Review

pubs.acs.org/CR

Orally Absorbed Cyclic Peptides

Daniel S. Nielsen,†,‡ Nicholas E. Shepherd,†,‡ Weijun Xu,†,‡ Andrew J. Lucke,†,§ Martin J. Stoermer,*,†
and David P. Fairlie*,†,‡
†
Division of Chemistry and Structural Biology, and ‡Australian Research Council Centre of Excellence in Advanced Molecular
Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia

ABSTRACT: Peptides and proteins are not orally bioavailable in mammals, although a
few peptides are intestinally absorbed in small amounts. Polypeptides are generally too
large and polar to passively diffuse through lipid membranes, while most known active
transport mechanisms facilitate cell uptake of only very small peptides. Systematic
evaluations of peptides with molecular weights above 500 Da are needed to identify
parameters that influence oral bioavailability. Here we describe 125 cyclic peptides
containing four to thirty-seven amino acids that are orally absorbed by mammals.
Cyclization minimizes degradation in the gut, blood, and tissues by removing cleavable
N- and C-termini and by shielding components from metabolic enzymes. Cyclization
also folds peptides into bioactive conformations that determine exposure of polar atoms
to solvation by water and lipids and therefore can influence oral bioavailability. Key
chemical properties thought to influence oral absorption and bioavailability are analyzed,
including molecular weight, octanol−water partitioning, hydrogen bond donors/
acceptors, rotatable bonds, and polar surface area. The cyclic peptides violated to different degrees all of the limits traditionally
considered to be important for oral bioavailability of drug-like small molecules, although fewer hydrogen bond donors and
reduced flexibility generally favored oral absorption.

CONTENTS 4.7. Nepadutant 8104

4.8. Bouvardin 8104
1. Introduction 8095 4.9. Pristinamycin and Related Antibiotics 8105
1.1. Absorption 8095 4.10. 1-NMe3 and Related Cyclic Hexapeptides 8105
1.2. Predicting Absorption 8095 4.11. Cyclo-[Arg-Arg-Arg-Arg-NaphthylAla-Phe] 8107
1.3. Metabolism 8096 4.12. Kahalalide F 8107
1.4. Oral Bioavailability versus Oral Activity 8096 5. Cyclic Heptapeptides 8107
1.5. Formulation and Pharmacokinetics 8097 5.1. Sanguinamide A and Danamides 8107
1.6. Review Scope 8097 5.2. Rhizonin A 8108
2. Cyclic Tetrapeptides 8098 5.3. Microcystin LR 8108
2.1. CJ-15208 8098 5.4. YM254890 8108
2.2. Apicidin and Chlamydocin 8098 5.5. CHEC-7 8108
2.3. Beauveriolides 8098 5.6. Polymyxin B1 and B2 8108
2.4. HIV Fusion Inhibitors 8098 5.7. Bacitracin A 8109
3. Cyclic Pentapeptides 8099 6. Cyclic Octapeptides 8109
3.1. DMP-728 8099 6.1. PF1022A and Emodepside 8109
3.2. Cyclochlorotine and Astin C 8099 6.2. WH1Fungin 8109
3.3. BL3020-1 8099 6.3. α-Amanitin 8109
3.4. Romidepsin 8100 6.4. Griselimycin and Synthetic Derivatives 8110
3.5. Largazole 8100 6.5. Dihydromycoplanecin and Mycoplanecin 8110
3.6. Complement C5aR Antagonists 8100 7. Cyclic Nona- and Deca-Peptides 8110
3.7. Actinomycin D 8100 7.1. CHEC-9 8110
3.8. Leucine Cyclic Peptides 8101 7.2. AFPep 8111
4. Cyclic Hexapeptides 8101 7.3. Antamanide and Cyclolinopeptide 8111
4.1. Desmopressin 8101 7.4. Cyclopeptolide 1 8112
4.2. Melanotan II 8101 7.5. Permetin A 8112
4.3. Oxytocin 8102 7.6. Synthetic N-Methyl β-Strand Decapeptides 8112
4.4. Anidulafungin, Caspofungin, and Micafun-
gin 8102
4.5. Somatostatin, Octreotide, and Analogues 8102 Received: December 20, 2016
4.6. Beauvericin and Enniatins 8104 Published: May 25, 2017

© 2017 American Chemical Society 8094 DOI: 10.1021/acs.chemrev.6b00838

Chem. Rev. 2017, 117, 8094−8128
Chemical Reviews Review

7.7. Surotomycin 8112 antibodies.18−20 They all need to be given by injection, with few
8. Cyclic Undecapeptides 8112 peptides known to be orally absorbed and hardly any being
8.1. Cyclosporin A and Synthetic Derivatives 8112 truly orally bioavailable. There is now growing interest in
8.2. THR-123 8114 developing molecules with molecular weights between those of
9. Cyclic Dodeca- and Tridecapeptides 8114 conventional drugs and antibodies.18 Here we describe some
9.1. Cerulide 8114 examples of orally absorbed peptides, all being cyclic peptides
9.2. L-Phenylalanine-Dipicolinate Macrocycle 8114 of 4−37 amino acid residues.
10. Cyclic Tetradecapeptides and Beyond 8115 1.1. Absorption
10.1. Conotoxins and Synthetic Derivatives 8115
10.2. Duramycin 8115 When a drug is administered by oral, buccal, sublingual, nasal,
10.3. Kalata B1 and Other Cyclotides 8116 dermal, intramuscular, subcutaneous, pulmonary, and rectal
10.4. Stapled α-Helix 8116 routes, it must be absorbed to enter the bloodstream where it is
11. Influences on Oral Bioavailability 8116 systemically circulated, distributed into tissues, metabolized,
12. Conclusions and Future Prospects 8118 cleared, and excreted. Absorption is the process by which a
Author Information 8120 drug moves unchanged from the site of administration to the
Corresponding Author 8120 site of measurement in the organism.21 Absorption from the
ORCID 8120 gastrointestinal (GI) tract after oral administration occurs
Present Address 8120 mainly from the small intestine. Small finger-like folds (villi)
Notes 8120 coating its surface generate a much larger surface area (200 m2)
Biographies 8120 for compound absorption compared to the stomach (1 m2).21
Acknowledgments 8120 The small intestine environment (pH 6−7.5) aids absorption
Abbreviations 8120 because many molecules are uncharged at this pH and can
References 8121 passively diffuse through intestinal membranes. Passive
diffusion is the most common way drugs and many nutrients
are absorbed from the GI tract into plasma. Passive diffusion of
1. INTRODUCTION drugs is most commonly transcellular (through cell membranes,
Proteins and peptides are the largest group of naturally > 90% of drugs) rather than paracellular (through tight
occurring mediators of biological and cellular processes, ranging junctions between enterocytes, 5−10% of drugs). Charged and
in size from small peptide hormones to large multidomain hydrophilic molecules such as peptides are not well absorbed
polypeptides and proteins.1 Proteins bind with high specificity via passive diffusion mechanisms. Membrane-bound transporter
and potency irrespective of whether interactions are localized to proteins also mediate absorption of amino acids, very small
one or more small binding pockets (“hot spots”)2−4 or more peptides, nucleosides, sugars, ions and hydrophilic molecules
dispersed across larger surface areas. When protein function is via facilitated diffusion and active transport.22−26 Studies on
localized, protein−protein interactions (PPIs) can potentially peptide and protein transport across gastrointestinal mucosal
be interfered with by small drug-like molecules.5 However, membranes is still in relative infancy, understanding still
more often than not the bioactive protein interfaces span large confined mainly to single amino acids and small peptides and
surfaces and rely upon multiple weak contacts for affinity and the biotin, transferrin, and glucose transporters. The human
selective recognition. In these cases, an alternative approach to intestinal peptide transporter SLC15A1 is one example of a
modulating PPIs is to mimic one of the interacting protein proton-dependent protein that transports very small peptides
surfaces by downsizing it to smaller peptides or peptidomi- (2−4 residues) via facilitated diffusion.27 SLC15A1 also
metics. These are larger than small molecule drugs, frequently facilitates absorption of certain peptide mimetics and peptide-
require molecular constraints to stabilize structure in water, and like drugs such as ACE inhibitors and β-lactam antibiotics.28
are usually not orally bioavailable.6−13 Conversely, some transporters in enterocytes (e.g., P-
During the last 20 years the pharmaceutical industry has glycoproteins) act as a barrier to absorption,29,30 actively
almost universally adopted drug-like property filters, such as the expelling peptides and drugs31 back into the GI tract.
“rule-of-five” (RO5)14−16 and related parameters.17 These have 1.2. Predicting Absorption
guided the design and development of orally bioavailable
modulators of macromolecules, deliberately focusing attention Predicting oral absorption of drugs in animals is complex and
on small molecules restricted to MW < 500. In the era of experimental measurements are low throughput. Attempts have
postgenomics, transcriptomics, and proteomics, it is time to been made to develop in vitro or ex vivo methods to assess
focus more attention on larger modulators of proteins that can membrane permeability. Although no in vitro or ex vivo model
span larger surfaces, access new therapeutic mechanisms of correlates well with in vivo parameters, some methods have
action, and provide greater target specificity.18 This has been allowed estimates of relative passive membrane permeability.
spectacularly demonstrated by antibodies and some proteins However, others factors (membrane uptake via endocytosis,
that have spearheaded a paradigm shift in the pharmaceutical transporter proteins, protein-binding, stability to proteolytic
industry toward larger therapeutic molecules.19 Proteins and and oxidative/reductive enzymes in the gut, intestine, tissues,
polypeptides are expensive to manufacture, chemically unstable plasma and liver, clearance rate and first pass metabolism) also
(degraded by pH, heat, oxidation, and proteases), difficult to contribute to oral bioavailability.
store, very flexible in water, immunogenic, have low membrane The parallel artificial membrane permeability assay
permeability, and poor oral bioavailability. Despite these (PAMPA)32 has been used widely in drug discovery as a high
limitations, many peptides and polypeptides are in clinical throughput assay to estimate passive diffusion across a
trials and a few are registered drugs, mainly naturally occurring membrane. The artificial membrane lacks transporter proteins
peptides, their semisynthetic derivatives, cyclic peptides, or and so only estimates permeability via passive diffusion.
8095 DOI: 10.1021/acs.chemrev.6b00838
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Chemical Reviews
Caco-2 cells are heterogeneous human epithelial colorectal
adenocarcinoma cells grown under conditions to mimic
absorptive cells of the small intestine.33 Their microvilli,
metabolic enzymes (e.g., peptidases and esterases), transporter
proteins, and bile salts better mimic the physiological
environment.34 PAMPA and Caco-2 assays can indicate
whether a compound is passively or actively transported across
epithelial cells. Madin-Darby canine kidney (MDCK) cells
isolated from dog kidney cortex35 retain many properties of the
kidney tubular epithelium. Morphologically, MDCK cells
exhibit apical microvilli, junctional complexes, and lateral
membrane infoldings36−39 characteristic of transporting epi-
thelia.40 Physiologically, MDCK cells transport sodium and
water in an apical-to-basal direction. When grown on
permeable substrates, MDCK cells generate transepithelial
electrical resistance indicating functioning tight junctions.41
These properties resemble Caco-2 cell and other intestinal tract
cells, making MDCK cells a viable model for measuring in vitro
permeability despite their anatomical origin. Like Caco-2,
MDCK cells have uptake and efflux transporter-proteins, but
their canine origin may endow different affinity, selectivity, and
activity for substrates compared to human proteins and cells.
Therefore, caution should be exercised in interpreting these
results.42 MDCK monolayers are faster to culture (3−7 days)43
than Caco-2 (14−28 days), making them useful for high
throughput permeability assays. A derived cell line, MDCKII-
LE (low efflux) was developed from subpopulations of MDCK
cells.44 MDCKII-LE have greatly reduced expression of canine
mRNA/protein and low active uptake/efflux properties making
them a live cell alternative to the artificial PAMPA assay. There
have been many recent reviews of cell and membrane
permeability of cyclic peptides and other macrocycles,45−50 so
we will not be covering that literature here. While this is of
importance, it is not the only contributor to oral absorption and
oral bioavailability. Many membrane and cell permeable
compounds show negligible oral bioavailability.
Instead of measuring transport in vitro across cells, an Ussing
chamber51 can be used to estimate oral absorption by
measuring ex vivo membrane transport of ions, nutrients, and
drugs across mouse52,53 or rat54,55 intestinal tissue. However,
such experiments also correlate poorly with oral bioavailability
(F%) in rodents and humans. Thus, the Ussing chamber has
become less popular in industry over the past decade.
1.3. Metabolism
Orally administered compounds face many obstacles en route
to the plasma. Compounds of high molecular weight, high
lipophilicity or low solubility are recognized and degraded by
metabolic enzymes. Orally ingested compounds are first
exposed to digestive amylases in the saliva, where glycosidic
bonds of starch and other carbohydrates are hydrolyzed. Then
in the stomach, acidic (pH 1−2) gastric juice containing
peptidases begins to degrade proteins and other nutrients.
Upon entering the duodenum, the pancreas excretes additional
enzymes (proteases, lipases, amylases), bile salts and pH-
neutralizing bicarbonate. Next, in the jejunum and ileum (small
intestine) the pH is 6−7.5 and most processed nutrients and
drugs are absorbed here. Intestinal P450 enzymes can
metabolize compounds even before they enter the bloodstream
and thus reduce measurable oral absorption. Once absorbed
from the GI tract, compounds enter the hepatic portal vein,
flow into the liver and are perfused into hepatocytes for first-
pass metabolism by cytochrome P450 (CYP450) enzymes and
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flavin-containing monooxygenases, monoamine oxidases and
NADPH−CYP450 oxidoreductases. At this stage the com-
pound can be (1) reabsorbed back into the bloodstream,
sometimes unchanged; (2) metabolized and then absorbed into
the bloodstream; or (3) combined with bile salts and excreted
back into the GI tract.
Metabolism of peptides can occur at all stages, while
traversing the GI tract, in intestinal tissues, in the bloodstream,
in the liver, and other organs and tissues. Peptides can be
hydrolyzed by a myriad of proteases in the gut, plasma, and
cells, for example, pepsin and HCl in the stomach; trypsin,
chymotrypsin, elastase, and carboxypeptidases in the small
intestine; and, by thrombin, plasmin and clotting factors in
blood plasma degrade peptides. The CYP450 monooxygenases
and oxidoreductases in the intestinal lining and in the liver
catalyze carbon hydroxylation and epoxidation, heteroatom
oxygenation and release. CYP450 enzymes account for > 75%
of drug metabolism within the liver.56 Substances with good gut
absorption can still have low oral bioavailability if metabolism in
the intestinal lining, liver, plasma, or elsewhere is high, or
clearance is rapid. It is a misconception that high gut
permeability equates to high oral bioavailability. Stability to
metabolic enzymes, especially in the gut, liver, and blood, is
important when predicting oral activity of membrane-
permeable compounds.
Finally, a relatively new consideration is the influence of the
microbiome on drug and nutrient metabolism. Differences in
oral bioavailability between people or animals are often
attributed to differences in metabolic enzymes or their
efficiencies that change with age, genetics and environment.
Until now, the influences of symbiotic bacteria inhabiting the
gut on oral drug stability, metabolism and absorption have not
been considered much and this is likely to be important to
study in the future.
1.4. Oral Bioavailability versus Oral Activity
Oral bioavailability (F) is the fraction of an orally administered
compound that reaches the systemic circulation intact.
Absorption alone is not a good predictor of oral bioavailability,
since extensive first-pass effects in the liver and intestine can
lead to poor systemic exposure. Thus, blood is usually sampled
from the jugular, rather than portal, vein to take into account
first pass metabolism. Oral bioavailability is defined as the ratio
of the amounts of drug found in plasma after intravenous (iv)
versus per oral (p.o.) dosing, with the iv dose representing
100% bioavailability.21
AUCpo dose iv
F(%) = 100 × ×
dose po AUCiv
For clinical trials, drugs normally need to have F > 20%.
Other parameters described in this review that relate to oral
bioavailability include the following: the area under the curve
(AUC), used to express the cumulative amount of drug found
in plasma over a period of time; the clearance rate (CL), the
volume of plasma from which a drug is completely removed per
unit time; the peak serum concentration (Cmax), the time to
reach peak serum concentration (Tmax); the half-life (t1/2), time
taken for serum drug concentration to decrease by half its
original amount; and the volume of distribution (VD), the
apparent volume in which the drug is distributed at steady state.
These parameters provide quantitative estimates of the
compound absorbed and surviving first pass metabolism.
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Chemical Reviews
Oral activity is often used as a surrogate to infer oral
bioavailability. Orally dosing an animal and observing a
therapeutic effect provides only qualitative evidence of oral
bioavailability. We have included reports for orally active
peptides in this review even though many such molecules are
poorly absorbed; most peptides and proteins are < 1% orally
bioavailable. We urge caution in drawing robust conclusions
about oral bioavailability of molecules from such studies. The
observed biological effect could occur by indirect mechanisms,
from metabolites or may be due to exceptional potency of trace
material absorbed. The main measures of oral activity used in
this review are the oral dose (mg/kg p.o.) required to induce a
measurable response; the effective dose for 50% of the maximal
response (ED50); and the lethal oral dose required to kill 50%
or 100% of the test population (LD50 and LD100).
1.5. Formulation and Pharmacokinetics
When comparing pharmacokinetic parameters for compounds
tested in separate studies, it is important to note possible
differences in experimental design. Compounds are usually
formulated with a solvent, vehicle, or matrix, which can
profoundly affect their solubility, rate and location of
dissolution, and permeability across intestinal membranes.
Formulation design is a crucial step in drug delivery. Some
formulations simply increase chemical stability or solubility at
different pH, in aqueous or lipophilic environments. Other
formulations are optimized to release the compound at a
specific location in the gut, in tissues or in target organs or cells.
Often an excipient is added to enhance membrane permeability
or oral absorption. Thus, formulation is a key determinant of
pharmacokinetic parameters, which are highly dependent on
the conditions under which they are measured.
Proteins and peptides have been formulated in many ways in
attempts to increase oral absorption57−63 including with (i)
enzyme inhibitors to reduce proteolysis in the GI tract (e.g.,
sodium glycocholate, trypsin inhibitors, camostat mesylate,
bacitracin, and ovomucoids); (ii) absorption enhancers to
improve intestinal permeability (e.g., detergents, surfactants,
bile salts, and chelating agents); (iii) mucoadhesive polymers to
improve delivery and permeability (PEGs, P(MAA-g-EG),
lectin microparticles, and thiolated polymers); (iv) formulation
vehicles to protect drug and improve permeability (emulsions,
liposomes, cyclodextrins, microspheres, micelles, and nano-
particles).
As shown above, absolute oral bioavailability (F%) is defined
as the dose-corrected ratio of accumulated concentrations in
plasma (AUC) following an iv injection (AUCiv: 100%
bioavailability by definition) and an oral dose (AUCpo). This
dose-corrected absolute value allows direct comparisons to be
made across different studies conducted at different doses. In
reality, as dose of the test compound is raised, physio-chemical
boundaries and saturation of the biological system will
increasingly affect solubility, absorption, active transport
mechanisms, and metabolism. Other pharmacokinetic param-
eters, including AUC and Cmax, are dose-dependent as they are
expressed in terms of compound concentration in plasma and
will therefore depend on the administered dose.
The animal model is an important consideration. Biological
variation allows different species, even different strains of the
same species, to metabolize drugs in different ways, making
comparisons across species and even strains difficult. For
technical reasons, it also may not be possible to perform
identical experiments in two species, even rodents like mice and
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rats. Pharmacokinetic investigations in rats often involve
sampling blood at multiple time points from the same animal
through a surgically implanted line. This is more difficult for
mice, for which studies usually involve larger groups of animals,
so that multiple mice can be sacrificed at each time point.
Pharmacokinetic parameters measured by the latter method
provide less accurate results due to variation within animals
(metabolism, total blood, body volume, individual weight, etc.)
or their environments. It is therefore prudent to carefully
consider parameters such as dose, species of test animal, and
experimental design before drawing conclusions.
1.6. Review Scope
Most proteins and peptides show negligible oral bioavailability
(F < 1% and usually F < 0.1%). Very few peptides are
sufficiently orally absorbed to produce some physiological effect
in an animal. Cyclic peptides are the most orally bioavailable
peptides known, but only a handful show F ≥ 10%. Other types
of macrocycles are orally absorbed, but the reader is directed to
other reviews for these.64−67 This review covers only orally
absorbed cyclic peptides reported up until 2016, including
some that are absorbed in only trace amounts but sufficient to
induce a physiological response. Cyclic peptides have been
classified herein according to the number of residues in their
macrocyclic portion. For some highly modified cyclic peptide
natural products, we have grouped them with cyclic peptides of
equivalent macrocycle size (e.g., largazole is grouped with
romidepsin). In cases where a related series of different sized
cyclic peptides were developed for the same target, we have
grouped them together based on the smallest cyclic peptide in
the series in order to streamline discussion and avoid repetition.
The main focus of this review is on cyclic peptides larger than
four residues because they violate most, often all, rule-of-five
(RO5)14−16 and associated17 parameters. We have also
included a few representative cyclic tetrapeptides, even though
these usually comply with RO5 parameters. We describe 125
cyclic peptides in total for which there is evidence of oral
absorption, oral activity, or oral bioavailability. Where possible,
we have indicated relevant doses, formulations, pharmacoki-
netic parameters, and pharmacological activities. In some cases,
there was evidence of solid state or solution conformations that
indicated rigidity or flexibility that likely influence absorption.
These are referenced to codes in the Protein Data Bank (PDB)
or Cambridge Crystallographic Data Centre (CCDC).
Our review concludes with an analysis of this compound set
for influences of physicochemical parameters normally
considered to affect oral bioavailability. A recent review68 on
bioactive linear and cyclic peptides from the ChEMBL database
only examined eight cyclic peptides that were orally
administered. For the cyclic peptides that follow, we have
calculated molecular weight (MW), the predicted octanol−
water partition coefficient (Molinspiration LogP, miLogP), the
number of hydrogen bond donors (HBD) and hydrogen bond
acceptors (HBA) as strictly defined by Lipinski and colleagues,
the number of rotatable bonds (RotB), and the topological
polar surface area (tPSA), all determined with the aid of the
Molinspiration webportal (http://www.molinspiration.com).69
We plot each of these six parameters against oral bioavailability
(F%), for those compounds where oral bioavailability has been
reported. We then discuss how the findings relate to the RO5
and associated guidelines commonly used to predict oral
bioavailability of drug-like small molecules. Systematic evalua-
tion of more peptides with MW > 500 are needed to determine
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Chemical Reviews
allowable upper limits for molecular properties (MW, logP,
HBA, HBD, RotB, and PSA) that influence (a) passive
(unassisted) permeability of cells and oral bioavailability and
(b) active or facilitated transport mechanisms that promote
uptake of peptides > 5 amino acids from the gut. This article
takes one important step toward identifying how these
molecular properties vary in orally absorbed cyclic peptides.
2. CYCLIC TETRAPEPTIDES
Cyclic tetrapeptides are generally RO5 compliant, with MW <
500, HBD = 4, ‘HBA’ = 8, and LogP = 0−5 if bearing
hydrophobic side chains. They generally have few rotatable
bonds and a small surface area and so, not surprisingly, they are
also often orally absorbed and orally bioavailable to some
extent. Cyclic tetrapeptides are typically used to stabilize a β-
turn conformation, with proline and D-amino acids often
promoting β-turn formation.
2.1. CJ-15208
Cyclic tetrapeptide CJ-15208 cyclo-(Phe-D-Pro-Phe-Trp) (1,
Figure 1), isolated from the fungus C. serratus ATCC 15502,
showed dose-dependent antinociceptive activity in mice
measured from 20 to 80 min after oral administration (1 mg/
kg p.o.). When given at a higher dose (10 mg/kg p.o.), 1
antagonized a centrally administered selective κ-opioid receptor
agonist, suggesting that 1 is brain permeable.70 This cyclic
tetrapeptide has only one RO5 violation (MW > 500), a D-
proline ring that removes one peptide NH hydrogen bond
donor, and hydrophobic side chains, making it suitable for oral
absorption.
Figure 1. Cyclic tetrapeptide CJ15208 (1). MW = 578; miLogP = 2.8;
HBD = 4; HBA = 9; RotB = 6; tPSA = 120 Å2.
2.2. Apicidin and Chlamydocin
Cyclic tetrapeptides 2 and 3 are analogues of 1 and are RO5
compliant except for MW (Figure 2). Apicidin (2) is a cyclic
tetrapeptide metabolite isolated from cultures of F. pallid-
oroseum. It is a histone deacetylase (HDAC) inhibitor and has
been shown to kill protozoa such as Plasmodium sporozoites.
When administered in DMSO/PEG400/saline (15:20:65) by
oral gavage, oral bioavailability varied slightly (10 mg/kg p.o.; F
= 14% (fasting), 19% (nonfasting); Cmax = 235 ng/mL, Tmax =
66 min, t 1/2 = 54 min, V D = 2.5 L/kg, CL = 62
mL.min−1.kg−1)).71 The apicidin-like natural product, chlamy-
docin (3, Figure 2), was found to inhibit P-815 cell growth in
mastocytoma mouse cells and glial tumor rat cells in vitro
(ED50 = 0.36 ng/mL). When 3 was administered to tumor-
inoculated mice (iv and p.o.) there was no antiproliferative
activity. The difference between cell and animal findings was
attributed to in vivo inactivation of the epoxide in blood.
Consistent with this notion, intraperitoneal injections were
more effective than intravenous injections in inhibiting tumor
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growth. For 3, LD50 was 226 (iv) and > 850 (p.o.) mg/kg in
mice and 66 (iv) and 141 (p.o.) mg/kg in rats.72 Both 2 and 3
contain one D-residue, a D-pipecolic acid in 2 and a D-proline in
3. In solution,73 two intramolecular hydrogen bonds defined a
γ- and β-turn in 2, but these were replaced in the crystal
structure from chloroform/methanol73 by intermolecular
interactions (CCDC code: HEWGOG).
Figure 2. Apicidin (2) and chlamydocin (3). 2: MW = 624; miLogP =
3.4; HBD = 3; HBA = 11; RotB = 12; tPSA = 139 Å2. 3: MW = 527;
miLogP = 0.9; HBD = 3; HBA = 10; RotB = 9; tPSA = 137 Å2.
2.3. Beauveriolides
Beauveriolides (Figure 3) are cyclic tetradepsipeptides, with
three amide bonds, one D-residue, and one ester bond, were
isolated from the fungus Beauveria sp. FO-6979. Beauveriolides
are potential antiatherosclerotic agents that reduce cholesteryl
ester synthesis in mouse macrophages via inhibition of acyl-
CoA:cholesterol acyltransferase (ACAT), leading to a reduction
in lipid droplet formation in macrophages. Lipid accumulation
is associated with the development of atherosclerosis, which can
be studied in apoE- or LDL-receptor knockout mice.
Beauveriolide III (5) was administered to apoE- or LDL-
receptor knockout mice for 2 months (25 mg/kg/day p.o.) and
was orally active in mouse models of atherosclerogenesis by
inhibiting ACAT activity.74 The ester bond in depsipeptides is
not as stable as the amide bond in vivo, but does promote cell
permeability before intracellular ester cleavage by esterases.
Figure 3. Beauveriolide I (4) and III (5). 4: MW = 488; miLogP = 4.1;
HBD = 8; HBA = 8; RotB = 12; tPSA = 114 Å2. 5: MW = 488;
miLogP = 4.3; HBD = 3; HBA = 8; RotB = 8; tPSA = 114 Å2.
2.4. HIV Fusion Inhibitors
A critical step in HIV-1 entry into host cells is the protein−
protein interaction between CD4 and gp120. Inhibition of this
protein−protein interaction (PPI) has been the target of many
research groups.75 A library of cyclic peptides was generated to
join two noncontinuous regions of CD4-containing key
residues Phe43 and Arg59 that interact with gp120.75 A six
residue mimic cyclized with succinic acid and ethylenediamine
gave cyclic heptapeptide-like 6 (Figure 4). Optimization of the
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Chemical Reviews
linker-length by inserting extra methylene units and replacing
residues Gln-Gly-Ser with a pimelic acid moiety enabled
inhibition of HIV-1 infection by analogue 7 (Figure 4), which
resembles a cyclic tetrapeptide. Despite retention of an
arginine, with its protonated guanidine at physiological pH
usually being an impediment to absorption, 7 still had
reasonable pharmacokinetic properties in rats (1 mg/kg iv:
Cmax = 102 ± 19 ng/mL, VD = 0.6 L/kg; 10 mg/kg p.o. in
water: Cmax = 866 ± 76 ng/mL, Tmax = 18 min, AUC = 21,770
± 63 min·ng/mL, t1/2 = 73 min, CL = 14.5 ± 0.3 mL/min·kg, F
= 10%).75 Converting 6 to 7 increased RO5 compliance for
MW, HBD, HBA, and RotB, halved the tPSA, and significantly
increased hydrophobicity. Although these changes make 7
smaller and less polar than 6, and are expected to enhance oral
bioavailability, no data for 6 has been reported to confirm this
point.
Figure 4. HIV fusion inhibitors (6) and cyclic tetrapeptide-like (7). 6:
MW = 775; miLogP = −5.2; HBD = 15; HBA = 22; RotB = 13; tPSA
= 363 Å2. 7: MW = 488; miLogP = −0.3; HBD = 8; HBA = 11; RotB
= 9; tPSA = 184 Å2.
3. CYCLIC PENTAPEPTIDES
Cyclic pentapeptides generally have HBD = 5, HBA = 10, LogP
= 0−5 if bearing hydrophobic side chains, but their molecular
weights usually exceed 500, resulting in more rotatable bonds
and larger surface areas. To be orally absorbed, they often
require synthetic modifications.
3.1. DMP-728
DMP-728 (8, Figure 5) is a potent and specific antagonist of
platelet glycoprotein IIb/IIIa complex (GPIIb/IIIa). Like 6 and
7, compound 8 contains an arginine residue that is normally
detrimental to oral absorption. It consists of four conventional
amino acids, including a D-Abu, residues with N- and C-termini
linked by a fifth, unnatural, hydrophobic amino acid, 3-
aminomethybenzoic acid. The pharmacokinetic profile for 8
was measured following oral administration with or without
absorption enhancers to rats (10 mg/kg iv, 8 mg/kg p.o. with
palmitoylcarnitine chloride; Cmax = 0.44 ± 0.21 μg/mL, Tmax =
0.75 h, AUC = 1.38 ± 0.56 μg·h/mL, t1/2 = 2.8 ± 2.1 h, F =
14.6%) and dogs (2 mg/kg iv, 2 mg/kg p.o. with
palmitoylcarnitine chloride; Cmax = 0.41 ± 0.10 μg/mL, Tmax
= 1.0 h, AUC = 1.09 ± 0.22 μg·h/mL, t1/2 = 3.3 ± 0.9 h, F =
20.5%).76 Absorption enhancers significantly improved phar-
macokinetic parameters compared to controls formulated in
microcapsules. The MW, LogP, HBD, and tPSA all violate
limits usually associated with passively diffusing, orally
bioavailable, drug-like small molecules. We speculate that the
N-methyl arginine might promote active transport of this
compound across the intestinal membrane.
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Figure 5. DMP-728 (8). MW = 561; miLogP = −2.3; HBD = 9; HBA
= 15; RotB = 8; tPSA = 236 Å2.
3.2. Cyclochlorotine and Astin C
Cyclochlorotine (9, Figure 6) is a hepatotoxic cyclic
pentapeptide that was isolated from the common rice-infecting
mold P. islandicum.77,78 Its structure consists of natural as well
as unnatural L-amino acids, including β-phenylglycine and 3,4-
dichloro-L-proline. LD50 values were determined in mice (LD50;
iv 0.3, sc 0.5, and p.o. 6.6 mg/kg), suggesting some oral
absorption.78
Figure 6. Structures of cyclochlorotine (9) and astin C (10). 9: MW =
572; miLogP = −2.5; HBD = 6; HBA = 12; RotB = 4; tPSA = 177 Å2.
10: MW = 570; miLogP = −1.0; HBD = 5; HBA = 11; RotB = 4; tPSA
= 157 Å2.
Astin C (or Asterin, 10, Figure 6), a close analogue of 9 is a
plant cyclic peptide isolated from the roots of A. tataricus. The
crystal structure of 10 from chloroform/methanol (CCDC
code: WILXEW) showed a single transannular hydrogen bond
from an Abu NH to the (i, i+3) carbonyl of the β-phenylglycine
carbonyl.79 The remaining hydrogen bond donors and
acceptors are highly solvent exposed. Inflammatory bowel
disease is characterized by activation of T lymphocytes. Astin C
has been shown to lower mouse serum concentrations of TNF,
IL-4, IL-17, and to induce apoptosis in activated T cells. Daily
oral dosing of astin C (2 or 4 mg/kg/day p.o. in 5%
methylcellulose in saline) was shown to protect mice against
TNBS-induced colonic inflammation.80
3.3. BL3020-1
α-Melanocyte stimulating hormone (α-MSH) is a 13-residue
peptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-
Pro-Val) that stimulates the release of melanin by skin
melanocytes. α-MSH also binds to the melanocortin 4 receptor
(MC4R), which modulates food intake and energy utilization.81
The tetrapeptide sequence His-Phe-Arg-Trp, and other
analogues derived from α-MSH, decrease food intake and
elevate energy utilization upon binding to MC4R, making them
attractive targets as antiobesity drugs.82 However, they suffer
from metabolic instability and poor intestinal permeability. A
backbone-cyclized α-MSH analogue based on Phe-D-Phe-Arg-
Trp-Gly-NH2, activated MC4R and had increased oral
bioavailability. One analogue, BL3020-1 (11, Figure 7) was
MC4R selective, had good permeability in the Caco-2 model,
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and enhanced metabolic stability in rat brush border membrane
vesicles (BBMVs). A single oral dose administered to rats (0.5
mg/kg p.o. dissolved in water) led to reduced food
consumption, while repetitive daily oral dosing reduced weight
gain in rats.82 Pharmacokinetic parameters measured in rats
following iv administration (1 mg/kg iv in H2O) indicated t1/2
> 105 min and VD = 2.1 L/kg. Oral administration (10 mg/kg
p.o. in H2O) to rats showed AUC = 24980 ng·min/mL, Cmax =
202 ± 39 ng/mL, Tmax = 37 ± 10 min, and F = 8.5%. We
speculate that charged residues in this compound may help
promote intestinal absorption via facilitated transport. Interest-
ingly, concentrations of ∼5 ng/mL were also found in brain
tissue, indicating some ability of 11 to cross the blood brain
barrier.82
Figure 7. Structure of BL3020−1 (11). MW = 836; miLogP = −0.8;
HBD = 12; HBA = 18; RotB = 13; tPSA = 287 Å2.
3.4. Romidepsin
Romidepsin (Istodax, FK-228, 12, Figure 8) is a cyclic peptide
HDAC inhibitor that possesses potent antitumor activity
against a variety of human cancer cell lines and xenografts.83,84
It contains D-cysteine, D-valine, and (3S,4E)-3-hydroxy-7-
mercapto-4-heptenoic acid residues. Romidepsin is an RO5-
compliant and orally active prodrug, reduction leading to a bis-
thiol, one of which covalently bonds to the catalytic zinc ion in
HDAC enzymes. It was administered orally to mice with
human prostate tumor xenographs (3 × 50 mg/kg/wk p.o.),84
and was also significantly absorbed after oral administration to
rats (10 mg/kg iv; Cmax = 1829 ± 359 ng/mL, VD = 22 ± 7 L/
kg, t1/2 = 5.9 ± 1.2 min, AUC = 25409 ± 8767 ng/mL·min.; 50
mg/kg p.o., AUC = 19712 ± 9168 ng/mL·min, F = 16 ±
7%).83
3.5. Largazole
Largazole (13, Figure 9) is a cyclic depsipeptide natural product
isolated from Symploca sp. collected from the Florida Keys.85
Largazole is one of the most potent HDAC inhibitors known,
with activity at low nM concentrations and selectivity for class I
Figure 8. Structure of romidepsin (12). MW = 541; miLogP = 1.6;
HBD = 4; HBA = 10; RotB = 2; tPSA = 143 Å2.
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HDACs. Largazole has been synthesized by multiple routes
allowing many derivatives to be produced.86−91 A crystal
structure of 13 bound to HDAC 8 (PDB code: 3RQD) shows a
single NH directed to the center of the macrocycle but no
strong evidence of intramolecular hydrogen bonding.92
Largazole analogues cocrystallized with HDAC 8 also do not
show any intramolecular hydrogen bonds (PDB codes: 4RN1,
4RN2).93 Pharmacokinetic parameters measured in rats showed
that 13 was unstable in vivo and rapidly cleared after a single iv
dose (10 mg/kg iv in EtOH/DMSO/PEG400/saline (1:1:1:2);
CL = 76 ± 18 L/h·kg, t1/2 = 0.5 ± 0.1 h, AUC = 134 ± 29 μg·
h/mL, VD = 27 ± 11 L/kg, Cmax = 280 ± 64).94 Largazole 13,
Boc-L-cysteine-largazole disulfide 14, and disulfide homodimer
15 (Figure 9) were administered orally (50 mg/kg p.o. in
polyethylene glycol/glycerol/EtOH/DMSO 60:15:15:15:10)
to female nude mice.95 Oral activities were measured through
in vivo hyperacetylation of harvested HCT116 tumors. Only 13
produced hyperacetylated histones, however largazole free thiol
was found in tumors from animals orally dosed with 13, 14, or
15, indicating that all three derivatives displayed some oral
absorption in mice.
Figure 9. Structures of largazole (13), its Boc-L-cysteine disulfide
derivative (14), and its disulfide dimer (15). 13: MW = 623; miLogP
= 5.3; HBD = 2; HBA = 9; RotB = 12; tPSA = 127 Å2. 14: MW = 716;
miLogP = 2.8; HBD = 4; HBA = 13; RotB = 12; tPSA = 185 Å2. 15:
MW = 991; miLogP = 3.6; HBD = 4; HBA = 16; RotB = 11; tPSA =
220 Å2.
3.6. Complement C5aR Antagonists
3D53 (16, Figure 10), later known as PMX53,96 is a derivative
of the C-terminal turn of human complement protein C5a and
is a potent antagonist of the human C5a receptor C5aR1 (IC50
3 nM against 3 nM C5a).96−100 The Fairlie group designed
3D53 and a range of cyclic pentapeptides97−99 featuring an
endocyclic D-cyclohexylalanine and L-arginine that are im-
portant for binding, an aromatic residue (L-tryptophan or L-
phenylalanine) that is required for antagonism, an L-proline
turn-inducing constraint, and an exocyclic aromatic ring (L-
phenylalanine in 16 or a phenyl propionyl group in 3D624
(later called PMX205)) for affinity.99 NMR structural studies of
16 and analogues in DMSO-d6 indicated a turn motif.99
Compound 16 is a classic example where oral activity is
observed despite low oral bioavailability, due to a long
residence time on the receptor (t1/2 ≈ 20 h) overcoming low
systemic availability (F = 1−2%, rat).100 This compound and its
analogues display efficacy in vivo in over 20 rat and mouse
models of human disease following oral administration.96
3.7. Actinomycin D
Actinomycin D (17, Figure 11) is an antibiotic natural product
from a family of actinomycins first isolated in 1940.101 It
features two cyclic pentapeptides, each with an L-proline and
two N-methyl amino acids, bridged by a phenoxazinone linker.
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p.o. in olive oil: AUC = 442 ng·h/mL, Cmax = 187 ng/mL, F =

4%), 19 (1 mg/kg iv in DMSO: CL = 4.7 mL/min·kg, VD =
0.19 L/kg, t1/2 = 1.0 h; 10 mg/kg p.o. in olive oil; AUC = 6289
ng·h/mL, Cmax = 1900 ng/mL, F = 18%), and 20 (1 mg/kg iv in
DMSO: CL = 24.1 mL/min·kg, VD = 0.75 L/kg, t1/2 = 1.2 h; 10
mg/kg p.o. in olive oil; AUC = 642 ng·h/mL, Cmax = 174 ng/
mL, F = 9%). Despite being larger with more RO5 violations
(Figure 12), 19 had better oral bioavailability than 18, and was
comparable to cyclosporin A (F = 21%) under the same
conditions.106
Figure 10. Structure of 3D53 (16). MW = 896; miLogP = 1.0; HBD =
11; HBA = 18; RotB = 14; tPSA = 273 Å2.

A crystal structure of actinomycin C in complex with

deoxyguanosine showed two intramolecular hydrogen bonds
between the two Val residues keeping the macrocycles close
together to help insert the phenoxazinone into DNA (CCDC
code: ACTDGU10).102 This preorganization minimized
solvent exposure to polar atoms. Similar structures in the
absence of nucleotides (CCDC codes: BEJXET, BRAXGU,
GIDNUC, POHMUU) show stacking of the cycles but the
Val···Val hydrogen bonds remain. These cycle−cycle inter-
actions loosened in crystal structures of 17 bound to short Figure 12. Cyclic pentaleucine (18) and cyclic hexaleucines (19, 20).
18: MW = 566; miLogP = 3.9; HBD = 5; HBA = 10; RotB = 10; tPSA
sequences of DNA (PDB codes: 4HIV, 1I3W).103,104 Actino-
= 145 Å2. 19: MW = 679; miLogP = 4.7; HBD = 6; HBA = 12; RotB =
mycin D suppresses transcription, is cytotoxic and effective in 12; tPSA = 174 Å2. 20: MW = 679; miLogP = 4.7; HBD = 6; HBA =
treating various tumors. It has an estimated bioavailability F = 12; RotB = 12; tPSA = 175 Å2.
5% based on adverse systemic effects after oral versus iv
administration over 2 weeks.105 LD50 values for 17 were
determined in mice (LD50 7.8 mg/kg p.o.) and rats (LD50 iv
0.4, p.o. 7.2 mg/kg). 4. CYCLIC HEXAPEPTIDES
Cyclic hexapeptides and larger macrocycles generally contra-
vene RO5 parameters, with MW > 500, HBD ≥ 6, HBA ≥ 12,
LogP = 0−5 if bearing hydrophobic side chains. They have
more rotatable bonds and larger surface areas than the smaller
cyclic peptides above.
4.1. Desmopressin
Desmopressin (21, Figure 13) was first reported in 1966 as an
analogue of vasopressin (antidiurectic hormone). Its deami-
nated cysteine at position 1 and D-arginine at position 8
enhanced metabolic stability and antidiuretic effects. The
bioavailability of desmopressin in normal healthy adults was
low following intranasal administration (F = 3−5%) and very
low after oral delivery (F = 0.08−0.16%).107−110
4.2. Melanotan II
Figure 11. Structure of actinomycin D, (17). MW = 1255; miLogP =
0.8; HBD = 6; HBA = 28; RotB = 8; tPSA = 360 Å2. Melanotan II (22, Figure 14) is a cyclic hexapeptide analogue
of α-melanocyte-stimulating hormone that was developed as a
tanning agent to help prevent skin cancer. It displayed some
3.8. Leucine Cyclic Peptides
A selection of three all-leucine cyclic peptides (Figure 12),
cyclo-[(L-Leu)5] (18), cyclo-[(L-Leu)6] (19), and cyclo-[(L-
Leu)5(D-Leu)] (20), were synthesized and orally administered
to rats.106 These compounds are at the boundary limits of RO5
guidelines for molecular weight, hydrogen bond donors/
acceptors. Their other properties (LogP, rotatable bonds,
tPSA) depend upon substituents on the cyclic peptide scaffold.
NMR, CD and X-ray structural studies on 18−20 did not show
intramolecular hydrogen bonds, but intermolecular interactions
for 18 were consistent with evidence for aggregation in
solution.106 Despite lacking N-methyl groups or depsipeptide
bonds to reduce the number of hydrogen bond donors, all three
compounds showed oral absorption: 18 (1 mg/kg iv in DMSO: Figure 13. Structure of desmopressin (21). MW = 1069; miLogP =
CL = 13.1 mL/min·kg, VD = 0.36 L/kg, t1/2 = 0.5 h.; 10 mg/kg −4.3; HBD = 18; HBA = 26; RotB = 20; tPSA = 435 Å2.

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bioavailability after oral administration to rats (0.3 mg/kg iv,
6.76 mg/kg p.o., F = 4.6%) despite violating all RO5 and
related parameters (Figure 14).111
Figure 14. Structure of melanotan II (22). MW = 1024; miLogP =
−0.1; HBD = 16; HBA = 24; RotB = 18; tPSA = 382 Å2.
4.3. Oxytocin
Oxytocin (23, Figure 15) is a disulfide-bridged cyclic
hexapeptide hormone with an additional three amino acids
outside the cycle. This mammalian neurotransmitter and
hormone is associated with numerous physiological responses.
It is currently used in the clinic to induce childbirth and
lactation.112,113 All physicochemical parameters listed in Figure
15 violate guidelines for oral bioavailability. An NMR structure
for oxytocin in water showed two transannular mainchain
hydrogen bonds (Asn NH···OC Tyr, Cys NH···OC Tyr),114
which likely reduce the 3D PSA from the nominal tPSA value.
A crystal structure of a des-amino derivative of oxytocin (PDB
codes: 1XY1, 1XY2; CCDC code: DUPFAV) showed a
hydrogen bond defining a β-turn for the YIQN motif, and a
Tyr NH···OC Asn transannular hydrogen bond along with the
disulfide bond bracing the structure.115 Nevertheless, the oral
bioavailability of oxytocin is very low in rats (F = 0.9%),
requiring it to be injected for efficacy.116
Figure 15. Structure of oxytocin (23). MW = 1007; miLogP = −3.7;
HBD = 16; HBA = 24; RotB = 17; tPSA = 400 Å2.
4.4. Anidulafungin, Caspofungin, and Micafungin
Anidulafungin (LY303366, Eraxis, 24, Figure 16) is a lipid-
modified cyclic peptide analogue of echinocandin B.117
Pharmacokinetic parameters were measured in female Beagle
dogs following oral and intravenous administration (5 mg/kg
iv: t1/2 = 15.6 h; CL = 0.10 ± 0.02 L/h·kg, VD = 1.76 ± 0.11 L/
kg, AUC0‑∞ = 49409 ± 10286 ng·h/mL; 5 mg/kg p.o.: Cmax =
307 ± 61 ng/mL, Tmax = 4.7 ± 1.2 h, AUC0‑∞ = 4477 ± 768 ng·
h/mL, F = 9%).118 Region-dependent intestinal absorption and
meal composition effects on Cmax and AUC were found for
dogs given the same dose via oral, duodenal, jejunal or colonic
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administration (250 mg/kg p.o.: AUC0−48h = 23.3, 26.5, 17.7,
and 6.9 μg·h/mL, respectively; Cmax = 1.6, 1.5, 0.6, and 0.3 μg/
mL, respectively). The effect of different meal treatments
(fasted, mixed meal, lipid meal, protein meal, or carbohydrate
meal) prior to identical oral dosing was also investigated (250
mg/kg p.o.; AUC0−48h = 21.2 ± 5.8, 8.9 ± 2.6, 7.5 ± 1.8, 8.9 ±
2.8, and 25.2 ± 5.1 μg·h/mL, respectively. Cmax = 1.1 ± 0.3, 0.5
± 0.17, 0.4 ± 0.13, 0.5 ± 0.2, and 1.6 ± 0.3 μg/mL,
respectively).117 Low oral absorption of 24 in humans was
reported in early clincal trials (F = 2−7%).119 A similar
compound, caspofungin (25), displayed poor oral absorption in
rats (50 mg/kg p.o., F < 0.2%).120 Micafungin (26) is an
antifungal agent approved for intravenous use by the USFDA in
2005 and in several other countries. It is a further modified
echinocandin analogue primarily used against Candida
infections in HIV-positive patients. It is an inhibitor of beta-
1,3-D-glucan synthase, essential for fungal cell wall synthesis. It
is poorly orally absorbed.121,122
Figure 16. Structures of anidulafungin (24), caspofungin (25), and
micafungin (26). 24: MW = 1140; miLogP = −0.8; HBD = 14; HBA =
24; RotB = 14; tPSA = 377 Å2. 25: MW = 1093; miLogP = −4.6; HBD
= 18; HBA = 25; RotB = 23; tPSA = 412 Å2. 26: MW = 1270; miLogP
= −5.3; HBD = 17; HBA = 32; RotB = 18; tPSA = 510 Å2.
4.5. Somatostatin, Octreotide, and Analogues
Somatostatin-14 (27) is a growth hormone-inhibiting peptide
resulting from cleavage of its 166 residue preproprotein that is
differentially expressed in tissues and regulates endocrine
function. Potential therapeutic applications of somatostatin-14
were quickly recognized, however its low half-life in plasma (∼2
min) made it an unsuitable drug candidate. Many analogues
(e.g., Figure 17) were developed to improve pharmacokinetic
properties. Bicyclic 28 displayed potent biological activity.
Contracting this cycle by removing nonessential amino acids
gave 29. These analogues, in particular 28, retained potency in
vitro and in rats. Their metabolic stability improved relative to
somatostatin-14, however, no oral bioavailability was re-
ported.123
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Figure 17. Structures of somatostatin and cyclic analogues (28−29).
Somatostatin (27): MW = 1638; miLogP = −5.4; HBD = 26; HBA =
37; RotB = 26; tPSA = 613 Å2. 28: MW = 1404; miLogP = −0.7; HBD
= 19; HBA = 27; RotB = 19; tPSA = 428 Å2. 29: MW = 894; miLogP
= 0.1; HBD = 11; HBA = 17; RotB = 9; tPSA = 266 Å2.
Replacing the -Cys-Aha-Cys- motif in 29 with the simpler
-Phe-Pro- motif gave analogue 30. Compounds 27 and 30 both
significantly reduced the effects of growth hormone following
sc injection to rats. Somatostatin-14, 27, and 30 were
administered orally to rats (25 mg/kg p.o.). Somatostatin
failed to lower growth hormone levels, 28 reduced levels at 1 h
but not at 2 h, while 30 reduced levels for at least 3 h. Peptide
28 was cleaved slowly by trypsin at a similar rate to 29.124 To
improve oral bioavailability and other pharmacokinetic proper-
ties, 30 N-methylated analogues of 30 were synthesized.125
Compound 30 and tri-N-methylated analogue 31 (Figure 18)
were administered orally to rats (10 mg/kg p.o.). Oral
bioavailability of 30 could not be determined, whereas 31
showed some oral absorption (F = 10%). Other pharmacody-
namic properties showed that 31 had an extended elimination
half-life (74 vs 15 min) and increased steady state VD (3.7 vs 0.3
L/kg) relative to 30.125
Bauer (Sandoz Ltd.) reported a series of somatostatin-14
analogues based on a disulfide cyclized hexapeptide 32
containing an L-Phe-D-Trp-L-Lys motif (Figure 19).126 Cyclic
hexapeptide 32 was much less active than somatostatin-14
when tested in rats and monkeys. To regain activity, D-Phe was
added to the N-terminus to mimic the natural Phe-6 and
significantly improved in vivo potency. Addition of threonol to
the C-terminus to mimic the natural Thr-12 gave octreotide
(33, Sandostatin, Figure 19). NMR solution structural studies
of octreotide127 and close analogues128 in DMSO-d6 (PDB
codes: 1SOC, 2SOC, 1YL8, and 1YL9) showed a mixture of β-
sheet and partially α-helical motifs. The β-sheet like structures
contained 5 internal hydrogen bonds, 2 transannular and 1 β-
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Figure 18. Cyclic somatostatin analogues. Cyclic hexapeptide (30)
and tri-N-methylated analogue (31). 30: MW = 807; miLogP = 0.5;
HBD = 9; HBA = 15; RotB = 11; tPSA = 228 Å2. 31: MW = 849;
miLogP = 1.3; HBD = 6; HBA = 15; RotB = 11; tPSA = 201 Å2.
turn in the macrocyclic ring and 2 hydrogen bonds between
side chains. The α-helical conformations contained 2 internal
and 2 side chain-side chain hydrogen bonds. In the solid state,
crystals of octreotide from aqueous oxalic acid (CCDC code:
YICMUS) contained two hydrogen bonds in linked β-turns
about the FwKT and wKTC motifs which, in conjunction with
the disulfide, produce a very rigid molecule.129 Octreotide
resisted degradation by enzymes and tissue homogenates.
When injected into rats and monkeys, octreotide showed the
greatest inhibition of growth hormone, insulin and glucagon
production. Furthermore, octreotide was better tolerated than
somatostatin and had ≥20 times longer duration of action.126
Octreotide is orally active126,130,131 but the dose-corrected
systemic oral bioavailability relative to subcutaneous admin-
istration is very low (F = 0.3%).130
Figure 19. Somatostatin analogue 32 and octreotide (33). 32: MW =
784; miLogP = −1.8; HBD = 13; HBA = 16; RotB = 10; tPSA = 277
Å2. 33: MW = 1019; miLogP = −2.0; HBD = 15; HBA = 20; RotB =
17; tPSA = 332 Å2.
SDZ CO 611 (Ilatreotide, 34, Figure 20) is a D(+)-maltose
N-terminally modified amadori derivative of octreotide 33. SDZ
CO 611 (34) displayed improved metabolic stability
supposedly leading to enhanced oral absorption in rats (F =
1.1%).132 Intravenous infusion of somatostatin and sc injection
of octreotide 33 suppress pancreatic and gastrointestinal
hormones and accelerate early gastric emptying. Octreotide
33 also prolongs mouth to cecum transit time (MCTT). When
administered orally (1 or 5 mg p.o. twice daily) to healthy
males, 34 was equally effective as sc 33 in suppressing
preprandial hormone levels, early gastric emptying and
prolonging MCTT.133
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Figure 20. SDZ CO 611 (ilatreotide, 34). MW = 1344; miLogP =

−4.9; HBD = 21; HBA = 32; RotB = 23; tPSA = 488 Å2.

4.6. Beauvericin and Enniatins

Beauvericin (35, Figure 21) is a symmetrical cyclic hexa- Figure 21. Beauvericin (35), enniatins A (R = Ile, 36), B (R = Val,
depsipeptide consisting of three repeating units of the 37), and C (R = Leu, 38), and enniatin B1 (39). 35: MW = 784;
dipeptides N-methyl-L-phenylalanine and the D-valine surrogate miLogP = 6.5; HBD = 0; HBA = 12; RotB = 9; tPSA = 140 Å2. 36:
MW = 682; miLogP = 6.0; HBD = 0; HBA = 12; RotB = 9; tPSA =
(R)-hydroxyisovaleric acid. There are no HBD in 35 or its
140 Å2. 37: MW = 640; miLogP = 4.5; HBD = 0; HBA = 12; RotB =
analogues 36−39, few rotatable bonds and low tPSA, but they 6; tPSA = 140 Å2. 38: MW = 682; miLogP = 6.1; HBD = 0; HBA =
do have RO5 violations for MW, HBA, and LogP (Figure 21). 12; RotB = 9; tPSA = 140 Å2. 39: MW = 654; miLogP = 5.0; HBD =
The crystal structure of beauvericin hydrate134 from n-heptane 0; HBA = 12; RotB = 7; tPSA = 140 Å2.
(CCDC code: BEVERC) did not show intramolecular
hydrogen bonds; however, solvent exposure of the hydrogen confirmed oral activity in infants by determining the
bond acceptors was evidenced in the solid state by complex- concentration of 40 in urine 24 h post oral dosage.148
ation of barium salts (CCDC code: BEAVBA).135 Compound
35 was first isolated from B. bassiana136 and inhibited
cytochrome P450 enzymes. Pharmacokinetic data following
oral administration at various doses to rats (0.5, 1.0, 2.0 mg/kg
p.o.: Cmax = 3.4, 5.4, 13.9 mg/L; Tmax = 4.1, 4.3, 5.4 h; CL =
0.29, 0.21, 0.32 mL/min·kg; AUC0‑∞ = 29, 80, 103 h·mg/L; t1/2
= 2.9, 3.6, 3.0 h, respectively)137 but no iv data was reported
making it difficult to estimate oral absorption. LD50 of 35 has
been reported in mice (LD50 ip >10 mg/kg, p.o. >100 mg/
kg).138,139 The beauvericin scaffold is shared by enniatins A-
C140 (Figure 21, 36−38), where L-phenylalanine is substituted
by L-isoleucine (36), L-valine (37), or L-leucine (38),
respectively. Several crystal structures of 37 and 38 have been
reported (CCDC entries DESYIJ, BICMEF, CIKJAH,
ZASQOZ, QEMCAM). As for beauvericin, there were no
intramolecular hydrogen bonds.141,142 However, 37 did Figure 22. Nepadutant (40). MW = 947; miLogP = −1.6; HBD = 11;
complex rubidium143 and potassium144 ions, indicating a HBA = 22; RotB = 11; tPSA = 348 Å2.
solvent accessible polar surface (CCDC codes: MVHIRB10,
PEKFEQ). Pharmacokinetic data were estimated for 37 from in
vitro assays.145 The toxicokinetic profile of enniatin B1 (39, 4.8. Bouvardin
Figure 21) was measured in piglets (0.05 mg/kg iv or p.o. in 2%
Bouvardin (41, Figure 23) is a cyclic hexapeptide containing
EtOH/water). Compound 39 was highly orally bioavailable (F
three N-methyl amides, one O-methylated tyrosine, one D-Ala,
91%) but was cleared rapidly (t1/2 = 0.15 h, CL = 2.00 L/h·kg)
and an additional diphenylether-bridge between two tyrosine
with low oral plasma exposure (AUC = 25.3 ng·h/mL).146
side chains. The crystal structure of 41 from methanol/water
4.7. Nepadutant (CCDC code: BOUVAR) did not show internal hydrogen
Nepadutant (40, Figure 22) is a glycosylated, bicyclic bonds but was in an extended conformation induced by the
hexapeptide tachykinin NK2 receptor antagonist. Cyclisation oxidatively fused tyrosine side chains. This bridged constraint
is achieved through both head-to-tail and side chain-to-side also introduced a cis-amide bond between the two residues.149
chain linkages and an aminohexose is attached to an asparagine It was isolated from B. ternifolia and showed in vitro
side chain. The aminohexose moiety gives the structure cytotoxicity against P388 lymphocytic leukemia and B16
amphiphilic character. Compound 40 was given orally to melanotic melanoma cell lines. Bouvardin and an acetylated
mice (38 mg/kg p.o. in castor oil; Tmax = 2.7 ± 0.7 h, Cmax = deoxybouvardin derivative 42 (Figure 23) had LD50 values of
1401 ± 220 nM, t1/2 = 11.7 ± 0.9 h, AUC = 8463 ± 2635 10 mg/kg (42, ip), 229 mg/kg (41, p.o.), and 20 mg/kg (42,
nmol·h/mL, F = 5%).147 Pediatric phase 1 clinical studies iv) in mice, indicating that 41 is orally absorbed.150 These
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compounds have few hydrogen bond donors and few rotatable
bonds, but they have high polar surface areas and low LogP.
Figure 23. Bouvardin (41) and acetylated derivative 42. 41: MW =
773; miLogP = 0.5; HBD = 5; HBA = 16; RotB = 3; tPSA = 207 Å2.
42: MW = 799; miLogP = 1.2; HBD = 3; HBA = 16; RotB = 5; tPSA
= 193 Å2.
4.9. Pristinamycin and Related Antibiotics
Pristinamycin is an oral antibiotic consisting of two synergistic,
but structurally unrelated, components. Separately, the
compounds exhibit bacteriostatic activity, while the combina-
tion of pristinamycin IA (43) and IIA (44, Figure 24) has
bactericidal activity. 43 has one L-Pro, one N-methylated amide,
a D-Abu, and a 4-oxopiperidine incorporated into its backbone.
44 bears a hybrid terpenoid/peptide backbone rigidified by
three alkenyl bonds, a 4,5-dehydroproline, and an oxazole ring.
A study in an Australian hospital investigated the effectiveness
of oral pristinamycin in patients with various bacterial
infections.151 Patients were dosed orally with 500−1000 mg
of 43 and 44 on multiple days. Of 46 patients, five were
excluded due to side effects, 10 were cured from infection, and
21 had infections suppressed.
Figure 24. Antibacterial combination drug, pristinamycin consisting of
pristinamycins IA (43) and IIA (44). 43: MW = 867; miLogP = 0.7;
HBD = 4; HBA = 18; RotB = 7; tPSA = 228 Å2. 44: MW = 526;
miLogP = 0.7; HBD = 2; HBA = 10; RotB = 1; tPSA = 139 Å2.
NXL103 (XRP2868; Figure 25) is also an antibiotic cocktail
made of linopristin 45 and floprestin 46 (30:70 mixture). These
compounds replace three hydrogen bond donor NHs with 5 or
6 membered rings and a depsipeptide ester, 45 also has an N-
methyl group. Pharmacokinetic parameters were measured after
oral administration at multiple doses to cyclophosphamide-
induced neutropenic mice infected with strains of S. pneumoniae
(NXL103: 10 mg/kg p.o.: Cmax = 0.32 mg/L; 40 mg/kg p.o.:
Cmax = 0.8 mg/L, t1/2 = 21 min; 80 mg/kg p.o.: Cmax = 4 mg/L,
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t1/2 = 55 min, AUC0−24h = 4.5 mg·h/kg; 160 mg/kg p.o.: Cmax =
10.4 mg/L, t1/2 = 76.2 min, AUC0−24h = 11.34 mg·h/kg).152
Figure 25. Antibacterial drug, NXL103 consisting of linopristin (45)
and floprestin (46). 45: MW = 950; miLogP = 1.6; HBD = 4; HBA =
19; RotB = 9; tPSA = 223 Å2. 46: MW = 532; miLogP = 1.5; HBD =
2; HBA = 9; RotB = 1; tPSA = 122 Å2.
4.10. 1-NMe3 and Related Cyclic Hexapeptides
To permeate through membranes, molecules must transition
from aqueous to hydrophobic and back to aqueous phases.
Conformational changes might facilitate this process by
lowering desolvation energies. To test this idea, molecular
modeling, H-D exchange experiments, and permeability
measurements were combined to produce passively permeable
cyclic hexapeptide diastereomers from cyclo-[Leu-Leu-Leu-
Leu-Pro-Tyr]153 (Figure 26). None of the diasteromers had N-
methyl groups; however, one compound, cyclo[Leu-D-Leu-Leu-
Leu-D-Pro-Tyr], showed a conformational change between
CDCl3 and water by molecular modeling. This diastereomer
was the least permeable in the initial series even though higher
permeability had previously been reported for the same
peptide.154 In further studies, tri-N-methylation yielded 1-
NMe3 47 which had good PAMPA permeability and oral
bioavailability in rats (1 mg/kg iv: CL = 4.5 mL/min·kg, VD =
1.1 L/kg; 10 mg/kg p.o.: AUC = 10.5 ng·h/mL, Cmax = 852 ng/
mL, F = 28%).155 Analogues of 47 varied a single side chain
substitution to learn effects on pharmacokinetic profiles.156 An
N-methyl-leucine was substituted for N-methyl-serine (48), N-
methyl-threonine (49), N-methyl-aspartate (50), or N-methyl-
lysine (51). Their pharmacokinetic profiles revealed that
increasing side chain polarity reduced oral absorption.
Interestingly, threonine analogue 49 (1 mg/kg iv: CL = 63.6
mL/min·kg, VD = 3.8 L/kg, t1/2 = 1.0 h; 10 mg/kg p.o.: AUC =
201 ng·h/mL, Cmax = 105 ng/mL, F = 24%) displayed similar
pharmacokinetics as 47, while serine analogue 48 had low oral
absorption (1 mg/kg iv: CL = 60.4 mL/min·kg, VD = 4.3 L/kg,
t1/2 = 1.5 h; 10 mg/kg p.o.: AUC = 42.3 ng·h/mL, Cmax = 9.86
ng/mL, F = 2%). Aspartate analogue 50 (1 mg/kg iv: CL =
56.4 mL/min·kg, VD = 0.36 L/kg, t1/2 = 0.4 h; 10 mg/kg p.o.:
AUC = 18.7 ng·h/mL, Cmax = 17.3 ng/mL, F < 1%) and lysine
analogue 51 (1 mg/kg iv: CL = 10.4 mL/min·kg, VD = 0.9 L/
kg, t1/2 = 1.0 h; 10 mg/kg p.o.: AUC = 18.1 ng·h/mL, Cmax =
18.3 ng/mL, F < 1%) had negligible oral bioavailability (F <
1%). NMR structural studies of this series of peptides153−155 in
CDCl3 and DMSO-d6 suggested that the pattern of internal
hydrogen bonds was strongly conserved.
The same orally bioavailable scaffold (47) inspired further
investigations to better understand and improve passive
permeability of cyclic hexapeptides. N-Methylamino acid-to-
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Figure 26. 1-NMe3 (47) and derivatives (48−51). 47: MW = 755;
miLogP = 4.1; HBD = 3; HBA = 13; RotB = 10; tPSA = 160 Å2. 48:
MW = 729; miLogP = 1.8; HBD = 4; HBA = 14; RotB = 9; tPSA =
180 Å2. 49: MW = 743; miLogP = 2.1; HBD = 4; HBA = 14; RotB =
9; tPSA = 180 Å2. 50: MW = 757; miLogP = 1.9; HBD = 4; HBA =
15; RotB = 10; tPSA = 197 Å2. 51: MW = 770; miLogP = 2.3; HBD =
5; HBA = 14; RotB = 12; tPSA = 186 Å2. Dotted lines indicate
hydrogen bonds observed in crystal structure of 47, and NMR solution
structures of analogues.
peptoid substitutions were explored in cyclic hexapeptide/
peptoid hybrids (Figure 27). Peptoid substitutions that
replaced one (52) or two (53) of the three N-methyl amides
facilitated permeability across a monolayer of low efflux
epithelial MDCK cells. Interestingly, replacing all three N-
methyl moieties with peptoid units (54) generated a macro-
cycle that was less permeable.157
β-Hydroxy-γ-amino acids are naturally occurring peptidomi-
metic surrogates that structurally resemble a peptide backbone
by replacing heteroatoms with carbon to minimize polarity.
These substructures may have evolved to allow molecules to
Figure 27. Peptoid substituted analogues 52−54 and β-hydroxy-γ-
amino acid analogue 55. 52: MW = 741; miLogP = 3.5; HBD = 3;
HBA = 13; RotB = 10; tPSA = 160 Å2; 53: MW = 727; miLogP = 3.6;
HBD = 3; HBA = 13; RotB = 10; tPSA = 160 Å2; 54: MW = 713;
miLogP = 3.6; HBD = 3; HBA = 13; RotB = 10; tPSA = 160 Å2; 55:
MW = 793; miLogP = 3.6; HBD = 4; HBA = 14; RotB = 10; tPSA =
180 Å2. Dotted lines indicate conserved hydrogen bonds observed by
NMR spectroscopy.
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enter cells and interact with intracellular targets. β-hydroxy-γ-
amino acids were used to create cyclic hexapeptides based on
47. Oral bioavailability in rats was reported for one compound,
55, displaying promising passive permeability (1 mg/kg iv: CL
= 10 mL/min·kg, VD = 1.1 L/kg, t1/2 = 1.6 h; 5 mg/kg p.o.;
AUC = 1697 ng·h/mL, Cmax = 324 ng/mL, F = 21%).158
Although good permeability was maintained in the less peptidic
55, its oral bioavailability and plasma concentration (AUC)
were worse than for 47. Interestingly, NMR evidence suggested
that, despite variability in the membrane permeability of some
peptoid and statin-type analogues of 47, these compounds still
retained the two internal hydrogen bonds shown in Figure
27.157,158
Other groups have used 47 and analogues to investigate
effects of N-methylation,159 correlating amide NH NMR
temperature coefficients with cell permeability160 and flexi-
bility/rigidity.161 A crystal structure of 47 (CCDC code:
AHELEG) indicated a saddle-like structure incorporating two
β-turns at either end, with one close hydrogen bond in each
(2.0, 2.5 Å, indicated by dotted lines, Figure 26).159 Novartis
researchers compared the original scaffold of 47 with its non-N-
methylated counterpart (56, Figure 28) and showed that N-
methylation was important for oral absorption. Permeability
enhancers did not improve oral bioavailability of 47.
Pharmacokinetics were measured in mice for non-N-methylated
analogue 56 (3 μM/kg iv: CL = 82 mL/min·kg, VD = 0.8 L·kg,
t1/2 = 0.2 h, AUCiv = 292 nM·h; 10 μM/kg p.o.: AUCpo = 5.7
nM·h, Cmax = 2.9 nM, Tmax = 0.6 h, F = 2%) and 47 (3 μM/kg
iv: CL = 1 mL/min·kg, VD = 0.4 L/kg, t1/2 = 22 h, AUCiv =
29618 nM·h; 10 μM/kg p.o.: AUC = 8967 nM·h, Cmax = 534
nM, Tmax = 3.8 h, F = 30%). These striking results highlight
good oral absorption for 47 in rodents.
Figure 28. Non-N-methylated analogue 56 and bis-N-methylated
analogue 57 with a different N-methylation pattern to 47 retained oral
bioavailability. 56: MW = 713; miLogP = 3.3; HBD = 6; HBA = 13;
RotB = 10; tPSA = 186 Å2. 57: MW = 741; miLogP = 3.8; HBD = 4;
HBA = 13; RotB = 10; tPSA = 168 Å2. Dotted lines indicate conserved
hydrogen bonds observed by NMR spectroscopy.
Cyclic hexapeptides based on 47 were examined with
shuffled N-methyl amino acids and inverted stereochemistries.
Correlations between amide NH NMR temperature coef-
ficients, Caco-2 and PAMPA permeability led to cyclic
hexapeptide 57 (Figure 28). Compared to 47, 57 had one
less N-methyl group and a modified N-methylation pattern but
was still orally bioavailable in rats (1 mg/kg iv: CL = 55 mL/
min·kg, VD = 1.1 L/kg, t1/2 = 29 min; 10 mg/kg p.o.: AUC =
1003 ng·h/mL, Cmax = 117 ng/mL, F = 33%).141
Peptide 47 was thought to be orally bioavailable due to
cyclosporin-like conformational flexibility; however, it showed
no conformational difference in polar versus nonpolar
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Chemical Reviews Review

solvents.161 Several analogues of 47 were synthesized and their

pharmacokinetic properties characterized to investigate con-
formational flexibility versus rigidity. The structure of 47 was
rigidified by introducing a proline (58, Figure 29) or relaxed by
introducing N-methylleucine (59, Figure 29). Both cyclic
hexapeptides 58 (1 mg/kg iv: CL = 11 mL/min·kg, t1/2 = 58
min; 10 mg/kg p.o.: AUC = 4320 ng·h/mL, Cmax = 879 ng/mL,
F = 30%) and 59 (1 mg/kg iv: CL = 10 mL/min·kg, t1/2 = 121
min; 10 mg/kg p.o.: AUC = 3713 ng·h/mL, Cmax = 768 ng·mL,
F = 18%) were appreciably orally bioavailable in rats.161 For
these compounds (59, 47, and 58) with identical HBD, HBA,
and tPSA (Figure 29), reducing the MW (785 > 755 > 725),
the number of rotatable bonds (12 > 10 > 8), and the Figure 30. Cyclic cell-penetrating hexapeptide (60). MW = 969;
hydrophilic surface area (FISA 127.5 > 122.5 > 110.4 Å2) all miLogP = −2.9; HBD = 22; HBA = 24; RotB = 24; tPSA = 422 Å2.
improved oral bioavailability (F% = 18−16 (28155) < 30, under
the same conditions161). These changes had little effect on the absorption. Preclinical toxicity studies determined LD50 in rats
overall structure of the macrocycle, as shown by NMR at 0.38−0.60 mg/kg iv.169
spectroscopy in CDCl3 and DMSO-d6, with two rigidifying
antiparallel β-sheets connected by type 1 β-turns at each end
(dotted lines, Figure 29).

Figure 31. Kahalalide F (61). MW = 1492; miLogP = 3.0; HBD = 15;

HBA = 30; RotB = 34; tPSA = 442 Å2.

Figure 29. Derivatives of 47 with increased (58, left) or decreased (59,

right) rigidity. 58: MW = 725; miLogP = 2.8; HBD = 3; HBA = 13;
RotB = 8; tPSA = 160 Å2. 59: MW = 785; miLogP = 5.3; HBD = 3;
HBA = 13; RotB = 12; tPSA = 160 Å2. Dotted lines indicate conserved
hydrogen bonds observed by NMR spectroscopy.
4.11. Cyclo-[Arg-Arg-Arg-Arg-NaphthylAla-Phe]
Cell penetrating peptides (CPPs) are a small group of short,
natural and synthetic, peptides that are often arginine rich,
positively charged, amphiphilic, and able to cross cell
membranes. How they cross membranes is still not fully
resolved but mechanisms include endocytosis and direct
translocation across the membrane.162−164 A series of
arginine-rich, cell-penetrating, cyclic hexapeptides were synthe-
sized, and their cellular uptake was measured. One analogue,
cyclic hexapeptide 60 (Figure 30), was orally administered to
mice and pharmacokinetic parameters were measured (1.5 mg/
kg iv: Cmax = 12174 nmol/L, t1/2 = 1.02 h, CL = 0.08 mL/min,
VD = 7.51 mL; 40 mg/kg p.o.: Cmax = 3156 nmol/L, AUC =
6357 nmol/L, F = 4%) showing it had some oral
bioavailability.165
4.12. Kahalalide F
Kahalalide F (61, Figure 31) is the largest depsipeptide in the
family of kahalalides (A-F) and was isolated from the
sacoglossan mollusk E. rufescens and the green alga Bryopsis.166
Compound 61 induced oncosis in human prostate and breast
cancer cells.167 Pharmacokinetic parameters were measured in
Phase I clinical trials (iv: t1/2 = 0.47 h, CL = 11.0 L/h, VD = 7.0
L).168 Kahalalide F had LD50 of 300, 980, and 3200 mg/kg p.o.
in mouse, rat, and rabbit, respectively, suggesting very low oral
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5. CYCLIC HEPTAPEPTIDES
5.1. Sanguinamide A and Danamides
Sanguinamide A (62, Figure 32) is a thiazole-containing cyclic
heptapeptide isolated from H. sanguineus.170 It has six natural L-
amino acids including two prolines that adopt a cis, trans
configuration, and an isoleucine-derived thiazole.171 No
biological activity is known for sanguinamide A, but it is orally
bioavailable in rats (1 mg/kg iv in DMSO: t1/2 = 23 min, CL =
70 mL/min; 10 mg/kg p.o.: Tmax = 60 min, Cmax = 40 nM, F = 7
± 4%).171 The three-dimensional structure helped rationalize
the pharmacokinetic profile of 62. Two amide protons formed
intramolecular hydrogen bonds and were shielded from solvent
by bulky side chains, while two prolines and thiazole removed 3
amide NH protons, lowering the HBD count. NMR studies on
analogues of 62 examined H-D exchange rates and amide
temperature coefficients and determined NMR solution
structures in d6-DMSO. These data guided introduction of
the bulky tert-butylglycine to minimize solvent exposed polarity,
resulting in danamide D (63, Figure 32) that was orally
bioavailable (1 mg/kg iv in DMSO: CL = 12.5 mL/min·kg, t1/2
= 65 min; 10 mg/kg p.o. in olive oil: Cmax = 352 ng/mL, Tmax =
240 min, AUC = 2647 ng·h/mL, F = 21 ± 2%). When an N-
methyl group was removed (danamide F, 64, Figure 32), even
better oral bioavailability was obtained (1 mg/kg iv in DMSO:
CL = 23.0 mL/min kg, t1/2 = 97 min; 10 mg/kg p.o. in olive oil:
Cmax = 726 ng/mL, Tmax = 240 min, AUC = 3372 ng·h/mL, F =
51 ± 9%). This result highlighted the point that N-methylation
of solvent accessible amides does not necessarily always
improve oral bioavailability.172
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Chemical Reviews
Figure 32. Sanguinamide A 62 (R1 = H, R2 = Me), danamide D 63
(R1 = Me, R2 = tBu), and danamide F 64 (R1 = H, R2 = tBu). 62: MW
= 722; miLogP = 2.0; HBD = 4; HBA = 13; RotB = 6; tPSA = 170 Å2.
63: MW = 778; miLogP = 3.6; HBD = 3; HBA = 13; RotB = 7; tPSA
= 161 Å2. 64: MW = 764; miLogP = 3.3; HBD = 4; HBA = 13; RotB =
7; tPSA = 170 Å2. Dotted lines represent hydrogen bonds as
determined by NMR spectroscopy.171
5.2. Rhizonin A
Rhizonin A (65, Figure 33) is cyclo-[N(Me)Ala-N(Me)D-
Ala(fur-3-yl)-D-aIle-D-Val-Val-N(Me)Ala(fur-3-yl)-Leu]. It was
isolated from fungi R. microspores, a known infection in fruits,
vegetables and malt products. This cyclic heptapeptide consists
of L- and D-amino acids, three N-methyl amides and two (2-
furyl)-alanines in both (R)- and (S)-configurations.173 In
crystals of 65 grown from ethyl acetate/hexane, the four
bulky side chains including three β-branched residues were at
the wide end of the ovoid macrocycle. The other end showed a
transannular hydrogen bond between the Ile NH and D-
(furylalanine)-carbonyl oxygen.173 Rhizonin A 65 was given
orally to male albino rats (70, 96, 131, or 180 mg/kg p.o.) to
determine the oral LD50. The lowest dose exceeded LD100 as all
rats died within 10 days.174 There was no data for iv
administration, so oral bioavailability could not be quantified.
Figure 33. Rhizonin A (65). MW = 812; miLogP = 2.6; HBD = 4;
HBA = 16; RotB = 10; tPSA = 204 Å2. Dotted line represents
hydrogen bond observed in crystal structure.
5.3. Microcystin LR
Microcystin-LR (66, Figure 34) from M. aeruginosa is one of
the most toxic of over 80 microcystins produced by
cyanobacteria in aquatic environments. Its amino acid sequence
is D-alanine, L-leucine, D-methylaspartic acid, variable L-amino
acid, a 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-
dienoic acid (Adda), D-glutamic acid, and N-methyldehydroa-
lanine. The extensively modified cyclic peptide is highly water-
soluble and a persistent contaminant that accumulates in fish
and displays effects on their liver, gastrointestinal tract, and
kidneys.175 Acute hepatotoxic effects especially apoptotic
hepatocyte death were observed in mice,176 although the oral
toxic dose (LD50 = 11 mg/kg p.o.) is ∼170 fold lower than the
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lethal dose (LD100 = 65 mg/kg ip). Distribution studies in
mice177 indicated uptake from the small intestine with
concentration in the liver, lung, and heart.
Figure 34. Microcystin-LR (66). MW = 995; miLogP = −3.5; HBD =
12; HBA = 22; RotB = 16; tPSA = 341 Å2.
5.4. YM254890
YM254890 (67, Figure 35) is a specific Gαq/11 inhibitor
produced by an isolate of the chromobacterium sp. QS3666. The
structure is a depsipeptide with 2 ester linkages and a D-
phenylalanine derivative. Gαq/11, a G protein implicated in
purine nucleotide-induced platelet aggregation, was inhibited
by 67 by targeting the transformation of GDP to GTP. In rats,
67 inhibited ex vivo platelet aggregation and in vitro thrombus
formation, suggesting it may lead to a new antithrombotic
agent. It has been used orally to treat mice with acute
thrombosis and chronic neointima formation after induced
vascular injury. Internal bleeding in mice was also effectively
treated with 67 at 0.03 mg/kg iv or 1 mg/kg p.o., indicating an
oral absorption < 3%.178
Figure 35. YM254890 (67). MW = 960; miLogP = −0.2; HBD = 5;
HBA = 22; RotB = 13; tPSA = 288 Å2.
5.5. CHEC-7
CHEC refers to a series of peptides of various sizes derived
from the natural survival anti-inflammatory protein dermicidin.
CHEC-7 (68, Figure 36) reduced the incidence of experimental
autoimmune encephalomyelitis in a disease model when
administered to rats (1.5 mg/kg p.o. or 0.1 mg/kg/day sc).179
5.6. Polymyxin B1 and B2
Polymyxin B (Figure 37) is a mixture of two natural product
cyclic peptides, polymyxin B1 69 and B2 70, containing multiple
unusual diaminobutyric acids and a D-Phe residue. Prolonged
treatment with polymyxin B mixture could not neutralize
lipooligosaccharide-induced immune cell activity in mice.
However, modulation of this response was observed by oral
administration in drinking water (29.5 mg/L p.o. suspension in
water).180 Human patients with liver disease have been treated
with enteric coated oral polymyxin B. Plasma endotoxin and
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Figure 36. CHEC7 (68). MW = 759; miLogP = −5.6; HBD = 3; HBA
= 21; RotB = 9; tPSA = 347 Å2.
ammonia levels were reduced, even though polymyxin B itself
could not be detected above 0.5 units/mL.181
Figure 37. Polymyxin B1 (69) and B2 (70). 69: MW = 1189; miLogP
= −5.6; HBD = 23; HBA = 29; RotB = 28; tPSA = 491 Å2. 70: MW =
1203; miLogP = −5.5; HBD = 23; HBA = 29; RotB = 29; tPSA = 160
Å2.
5.7. Bacitracin A
Bacitracin A (71, Figure 38) is a cyclic thiazoline-containing
peptide from a family of cyclic peptides isolated from the
“Tracy I” strain of B. subtilis. Bacitracins are an intriguing group
of non-ribosomal peptides containing a sequence of alternating
L - and D-amino acids. Bacitracin A was compared to
vancomycin for treating antibiotic-associated diarrhoea. Plasma
and urine concentrations were measured to quantify unwanted
systemic absorption. 71 was found in plasma at low non-toxic
concentrations after oral administration.182 Another study in
which 71 was administered orally to dogs found significant
levels in blood and urine, confirming absorption from the
intestinal tract.183 Bacitracin A has been investigated for the
treatment of vancomycin-resistant E. faecium in humans. In
combination with doxycycline, 71 was not efficacious beyond
the 2-week interval during which they were given.184 71 has
oral toxicity in mice (LD50 > 3750 mg/kg p.o.).185
6. CYCLIC OCTAPEPTIDES
6.1. PF1022A and Emodepside
PF1022A (72, Figure 39) is a cyclic octapeptide natural product
with anthelmintic activity first isolated in 1992 from the fungus
M. sterilia. It contains four N-methyl-L-leucine residues
alternating with D-lactic acid or D-phenyllactic acid. PF1022A
(72) was orally active (0.5−2.0 mg/kg p.o.) and effective
against A. galli nematode (ring worm) infections in chickens
(0.5−2.0 mg/kg p.o.)186 and nematode infections in rats (5 or
10 mg/kg p.o.),187 sheep (1 mg/kg p.o.),188 and other
species.189,190 These data gave no insights into intestinal
absorption of 72 following oral dosing, since the nematodes
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Figure 38. Bacitracin A (71). MW = 1437; miLogP = −1.8; HBD =
20; HBA = 33; RotB = 32; tPSA = 531 Å2.
inhabited the gut of the test animals. Further work on
analogues of 72 in chickens suggested that certain functional
groups, lipophilicity, and PSA influenced anthelmintic activity
in vivo.191
Figure 39. PF1022A (72) and emodepside (73). 72: MW = 949;
miLogP = 7.9; HBD = 0; HBA = 16; RotB = 12; tPSA = 186 Å2. 73:
MW = 1119; miLogP = 7.8; HBD = 0; HBA = 20; RotB = 14; tPSA =
211 Å2.
Emodepside (BY 44-4400, 73, Figure 39), a dimorpholine
derivative of 72, has been approved for use in animals. Four
different crystal forms have been reported from methanol.192
With no possibility of forming transannular hydrogen bonds,
the carbonyl groups are directed above and below the plane of
the macrocycle and are highly solvent exposed, one D-lactate
side chain occupying the interior (CCDC code: DOMZOW).
Compound 73 was orally active against nematodes and orally
bioavailable (F = 47−54%). It had a long elimination half-life
following intravenous administration to rats (t1/2 = 39−51
h).189,190 A phase I clinical trial investigating oral dosing of 73
in normal healthy male volunteers is ongoing.193
6.2. WH1Fungin
WH1Fungin (74, Figure 40) is a surfactin type lipo-
depsipeptide extracted from B. amyloliquefaciens WH1. As an
immunoadjuvant, 74 was orally active in mice (0.2 mg) and
enhanced the immune response to coadministered protein
antigens (chicken ovalbumin or glutathione S-transferase).194
74 was also shown to reduce blood glucose and increase serum
insulin in non-obese diabetic mice following oral administration
at two doses (5 or 25 mg/kg p.o. in 100 μL PBS).195
6.3. α-Amanitin
The poisonous mushrooms of the Amanita genus contain a
number of related constrained cyclic peptides of MW = 770-
920. These are toxic after ingestion and include phallotoxins,
amatoxins, and virotoxins.196,197 Some of these toxins are heat
stable and are not destroyed by cooking. Phallotoxins from A.
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Figure 40. WH1Fungin (74). MW = 1064; miLogP = 8.0; HBD = 9;
HBA = 20; RotB = 27; tPSA = 345 Å2.
phalloides are bicyclic heptapeptides, amatoxins (A. phalloides)
are bicyclic octapeptides, and virotoxins (A. virosa) are
monocyclic heptapeptides.198 Despite extensive modifications
(e.g., leucine, proline hydroxylation, D-configuration, thia-cross-
link) these are ribosomally generated peptides.199 A systematic
examination of their oral absorption has not been carried out
but they display high toxicity.
α-Amanitin (75, Figure 41) is a member of the group of
poisonous peptides called amatoxins; all L-configured bicyclic
octapeptides found in Amanita species of mushrooms.
Amatoxins contain an oxidized tryptathionine linkage in
addition to backbone cyclization. Multiple crystal structures
(PDB codes: 1K83, 2VUM, and 3CQZ)200−202 of α-amanitin
(75) bound to RNA polymerase II showed, in addition to six
hydrogen bonding interactions to the enzyme, two transannular
hydrogen bonds and two more describing β-turns within the α-
amanitin framework. Although native unbound α-amanitin
crystal structures are not available, simple OMe and sulfone
analogues (CCDC entries CAZFIS, CAZFIS10, CAZFOY,
CAZFOY10) show similar structures to the protein bound
forms above, with two β-turns, one transannular hydrogen
bond is lost in the free form.203,204 Interestingly, it appears that
the toxicity of the amanitins can be modulated by the degree
and stereochemistry of oxidation at the bridging sulfur, with the
sulfone analogues being the most toxic, while retaining nearly
all structural features of the peptidic backbone. α-Amanitin
causes severe liver damage and in some cases death (50−100
fatalities per year in Europe) through acute liver failure.205 75 is
orally active only in certain species. The lethal oral dose in
humans has been estimated at 0.1 mg/kg from accidental
deaths. In guinea pigs, identical toxicity following oral,
intravenous and intraperitoneal administration (LD50 = 0.1
mg/kg) indicated good intestinal absorption. By contrast,
toxicity in mice (LD50 = 0.4−0.8 mg/kg iv) and rats (LD50 =
3.5−4.0 mg/kg iv) is only observed following iv administration.
Cats and dogs absorb amatoxins following oral administration,
but the absorption rate is slow. In dogs, the toxic oral dose is
five times higher than the toxic intravenous dose (LD50 = 0.5
mg/kg p.o. vs 0.1 mg/kg iv). In cats, the toxic oral dose was
more than ten times higher than the toxic intravenous dose.206
6.4. Griselimycin and Synthetic Derivatives
Griselimycin (76, Figure 42) is a macrocyclic poly-N-
methylated depsipeptide isolated from Streptomyces bacteria
cultures.207 Griselimycin derivatives exhibit antibiotic activity,
oral bioavailability, metabolic stability, and antitubercular
activity.208,209 Griselimycin has impressive pharmacokinetics
for its chemical class and size (3 mg/kg iv: CL = 40 mL/min·
kg, VD = 1.2 L/kg, t1/2 = 120 min; 30 = mg/kg p.o.: AUC =
5100 ng·h/mL, C max = 3900 ng/mL, F = 48%). 208
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Figure 41. α-Amanitin (75). MW = 889; miLogP = −4.1; HBD = 13;
HBA = 23; RotB = 7; tPSA = 361 Å2.
Pharmacokinetics were improved by simple structural mod-
ifications. Griselimycin contains an N-methyl-D-leucine, one
proline, and two 4-methylproline residues. Modification of the
4-position of proline gave methyl-griselimycin 77 (3 mg/kg iv:
CL = 35 mL/min·kg, VD = 1.6 L/kg, t1/2 = 144 min; 30 = mg/
kg p.o.: AUC = 6600 ng·h/mL, Cmax = 2820 ng/mL, F = 47%),
dimethyl-griselimycin 78 (3 mg/kg iv: t1/2 = 132 min; 30 mg/
kg p.o.: AUC = 17000 ng·h/mL, Cmax = 4570 ng/mL, F =
59%), (S)-fluoro-griselimycin 79 (3 mg/kg iv: t1/2 = 120 min;
30 mg/kg p.o.: AUC = 5100 ng·h/mL, Cmax = 1490 ng/mL, F =
55%), and cyclohexyl-griselimycin 80 (3 mg/kg iv: CL = 18.3
mL/min·kg, VD = 5.5 L/kg, t1/2 = 258 min; 30 mg/kg p.o.:
AUC = 23000 ng·h/mL, Cmax = 2620 ng/mL, F = 89%); all
apparently with improved oral bioavailability and pharmacoki-
netic properties.208 Griselimycin and the cyclohexyl analogue
80 have each been crystallized in complex with the sliding
clamp of Mycobacterium tuberculosis (PDB code: 5AH2, 5AGU,
5AH4, and 5AGV).208 The bound griselimycins showed two
internal hydrogen bonds, one γ-turn from the Leu CO
preceding MePro to the Leu NH after it, one transannular
hydrogen bond between the Gly NH and the N-Me-Leu
carbonyl group, and one transannular hydrogen bond between
the Gly NH and the N-Me-Leu carbonyl group.
6.5. Dihydromycoplanecin and Mycoplanecin
Dihydromycoplanecin A (81, Figure 43) was discovered as an
active metabolite in the urine of dogs and mice treated with the
antibiotic mycoplanecin A (82, Figure 43). Both 81 and 82 are
cyclized through a depsipeptide linkage between the C-
terminus and a threonine residue. Further modifications include
four N-methylated amides, 4-methyl and 4-ethylsubstituted
prolines, L-homoleucine and α-hydroxy (81) or α-ketobutanoyl
(82) N-terminal caps. No oral pharmacokinetic data has been
reported for 82, but oral toxicity has been reported (LD50 > 3
g/kg p.o.).210 Pharmacokinetic parameters for 81 have been
measured in mice (t1/2 = 0.5 h; 50 mg/kg p.o.: Cmax = 10 μg/
mL at 2 and 4 h) and dogs (t1/2 = 5.5 h; 25 mg/kg p.o.: Cmax =
9 μg/mL at 3 h; 12.5 mg/kg p.o.: Cmax = 5 μg/mL at 3 h).
Quantification of 81 from urine led to estimates of F = 21% in
mice and 22−25% in dogs.211
7. CYCLIC NONA- AND DECA-PEPTIDES
7.1. CHEC-9
Disulfide-cyclized nonapeptide CHEC-9 (83, Figure 44) is a
putative inhibitor of Heat Shock Protein 70 and secreted
phospholipase A2. Following oral administration to rats (1 mg/
kg p.o. in gelatin solution) it was found in cytosolic fractions of
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Figure 42. Natural product griselimycin (76, R1 = R2 = H) and
synthetic derivatives: methyl-griselimycin (77, R1 = Me, R2 = H),
dimethyl-griselimycin (78, R1 = R2 = Me), (S)-fluoro-griselimycin (79,
R1 = F, R2 = H), and cyclohexyl-griselimycin (80, R1 = cyclohexyl, R2
= H). 76: MW = 1113; miLogP = 4.3; HBD = 3; HBA = 22; RotB =
12; tPSA = 256 Å2. 77: MW = 1127; miLogP = 5.6; HBD = 3; HBA =
22; RotB = 12; tPSA = 256 Å2. 78: MW = 1142; miLogP = 5.1; HBD
= 3; HBA = 22; RotB = 12; tPSA = 256 Å2. 79: MW = 1131; miLogP
= 4.3; HBD = 3; HBA = 22; RotB = 12; tPSA = 256 Å2. 80: MW =
1196; miLogP = 7.5; HBD = 3; HBA = 22; RotB = 13; tPSA = 256 Å2.
Figure 43. Dihydromycoplanecin (81) and mycoplanecin (82). 81:
MW = 1186; miLogP = 5.2; HBD = 4; HBA = 23; RotB = 16; tPSA =
276 Å2. 82: MW = 1184; miLogP = 5.0; HBD = 3; HBA = 23; RotB =
16; tPSA = 273 Å2.
the frontal cortex tissues. This indicated both oral absorption
from the gut and blood-brain-barrier penetration,212 despite
violating all RO5 and like parameters (Figure 44).
Figure 44. CHEC-9 (83). MW = 917; miLogP = −6.0; HBD = 16;
HBA = 26; RotB = 10; tPSA = 425 Å2.
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Review
7.2. AFPep
AFPep (84, Figure 45) is a cyclic analogue of the 472−479
fragment from alpha-fetoprotein (AFP). AFP inhibits estrogen-
stimulated growth of immature mouse uterus and estrogen-
dependent proliferation of human breast cancer cells.213 The
linear peptide fragment has a bench life of a few weeks and an
undesirable biphasic dose−response curve. Cyclization, sub-
stitution of proline for hydroxyproline, and addition of
asparagine to the C-terminus gave AFPep (84).214 AFPep
was orally active and arrested growth of human tumor
xenografts in mice (10 μg/mouse p.o.). It also decreased the
incidence and multiplicity of breast cancers in carcinogen-
exposed rats (100 μg/rat p.o.). In these animal models, AFPep
had similar effects when administered via oral, ip or sc
routes.215,216
Figure 45. AFP fragment 472−479: MW = 844; miLogP = −5.1;
HBD = 12; HBA = 22; RotB = 23; tPSA = 350 Å2. AFPep (84): MW =
969; miLogP = −5.4; HBD = 17; HBA = 28; RotB = 13; tPSA = 455
Å2 .
7.3. Antamanide and Cyclolinopeptide
Antamanide (85, Figure 46) is a cyclic decapeptide derived
from the same fungus as α-amanitin 75 (A. phalloides).
Antamanide protects against phalloidin poisoning198 by block-
ing the organic anion transporting polypeptide mechanism.
This inhibits the uptake of phalloidins into hepatocytes. Thus,
antamanide functions by a mechanism similar to immunosup-
pressants cyclosporin A and FK506.217 A related hydrophobic
cyclic nonapeptide cyclolinopeptide (86) found in linseed oil
displayed immunosuppressive activity in a mouse model
following oral administration (10 or 100 μg/mouse p.o. in
olive oil).218 A crystal structure of 85 from acetonitrile/water
(CCDC code: ANTAHC10) showed two transannular hydro-
gen bonds in a large bowl-shaped macrocycle. The concave face
displayed most of the polar atoms, including all hydrogen bond
donors. These NH groups were almost entirely shielded by the
hydrophobic side chains.219 Two structures of 86 (CCDC
codes: GIPKAR10, POWWEE) from isopropanol/water and
methanol/water both showed three internal hydrogen
bonds.220,221 One formed a β-turn around residues LIIL and
two transannular interactions. A conserved water molecule was
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Chemical Reviews
tightly held to a polar patch composed of 2 phenylalanine NH
and two proline carbonyl oxygens.
Figure 46. Antamanide (85) and cyclolinopeptide (86). 85: MW =
1147; miLogP = 2.4; HBD = 6; HBA = 20; RotB = 9; tPSA = 256 Å2.
86: MW = 1040; miLogP = 4.6; HBD = 7; HBA = 8; RotB = 13; tPSA
= 244 Å2.
7.4. Cyclopeptolide 1
Cyclopeptolide 1 (87, Figure 47) is a cyclic 10-residue peptide
featuring five N-methyl amides, a depsi-peptide, an O-methyl
tyrosine, and a pipecolic acid. Isolated from the fungus Septoria
sp., 87 was used to treat systemic and vaginal candidosis in rats
by twice daily oral dosing for 4 days (8 × 200 mg/kg p.o.). A
90% cure rate was observed compared to 100% for the
antifungal agent Ketoconazole.222
Figure 47. Cyclopeptolide 1 (87). MW = 1126; miLogP = 2.6; HBD =
4; HBA = 23; RotB = 12; tPSA = 282 Å2.
7.5. Permetin A
The decadepsipeptide permetin A (88, Figure 48) has two D-
residues and an unusual β-hydroxyisoheptanoic acid moiety. It
displayed broad antibacterial activity in vitro. LD50 was
determined in mice (LD50 36 mg/kg ip; LD50 2100 mg/kg
p.o.).223 From these data, the oral absorption of 88 can be
estimated at around 1−2%.
7.6. Synthetic N-Methyl β-Strand Decapeptides
Guided by known permeable and orally bioavailable peptides,
researchers from Novartis prepared a library of highly N-
methylated cyclic decapeptides (89−107, Table 1).224,225 NMR
studies showed extensive and varied intramolecular hydrogen
bonds. For 94, a crystal structure from dichloromethane/
heptane (CCDC code: ILOWAJ) showed two beta turns and 2
transannular hydrogen bonds.224 N-Methylation and stereo-
chemical patterns were preserved while specific side chains
8112
Review
Figure 48. Permetin A (88). MW = 1101; miLogP = −1.8; HBD = 16;
HBA = 24; RotB = 18; tPSA = 386 Å2.
were modified. Their in vitro permeability, metabolic stability
and other pharmacokinetic parameters were evaluated in rats
(Table 1). These were the first examples of cyclic peptides of
comparable size to cyclosporin A (109) with comparable or
greater oral bioavailability.
7.7. Surotomycin
Surotomycin (CB-183315, 108, Figure 49) is a macrocyclic
antimicrobial lipodepsipeptide, containing multiple unusual and
D-amino acids. Originally from Cubist Pharmaceuticals, 88 was
in development by Merck for treatment of C. dif f icile infections
(CDI) and associated diarrhea. In hamsters, 108 was as
effective as vancomycin when administered orally 12 h
postinfection twice-daily for five consecutive days (2, 10, or
25 mg/kg p.o. in water).226 In clinical trials, orally administered
108 (125 and 250 mg p.o.) was safe and well tolerated in
humans and proved more effective than vancomycin in the
treatment of CDI.227 CDI occur in the gut, therefore low
intestinal absorption and systemic availability is desirable. Low
oral absorption was reported in rats (F < 1%)228 and
pharmacokinetic parameters from clinical trials also indicated
low systemic absorption in humans at various doses (500 mg
p.o.: Cmax = 10.5 ng/mL, AUC0‑∞ = 317 ng h/mL; 4000 mg
p.o.: Cmax = 86.7 ng/mL, AUC0‑∞ = 2572 ng h/mL).229
8. CYCLIC UNDECAPEPTIDES
8.1. Cyclosporin A and Synthetic Derivatives
Cyclosporin A (Cyclosporine, CSA, 109, Figure 50) is an orally
bioavailable cyclic peptide natural product with a D-Ala and a
butenyl-methyl-L-threonine. It was first isolated from the fungus
T. inf latum in the early 1970s by scientists at Sandoz (now
Novartis). CSA is most often used in the clinic as an injectable
immunosuppressive drug to combat organ transplant rejection,
but it is also used as an oral treatment for graft versus host
disease. It binds to cyclophilin in lymphocytes.230 CSA has
interesting pharmacokinetics and is a rare example of a large
cyclic peptide (11 residues, MW = 1202 Da) with appreciable
oral bioavailability (F = 20−70% depending upon formulation
and species; 22−29% in rat).106,231 CSA has been used as a
model for developing peptides into orally deliverable drugs.
The crystal structure of CSA from acetone, and the NMR
solution structure determined in CDCl3, both show that it is
stabilized by four intramolecular hydrogen bonds between the
backbone amide NH and the backbone carbonyl oxygen atoms
(CCDC code: DEKSAN).232 In polar solvents like methanol,
CSA exists in at least four conformations, but it is insoluble in
water. Co-crystallization of CSA with cyclophillin (PDB code:
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Table 1. Highly N-Methylated Model Cyclic Decapeptides

no. AA 1 + 8 AA 2 + 7 AA 3 + 6 AA 4 + 9 AA 5 + 10 CLa AUCb (iv/p.o.) F% MW miLogP HBD HBA RotB tPSA (Å2)
89 L L L a A 66 256/69 27 1047 3.9 4 20 12 238
90 L A L a A 64 277/28 10 963 1.2 4 20 8 238
91 L L L G P 121 144/4 3 1043 3.3 4 20 12 238
92 A A A G P 56 377/5 1 791 −4.4 4 20 0 288
93 L A L f P 5 4532/767 18 1139 4.3 4 20 12 238
94 L A L p F 7 2206/1006 46 1139 4.3 4 20 12 238
95 L A L p A 30 579/912 130c 987 1.4 4 20 8 238
96 L A L p V 43 379/40 11 1043 2.9 4 20 10 238
97 L F L p A 4 3673/608 17 1139 4.3 4 20 12 238
98 L A L f A 5 5773/487 10 1115 4.2 4 20 12 238
99 L A L l F 1 12368/491 4 1200 6.8 4 20 16 238
100 L A L p F+X 5 3219/1317 40 1140 3.1 4 21 12 251
101 L A L p F+T 16 12368/491 73 1093 2.2 5 21 11 258
102 L G L p F 10 1490/322 22 1111 4.6 4 20 12 238
103 L L G p F 21 723/214 29 1111 3.6 4 20 12 238
104 L A+L L p F 7 2470/788 32 1181 5.6 4 20 14 238
105 L A+D L p F 12 1192/32 2 1184 3.5 5 22 14 276
106 L A+K L p F 8 1700/8 0 1197 3.8 6 21 16 264
107 L A+T L p F 3 4354/728 15 1170 3.7 5 21 13 258
a
Units = mL/min·kg. bUnits = nM·h. X= 3-pyridylalanine. cValue exceeds 100% but has no SD reported.224

Figure 50. Cyclosporin A (109) and KI-306 (110). 109: MW = 1203;

Figure 49. Surotomycin (108). MW = 1681; miLogP = −4.6; HBD =
25; HBA = 43; RotB = 33; tPSA = 702 Å2.
1CWA) revealed a single bound conformation that differed
from its unbound crystal state.233,234 NMR solution structures
of the CSA-cyclophilin complex in aqueous solution, and 13C
enriched CSA bound to cyclophilin, supported the unusual
binding conformation (PDB codes 1CYA, 1CYB).235,236 When
bound to cyclophilin, CSA was not stabilized by intramolecular
hydrogen bonds. Instead, the four-amide protons were
orientated toward the exterior of the macrocycle. The existence
of multiple different conformers of CSA suggests that it has a
flexible structure. This has been hypothesized to play an
important role in its unusual passive membrane permeability
and oral bioavailability, although this has not been proven.
KI-306 110 (Figure 50) is a monomethoxypoly(ethylene
glycol) modified CSA prodrug developed to improve water
solubility relative to CSA. The oral bioavailability of 110 and
CSA (Sandimmune Neoral solution) were compared in rats.
PEG-modified 110 (7 mg/kg p.o.: AUC = 32.8 μg/mL·h, Cmax
8113
miLogP = 3.6; HBD = 5; HBA = 23; RotB = 15; tPSA = 279 Å2. 110:
MW = 1391; miLogP = 3.7 (mPEG not counted); HBD = 4; HBA =
29; RotB = 24; tPSA = 347 Å2.
= 1.8 μg/mL, Tmax = 1.43 h) had higher bioavailability than
CSA (Sandimmune Neoral, 7 mg/kg p.o.: AUC = 21.4 μg/mL·
h, Cmax = 1.1 μg/mL, Tmax = 2.6 h).237
NIM811 (111, Figure 51) is a CSA analogue modified at
position 4 with N-methylisoleucine and a D-Ala at position 8,
and has been investigated as a therapeutic agent for the
treatment of hepatitis C. 111 suppresses hepatitis C virus
replication but, unlike CSA, was not immunosuppressive. It was
found to have similar oral bioavailability to CSA in mice (10
mg/kg p.o.: Cmax = 2.1 μg/mL, Tmax = 2−5 h, AUC0−24 = 23.7
μg/mL·h), rats (10 mg/kg p.o.: Cmax = 1.4 μg/mL, Tmax = 4−8
h, AUC0−24 = 13.1 μg/mL·h), dogs (20 mg/kg p.o.: Cmax = 5.2
μg/mL, Tmax = 1 h, AUC0−24 = 38.2 μg/mL·h), and monkeys
(10 mg/kg p.o.: Cmax = 1.7 μg/mL, Tmax = 3−8 h, AUC0−24 =
11.3 μg/mL·h).238
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Figure 51. NIM811 (111). MW = 1203; miLogP = 3.6; HBD = 5;

HBA = 23; RotB = 15; tPSA = 279 Å2.

SCY-635 112 (Figure 52) is a CSA analogue recently

developed as a potent inhibitor of hepatitis C virus RNA
replication and showed oral bioavailability in rats (5 mg/kg
p.o.: Cmax = 900 ng/mL, Tmax = 4.0 h, AUC0‑∞ = 13000 ng·h/
mL, t1/2 = 19.2 h, F = 23.1%) and monkeys (5 mg/kg p.o.: Cmax
= 1810 ng/mL, Tmax = 2.0 h, AUC0‑∞ = 27900 ng·h/mL, t1/2 =
24.6 h, F = 17.7%).239 Alisporivir (also Debio-025, DEB025,
UNIL025, 113, Figure 52) is an orally bioavailable CSA
analogue with N-methyl-D-alanine at position 3 and N-ethyl-
valine at position 4.240 Alisporivir completed Phase I and II
clinical trials as a HCV antiviral drug that blocks viral
replication. It has excellent pharmacokinetic and safety profiles
and significantly decreased viral load in HCV-infected patients
(1200 mg/day p.o.: Cmax = 1052 ng/mL, Tmax = 2.0 h,
AUC0−12h = 3858 ng·h/mL).241,242
Figure 52. SCY-635 (112) and alisporivir (113). 112: MW = 1322;
miLogP = 2.9; HBD = 6; HBA = 25; RotB = 19; tPSA = 302 Å2. 113:
MW = 1217; miLogP = 3.8; HBD = 5; HBA = 23; RotB = 15; tPSA =
279 Å2.
Figure 53. THR-123 (114). MW = 1926; miLogP = −6.2; HBD = 35;
HBA = 49; RotB = 42; tPSA = 833 Å2.
including 3 L-lactic acids and 3 D-alpha-hydroxyisovaleric acids.
The 36-membered macrocycle is a potassium-selective
ionophore (LD50 4 mg/kg p.o, rat).244 Unfortunately, there is
little pharmacokinetic data available for most dodecapeptides.
9.1. Cerulide
The cyclic dodecadepsipeptide cerulide (115, Figure 54)
contains seven L-residues, five D-residues, six depsipeptide
bonds, and six peptide bonds. It is an emetic toxin (induces
vomiting) produced by the bacteria B. cereus.245 Cerulide was
administered (p.o. or ip) to the S. murinus rodents to test for
emetic effects at doses from 2 to 32 μg/kg. The maximal oral
dose (32 μg/kg p.o.) resulted in emesis in 5 of 5 test animals.
115 (50 μg/kg p.o.) in combination with 5-HT3 antagonist,
ondansetron (iv), had no emetic effect. The authors concluded
that cerulide caused emesis through central 5-HT3 receptor
stimulation of the vagus afferent. Cerulide had ED50 12.9 μg/kg
(p.o.) and 9.8 μg/kg (ip) for inducing emesis in S. murinus
rodents.245
9.2. L-Phenylalanine-Dipicolinate Macrocycle
A series of linear and macrocyclic L-phenylalanine-dipicolinic
acid based compounds were synthesized and tested for anti-
inflammatory activity. Anti-inflammatory activity was evaluated
by oral administration (7 mg/kg p.o.) to rats with carrageenan-

8.2. THR-123
The 16-residue disulfide-bridged cyclic peptide THR-123 (114,
Figure 53) was designed to mimic a loop in the structure of
human BMP7; a protein from the TGF-β superfamily that
binds Alk2/3/6 and antagonizes TGF-β−mediated activity.
The disulfide bridge stabilizes the conformation. Compound
114 was active against kidney fibrosis by inhibiting the Alk3
receptor after oral administration (5−15 mg/kg p.o.) to
mice.243

9. CYCLIC DODECA- AND TRIDECAPEPTIDES

There are a number of cyclic dodecapeptides that are orally
active. One example is the antibiotic valinomycin, which Figure 54. Cereulide (115). MW = 1153; miLog P = 6.6; HBD = 6;
contains 3 L-valines, 3 D-valines, 6 depsipeptide components HBA = 24; RotB = 12; tPSA = 332 Å2.

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induced paw edema. Two known nonsteroidal anti-inflamma-
tory drugs (NSAIDs), indomethacin and diclofenac, were used
as controls. Compound 116 (Figure 55) caused 72% inhibition
of paw inflammation compared to 82% and 61% for
indomethacin and diclofenac.246
Figure 55. L-Phenylalanine-dipicolinic acid based macrocycle (116).
MW = 1930; miLogP = 8.6; HBD = 12; HBA = 36; RotB = 16; tPSA =
505 Å2.
10. CYCLIC TETRADECAPEPTIDES AND BEYOND
10.1. Conotoxins and Synthetic Derivatives
Linaclotide (117, Figure 56) is a 14-residue peptide toxin that
is cyclized through three cysteine disulfide bridges. It is a
treatment for irritable bowel syndrome with constipation and
for chronic constipation. 117 was stable in simulated gastric
fluid up to 3 h and was not hydrolyzed by pepsin. Following
oral administration to rats, 117 was found in plasma despite
minimal oral absorption (10 mg/kg p.o.: AUC0−6h = 19.7
ng·h/mL, F = 0.1%).247
Figure 56. Linaclotide (117). MW = 1527; miLogP = −5.7; HBD =
21; HBA = 36; RotB = 13; tPSA = 574 Å2.
α-Conotoxin MII (118) and lipidated analogue 119 (Figure
57) are 16-residue bis-disulfide bridged natural peptides that are
orally absorbed. Following oral administration of 118 and a
lipophilic analogue 119 (1 mg/kg p.o.) to male Sprague−
Dawley rats, ∼6% of both peptides crossed the GI tract and
were found in a variety of body tissues based on radioactive
labeling.248
A head-to-tail cyclized analogue of α-conotoxin Vc1.1 (120,
Figure 58) from the cone snail C. victoriae was administered
8115
Review
Figure 57. α-Conotoxin MII (118) and lipidated analogue (119). 118:
MW = 1711; miLogP = −5.8; HBD = 27; HBA = 45; RotB = 21; tPSA
= 718 Å2. 119: MW = 1837; miLogP = −4.6; HBD = 27; HBA = 45;
RotB = 29; tPSA = 718 Å2.
orally to rats (0.3 or 3.0 mg/kg p.o.) and produced significant
dose-dependent relief of neuropathic pain.249
Figure 58. Head-to-tail cyclized analogue of α-conotoxin Vc1.1 (120).
MW = 2160; miLogP = −6.3; HBD = 32; HBA = 59; RotB = 21; tPSA
= 909 Å2.
10.2. Duramycin
Duramycin (lancovutide, Moli1901, 121, Figure 59) is a highly
cross-linked 19-residue peptide with three lanthione bridges
and one lysoalanine bridge, all derived from L-cysteine. 121 was
given to rats via pulmonary, intravenous or oral administration.
Faeces, urine, blood and tissue were collected over a 7-day
period. Analysis indicated minimal systemic availability
following oral administration with almost 100% of the oral
dose recovered from faeces. Nevertheless, 121 was detected in
serum of rats 7 days after oral administration (231 mg/kg
p.o.).250
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Chemical Reviews
Figure 59. Duramycin (121). MW = 2013; miLogP = −6.1; HBD =
29; HBA = 48; RotB = 19; tPSA = 760 Å2.
10.3. Kalata B1 and Other Cyclotides
The cyclotides are a rapidly expanding family of cyclic peptides,
which owe their stability to a remarkably conserved cyclic
cysteine knot (CCK) motif. Cyclotides have a range of
biological activities (e.g., antiviral, antitumor, antibacterial)
and are actively being investigated for their potential in drug
development.251 The prototypic cyclotide, kalata B1 (122,
Figure 60) is a 29-residue peptide from the African medicinal
plant O. af f inis. Kalata B1 is a uterotonic agent252 and by virtue
of its knotted structure is chemically and enzymatically stable
and can be made into tea by infusing its parent plant with
boiling water. This tea is an indigenous medicinal agent used to
accelerate childbirth by inducing uterine contractions. Recently,
orally active peptidic bradykinin B1 receptor antagonists were
grafted onto kalata B1 to replace the entire loop 6 unit.253 The
grafted analogue CKB-KAL (123, Figure 60) was the most
potent compound studied and inhibited acetic acid induced
abdominal contraction (the writhing assay) after injection (1
mg/kg ip) or oral administration (10 mg/kg p.o.). Maximum
inhibition was similar for both routes (49% ip and 42% p.o.).253
All the cysteine cross-links have L-stereochemistry.
Figure 60. Cyclotides kalata B1 (122), CKB-KAL (123), and
KB1[T20K] (124). 122: MW = 2892; QPLogP = −15.8*; HBD =
42; HBA = 74; RotB = 23; tPSA* = 1207 Å2. 123: MW = 3135;
QPLogP = −17.6*; HBD = 42; HBA = 77; RotB = 26; tPSA* = 1224
Å2. 124: MW = 2919; QPLogP = −16.6*; HBD = 43; HBA = 74;
RotB = 26; tPSA* = 1212 Å2. “*”: Data calculated with QikProp256
due to structures exceeding Molinspiration maximum size.
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Review
Recently a lysine mutant of kalata B1, KB1[T20K] (124,
Figure 60),254 was investigated in an experimental autoimmune
encephalomyelitis model of multiple sclerosis. 124 was
efficacious when administered orally to mice (10 or 20 mg/
kg p.o.) and impeded disease progression at 20 mg/kg p.o.
compared to the control group dosed with an inactive kalata B1
derivative [V10K].255
10.4. Stapled α-Helix
SAH-gp41(626−662) (125) is a doubly stapled 37 residue peptide
reported to be a stable α-helix and resistant to pepsin and
chromtrypsin. The bridging residues are of normal S-
configuration but possess an additional α-methyl group.
When mice were given 125 by oral gavage (10 and 20 mg/
kg p.o.; Figure 61), it was found in plasma in a dose dependent
manner (10 mg/kg p.o.: Cmax = 1.5 μg/mL; 20 mg/kg p.o.: 2.3
μg/mL). The nonstapled linear analogue was not detected in
plasma after oral administration.257 We are not aware of any
other similar helix-constrained peptide reported to show any
significant oral absorption at all.
Figure 61. HIV fusion inhibitor helical peptide SAH-gp41(626−666)
(125). B = Norleucine, X = (S)-α-methyl(4-pentenyl)alanine. 125:
MW = 4589; QPLogP = −23.1*; HBD = 72; HBA = 117; RotB = 132;
tPSA* = 2173 Å2. “*”: Data calculated with QikProp256 due to
structures exceeding Molinspiration maximum size.
11. INFLUENCES ON ORAL BIOAVAILABILITY
Oral bioavailability is a specific term that indicates how much
intact peptide can be measured in the circulation after oral
ingestion. By definition this parameter is dictated not just by
compound absorption through an intestinal membrane but also
by compound stability, interactions, and metabolism before
intestinal absorption as well as compound stability, metabolism,
tissue distribution, and clearance after uptake. Most proteins
and peptides simply do not get absorbed from the GI tract, and
those that do are often rapidly metabolized, cleared, or
distributed into tissues. Key problems for peptides are their
high polarity and large polar surface area, large size, low
membrane permeability, high clearance, and rapid metabolism.
Cyclization of peptides frequently aids formation of intra-
molecular hydrogen bonds and orientation of side chains,
properties that can help shield polar atoms from the solvent
medium (water/lipid) and protect against proteolytic degrada-
tion, reduce flexibility, reduce polar surface area, and promote
permeation through cell membranes. We describe 125 cyclic
peptides above that are orally absorbed, orally active or have a
measured oral bioavailability (F%). Physicochemical parameters
traditionally associated with limits on oral bioavailability have
been calculated here for each compound (see Figure legends).
For cyclic peptides with reported oral bioavailability, we now
plot key molecular properties, such as MW (Figure 62A),
miLogP (Figure 62B), HBD (Figure 63A), HBA (Figure 63B,
Figure 64), RotB (Figure 65A) and tPSA (Figure 65B), against
F%.
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Chemical Reviews
Figure 62. Plots of oral bioavailability (F%) versus (A) molecular
weight, and (B) octanol−water partition coefficient (miLogP), of
orally absorbed cyclic peptides 2−117.
The plot of molecular weight versus oral bioavailability
(Figure 62A) shows that cyclic peptides with molecular weights
ranging from 500 to 1350 can have some oral bioavailability (F
= 0.1−10%). Cyclic peptides with higher oral bioavailability
clustered into two distinct MW ranges 700−800 and 1000−
1200, the latter possibly skewed by the high number of
decapeptide- and cyclosporine-like cyclic peptides. It is
encouraging that 14 cyclic peptides with MW = 500−850 (2,
8, 12, 19, 31, 39, 47, 49, 55, 57−59, 63, and 64) had > 10%
oral bioavailability. A further 23 cyclic peptides (73, 76−81, 89,
90, 93−98, 100−104, 107, 109, and 112) with MW = 960−
1350 had ≥10% oral bioavailability. These data demonstrate
that a higher MW (> 500) does not necessarily preclude oral
bioavailability for cyclic peptides, even though it is important
for most small molecules.17,64,65,123,124
Oral bioavailability for each cyclic peptide in this review was
next plotted against the predicted octanol−water partition
coefficient (miLogP) in Figure 62B. The calculated miLogP
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Review
Figure 63. Plots of oral bioavailability (F%) versus (A) hydrogen bond
donors (HBD) or (B) hydrogen bond acceptors (HBA), for orally
absorbed cyclic peptides 12−112.
value is one of the better predictors of octanol−water partition
coefficients.258 The partition coefficient P (or distribution
coefficient D) is a ratio of hydrophobicity : hydrophilicity and a
measure of relative compound solubility in the two solvents. To
be orally absorbed, a cyclic peptide must permeate intestinal
membranes by partitioning into the lipid, but it must not be so
lipophilic that it cannot partition out. The compromise for
small molecule drugs is LogP = 0−5, with some support for the
variation −0.4 to 5.6.259
Figure 62B shows that most cyclic peptides in this review
with F ≥ 10% oral bioavailability also had miLogP 1−5 (e.g., 2,
12, 19, 31, 39, 47, 49, 57, 58, 63, 64, 76, 79, 89, 90, 93−98,
100−103, 107, 109, and 112). The extent of oral bioavailability
did not correlate with miLogP within this range. Seven orally
bioavailable (F > 10%) cyclic peptides (59, 73, 77, 78, 80, 81,
and 104) had miLogP > 5. Eleven cyclic peptides with miLogP
< 0 had measurable oral bioavailability but this was limited to F
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Chemical Reviews
Figure 64. Plot of oral bioavailability (F%) versus hydrogen bond
acceptors (HBA), where the amide nitrogen is not counted as a HBA
atom, for orally absorbed cyclic peptides 12−112.
< 10% (7, 8, 21−25, 33, 34, 40, and 92). These data suggest
that hydrophobicity (and lipophilicity) is likely an important
contributor to oral bioavailability of these cyclic peptides. This
data shows that this parameter is more accommodating for
cyclic peptides (miLogP = 1−8) than it is for small molecules
(LogP = 0−5).
Two RO5 criteria that guide oral bioavailability of drug-like
small molecules are the numbers of hydrogen bond donors
(HBD) and acceptors (HBA). A small molecule is considered
more likely to be orally bioavailable if it contains ≤ 5 HBD and
≤ 10 HBA,14−16 This implies that hydrogen bond donors are
more detrimental to oral bioavailability. Figure 63A shows that
cyclic peptides with 1−6 HBD are much more likely to be
orally bioavailable than those with HBD > 6. A few compounds
(19, 20, 31, and 112) with HBD = 6 had F = 9−25%, but all
compounds with HBD > 6, except 8, (15%) had F ≤ 10%. If
this data set is representative of all cyclic peptides, HBD does
indeed limit oral bioavailability for this class of molecules.
Figure 63B shows that all cyclic peptides examined had HBA
> 10, and those with F > 50% had HBA = 12−22. Thus, almost
all of the orally bioavailable cyclic peptides in this review
apparently violate the RO5 limit on HBA (Figure 63B), a
guideline developed from small molecule drugs. This suggests
that optimizing cyclic peptides to reduce HBD, rather HBA, is
more likely to improve oral bioavailability. However, Figure
63B uses the original definition of HBA,14,15 which was simply
the total count of all nitrogen and oxygen atoms, irrespective of
their actual hydrogen bond accepting properties. This is
important because the amide nitrogen is not a hydrogen
bond acceptor due to electron delocalization of the nitrogen
lone pair, indeed protonation always occurs preferentially on
the amide oxygen, not the nitrogen.260 Thus, if HBA alone is
re-counted without amide nitrogens, the result changes
dramatically (Figure 64). As shown, 24 of the cyclic peptides
for which F ≥ 10% now have HBA ≤ 10 (e.g., 2, 7, 8, 12, 19,
31, 39, 47, 49, 55, 57−59, 63, 64, 89, 93, 95−98, and 102−
104). Only 12 cyclic peptides with F ≥ 10% now have HBA >
10 (e.g., 73, 76-81, 100, 101, 107, 109, and 112). Thus, the
HBA count is still violated by one-third of the cyclic peptides
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Review
with F ≥ 10% oral bioavailability, but two-thirds of the cyclic
peptides now conform to conventional RO5 guidelines. We
conclude that HBA count is somewhat important for oral
bioavailability of cyclic peptides but not as restrictive as the
HBD count.
Rotatable bonds (RotB) reflect the degree of conformational
flexibility in a molecule. RO5 compliant drug-like compounds
typically have ≤ 10 rotatable bonds.17 However, care must be
taken in the use of RotB itself, or as part of any prediction
model for oral bioavailability. First, it is notable that strict RO5
rules describe any bond in a cycle of any ring size as being not
rotatable. However, as cyclic peptide ring size increases, the
energy required to rotate Φ/Ψ bonds within the macrocycle
decreases, reducing the utility of this RotB metric. As observed
above, from cyclic tetrapeptide 2 to cyclosporin A, solution
structures and crystal structures can often differ greatly due to
such rotations or ring flips. Second, studies in rats and humans
have shown that the calculation method, therapeutic class of
compounds, and desired F% all influence the acceptable
number of RotB. In rats, certain therapeutic classes of drugs
had acceptable F% with RotB > 12.261 Recently, a study on the
oral bioavailability of 1014 molecules in humans found the
RotB limit to be 13.262 Figure 65A shows that most of the cyclic
peptides examined here with oral bioavailability had 6−16
rotatable bonds. Compounds 12 (RotB = 2, F = 16%) and 112
(RotB = 19, F = 23%) were outside this range. Twenty three
compounds exceeded RotB ≤ 10; compounds 2, 19, 31, 93, 97,
98, and 107 had F > 10%, while 36, 73, 76, 77, 78, 79, 80, 81,
89, 94, 100, 101, 102, 103, 104, 109, and 112 had F ≥ 20%.
This is consistent with a degree of conformational rigidity being
favorable for oral bioavailability of cyclic peptides.
The plot of topological polar surface area (tPSA) versus oral
bioavailability (Figure 65B) indicated that large polar surfaces
(tPSA > 300 Å 2) were a barrier to appreciable oral
bioavailability (F > 10%), likely due to low absorption and
high clearance. Fifteen of the smaller cyclic peptides with still
large polar surfaces (tPSA 100−205) had F = ≥ 5% (2, 7, 12,
18−20, 31, 39, 47, 49, 55, 57, 62, 63, and 64) and three had F
> 30% (39, 57, and 64). Twenty four compounds with tPSA
200−300 were still orally bioavailable with F ≥ 10% (8, 31, 73,
76−81, 89, 93−98, 100−104, 107, 109, and 112), and violate
the prediction limit17 for most drug-like small molecules (tPSA
< 140 Å2).
12. CONCLUSIONS AND FUTURE PROSPECTS
This compilation is based on data on oral activity, oral
absorption, and oral bioavailability for 125 cyclic peptides,
composed of 4 to 37 amino acids and derivatives. It indicates
that there are opportunities to expand the molecular weight
range beyond the RO5 limit of small molecules (MW ≤ 500)
for cyclic peptides and still obtain appreciable oral bioavail-
ability. This is encouraging for therapeutic targeting of
protein−protein interactions that may require modulators
with larger surface areas. The cyclic peptides show great
variation in the parameters (MW, HBD, HBA, LogP, RotB, and
tPSA) conventionally associated with limiting oral bioavail-
ability (F%) of drug-like molecules. Such parameters are of
course all interdependent. As molecules get bigger, they either
increase in the number of polar components (increasing HBD
and HBA) or nonpolar components (increasing LogP),
generally become more flexible (increasing RotB), and thus
expose more of their surface area to water (tPSA) and
membranes. The RO5 has been used very successfully to guide
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Chemical Reviews Review

development of orally bioavailable small molecule drugs over

the past two decades. However, it is clear from Figures 62−65
that orally bioavailable cyclic peptides can violate all of these
parameters, especially MW, tPSA, HBA, and, but not to the
same extent, HBD, RotB, and LogP. Interestingly, Figures 62,
63, and 65 show this to be true for the four most orally
bioavailable (F > 70%) cyclic peptides (39, 80, 95, and 101),
which respectively had MW > 500 (724, 1196, 987, and 1079),
HBA > 10 (12, 22, 20, and 21), and three had tPSA > 200 Å2
(140, 256, 238, and 258). On the other hand, all four
compounds respectively had HBD ≤ 5 (0, 3, 4, and 5), three
had RotB ≤ 12 (12, 13, 8, and 10), two had milogP < 5 (6.8,
7.5, 1.4, and 2.0) and, without counting amide nitrogens, two
had HBA ≤ 10 (9, 12, 10, and 11). Further systematic
evaluations of new orally bioavailable cyclic peptides with MW
> 500 are needed to support these observations and better
define limits of these parameters on oral bioavailability. This
may better enable prediction and optimization of orally
bioavailable cyclic peptides larger than 5 amino acids.
The above data set supports current and new chemical
strategies to enhance oral absorption. These include reducing
HBD by removing backbone amide NH protons through
substitution with N-alkyl groups, esters, ethers, or hydro-
carbons; incorporating the nitrogen into a heterocycle; or
substituting amides with other surrogates. Thus, cyclic
hexapeptide 39 has 3 depsipeptide linkages, 3 NMe
substituents, and no NH protons; cyclic octapeptide 80 has 1
depsipeptide linkage, 2 NMe substituents, 2 prolines, and 3 NH
protons; while cyclic decapeptides 90 and 101 both have 6
NMe substituents and 4 NH protons. However, these
approaches may not be a panacea to the problem of poor
oral bioavailability. Biological activity is usually dependent on
three-dimensional structure, and N-methylation or insertion of
amide isosteres263 that reduce NH protons (HBD) can alter
peptide structure, which may negatively impact function. For
example, amide protons might be required for direct binding to
a target or for maintaining the bioactive conformation through
intramolecular hydrogen bonds. N-Methylation or amide
isosteres can disrupt either or both of these properties.
Intramolecular hydrogen bonds are often desirable because
they reduce the exposed polar surface of a peptide. Introducing
an amide isostere also normally requires additional synthetic Figure 65. Plots of oral bioavailability (F%) versus: (A) rotatable
steps, the value of which must be weighed up against potential bonds (RotB) or (B) topological polar surface area (tPSA), for orally
absorbed cyclic peptides 12−112.
pharmacokinetic benefits. Ester bonds are present in many
naturally occurring cyclic depsipeptides and are thought to be
evolutionary adaptions that facilitate permeability through Two important considerations missing from the above
biological membranes. Romidepsin 12, largazole 13, kahalalide analysis are three-dimensional polar surface area and metabolic
F 61, and Gq inhibitor YM254890 67 are among many stability. There were only a few reported three-dimensional
examples of depsipeptides that are active in mammalian cells structures for cyclic peptides in this review, but the effect of
and bind to intracellular targets, supporting this hypothesis. three-dimensional structure and conformational preference of
However, ester bonds are also unstable and readily hydrolyzed cyclic peptides in water versus lipid membranes is very
by esterases in cells and in vivo. Although esterification has important. Three-dimensional structure can significantly change
been widely exploited for delivering carboxylic acids as the solvent-exposed polar surface area. Large macrocycles might
prodrugs, ester substitution can also jeopardize in vivo stability. be folded through intramolecular hydrogen bonds that are
For example, orally bioavailable romidepsin 12, largazole 13 shielded from solvent. This could produce much smaller
and kahalalide F 61 have very short half-lives at 37 °C (t1/2 = 6, exposed polar surface areas than suggested by tPSA calculations
0.3, 28 min), probably due to poor in vivo stability of their on 2D structures. Structural compression may enable larger
cleavable ester moieties. In enniatin B1 39, every second amino cyclic peptides (MW) with more polar atoms (HBD/HBA)
acid linkage is a depsipeptide bond. It displays unusually high and larger tPSA (but low 3D-PSA) to be tolerated for oral
oral bioavailability (F = 91%), but also has a very short half-life absorption. Compaction or hydrophobic collapse is a dominant
(t1/2 = 9 min) leading to minimal exposure in plasma (AUC feature of protein folding that enables proteins to shield their
25.3 ng.h/mL). hydrophobic residues in their interiors away from water.
8119 DOI: 10.1021/acs.chemrev.6b00838
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Chemical Reviews
Conversely, passive diffusion through a lipid membrane would
be favored by the reverse effect, structural compression to
shield polar residues in the interior of a cyclic peptide and
expose outer hydrophobic substituents to lipid bilayers.
Compact structures that undergo minimal change when going
from water through a lipid membrane to water again should pay
a lower entropy penalty to overcome this transition. Thus,
rigidity may be more desirable than flexibility for oral
bioavailability, although this brings an accompanying problem
of lower aqueous solubility.
The nature and extent of water solvation is likely to be a key
consideration for oral absorption. Water molecules need to be
stripped away from polar surfaces to facilitate uptake from the
gut via passive diffusion or for interaction with proteins that
promote active transport. Experimental determination and
comparison of solvation energies can be very helpful for
predicting oral absorption as well as solubility. The nature of
amino acid substituents and the degree of flexibility in the
macrocycle not only dictates solvation but also susceptibility to
oxidative and degradative metabolism. Amino acid substituents
and peptide backbone conformation also determine inter-
actions with proteins, including transporters and efflux
promoters, metabolizing enzymes, albumin, plasma, and cellular
proteins. All of these factors contribute to oral bioavailability
and need to be studied to better understand how metabolism
and clearance, in addition to absorption, are affected by
structural modifications to cyclic peptides.
This collection of cyclic peptides has the potential to
stimulate chemists to reach for new horizons in the design,
synthesis, and application to humans of larger, biologically
active, compounds including macrocycles. To date extensive
research has focused on membrane and cell permeability of
peptides and macrocycles but, as emphasized in this review,
membrane permeability is only one of many contributors to
oral bioavailability. More details on solvation, absorption,
metabolism, tissue distribution, clearance and three-dimen-
sional structures of cyclic peptides can provide a deeper
understanding of how to better exploit the different factors that
influence their oral bioavailability and their promise as new oral
therapeutics.
AUTHOR INFORMATION
Corresponding Author
*Fax: +61-733462990. E-mail: d.fairlie@imb.uq.edu.au.
ORCID
Martin J. Stoermer: 0000-0003-3445-2104
David P. Fairlie: 0000-0002-7856-8566
Present Address
§
La Trobe Institute of Molecular Sciences, La Trobe University,
Melbourne, VIC 3083, Australia.
Notes
The authors declare no competing financial interest.
Tables of physicochemical parameters, pharmacological param-
eters, formulation vehicles for compounds in this review can be
provided upon request from the authors.
Biographies
Daniel S. Nielsen obtained a B.Sc and M.Sc from the Faculty of
Pharmaceutical Sciences at University of Copenhagen (2011). He
received a University of Queensland International Ph.D. Scholarship
(2012) and obtained a Ph.D. (2016) on orally bioavailable cyclic
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Review
peptides with Professor Fairlie. He conducted postdoctoral research in
the same lab. In 2017, he is a postdoctoral fellow in Professor Meldal’s
group at University of Copenhagen. Research interests are in
pharmaceutical sciences, natural products, cyclic peptides, organic
synthesis, and drug discovery.
Nicholas E. Shepherd developed bioactive helical peptide mimics
during his Ph.D. at the University of Queensland. As a postdoctoral
researcher at the University of Tokyo, he identified novel bimetallic
catalysts to enantioselectively synthesize pharmaceutically important
chiral small molecules. As an ARC DECRA fellow at the University of
Sydney, he used chemical tools to define the structural basis for R/
DNA-binding proteins and epigenetic multiprotein complex function.
He is currently a senior research officer at the University of
Queensland.
Weijun Xu was a lecturer at the School of Chemical and Life Sciences
(Singapore Polytechnic), an undergraduate (B.Sc. Hons. Biochemistry,
2006) and postgraduate of University of Queensland (Ph.D., 2013−
2016), where he is a research assistant. He was an International
Postgraduate Research Scholar and University of Queensland
Advantage Scholar with Professor Fairlie on computer-aided molecular
modeling of protein−ligand and protein−protein interactions,
involving discovery of ligands for GPCRs, proteases, enzymes, and
other proteins involved in human immune systems.
Andrew J. Lucke obtained a Ph.D. in organic chemistry at Griffith
University, followed by postdoctoral research in the United Kingdom
and Australia. He has published in organic, medicinal, physical, and
macromolecular chemistry. His recent research has used molecular
simulation techniques to model interactions between small organic
molecules and large biomolecules. He is a molecular modeller at La
Trobe University with interests in organic synthesis, medicinal
chemistry (drug design and development), peptidomimetics, and
protein molecular dynamics.
Martin Stoermer obtained a B.Sc. Hons (1986) and Ph.D. (1991)
from University of Sydney in organic synthesis and organometallic
reaction mechanisms. He worked in drug design at the Centre for
Drug Design and Development, University of Queensland (1991−
1995) and in the Institute for Molecular Bioscience since 2001 as a
Senior Research Officer. He has also collaborated with the
pharmaceutical industry while at the Technical University of
Clausthal-Zellerfeld, Germany (1995−1996) and the Victorian College
of Pharmacy, Monash University (1996−2000).
David Fairlie studied at Adelaide, Australian National, New South
Wales, Stanford and Toronto Universities. At University of Queens-
land, he led the Chemistry Group in the Centre for Drug Design and
Development and is Head of the IMB Division of Chemistry and
Structural Biology. Interests are medicinal/organic chemistry, protein
mimics, and modulators of GPCRs, PPIs and enzymes in
inflammation, infection, neurodegeneration and cancer. He studies
mechanisms of chemical, immunological and biological reactions,
disease development, and drug action.
ACKNOWLEDGMENTS
We acknowledge grants from the National Health and Medical
Research Council (Senior Principal Research Fellowships
1027369 and 1117017 to D.P.F.) and the Australian Research
Council (DP150104609, DP130100629, DP160104442, and
CE140100011).
ABBREVIATIONS
AFP alpha-fetoprotein
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Chemical Reviews Review

AFPep alpha-fetoprotein 472−479 cyclic analogue Tmax time to reach the peak serum concentration
Aha 6-aminohexanoic acid TNBS trinitrobenzenesulfonic acid
Alk3 bone morphogenetic protein receptor, type IA tPSA topological polar surface area
AUC area under the curve, the integral of the VD volume of distribution, the apparent volume in
concentration time curve (0-∞ = after a single which the drug is distributed at steady state
dose or τ,ss = at steady state)
BBMV brush border membrane vesicles
BMP7 bone morphogenic protein 7 REFERENCES
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8128 DOI: 10.1021/acs.chemrev.6b00838

Chem. Rev. 2017, 117, 8094−8128