinetic properties.

6. The forefront of the field is the rational design of non-peptidomimetics using

computer modeling, database searching, combinatorial chemistry and screening
approaches, and analysis of X-ray structure data.

39-conclusion 4/12/01 12:30 PM

 Lead Reviews and References

Giannis, A.; Rubsam, F. Adv. Drug. Res. 1997, 29, 1-78 Excellent coverage of design principles
and recent examples of the major classes of
non-peptidomimetics

Gante, J. Angew. Chem. Int. Ed. Eng. 1994, 33, 1699-1720 Overview of peptide isosteres and review of
Giannis, A.; Kolter, T. Angew. Chem. Int. Ed. Eng. 1993, 32, 1244-1267 peptidomimetics designed for specific biological receptors

Goodman, M.; Ro, S. Burger's Medicinal Chemistry and Drug Comprehensive coverage of constrained
Discovery. Ed. M. E. Wolff. New York, John Wiley & Sons, Inc.; amino acids and peptide isosteres
1995, 803-861.

Liskamp, R. M. J. Recl. Trav. Chim. Pays-Bas 1994, 113, 1-19 Thorough compliation of constrained amino acid
syntheses and secondary structure mimetics

Rieger, D. Evans Group Seminar, 1991 Review of peptide bond isosteres

Ripka, A. S.; Rich, D. H. Curr. Opin. Chem. Bio. 1998, 2, 441-452 Most recent coverage of biologically
active peptidomimetics

Fauchere, J.-L.; Thurieau, C. Adv. Drug. Res. 1992, 23, 127-159 Review of peptidomimetic biological stability

Marshall, G. R. Tetrahedron 1993, 49, 3547-3558 Concise explanation of peptidomimetic design process

Hirshmann, R. Angew. Chem. Int. Ed. Eng. 1991, 30, 1278-1301 Overview of medicinal chemistry with a section on the
beginning of peptidomimetics

40-reviews 4/10/01 8:22 PM