Paired Utility of Aza-Amino Acyl Proline and Indolizidinone Amino Acid Residues for Peptide Mimicry: Conception of Prostaglandin F2α Receptor Allosteric Modulators That Delay Preterm Birth

Article abstract of DOI:10.1021/acs.jmedchem.9b00056

Peptide mimicry employing a combination of aza-amino acyl proline and indolizidinone residues has been used to develop allosteric modulators of the prostaglandin F2α receptor. The systematic study of the N-terminal phenylacetyl moiety and the conformation and side chain functions of the central turn dipeptide residue has demonstrated the sensitive relationships between modulator activity and topology. Examination of aza-Gly-Pro and aza-Phe-Pro analogs 2a and 2b in a murine preterm labor model featuring treatment with lipopolysaccharide demonstrated their capacity to extend significantly (>20 h) the average time of delivery offering new prototypes for delaying premature birth.

Full text of DOI:10.1021/acs.jmedchem.9b00056

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Article
Paired Utility of Aza-Amino Acyl Proline and Indolizidinone Amino
Acid Residues for Peptide Mimicry: Conception of Prostaglandin
F2# Receptor Allosteric Modulators that Delay Preterm Birth
Fatemeh M. Mir, N. D. Prasad Atmuri, Carine B Bourguet, Jennifer
Rodon Fores, Xin Hou, Sylvain Chemtob, and William D. Lubell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00056 • Publication Date (Web): 01 Apr 2019
Downloaded from http://pubs.acs.org on April 1, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
Paired Utility of Aza-Amino Acyl Proline and
Indolizidinone Amino Acid Residues for Peptide
Mimicry: Conception of Prostaglandin F2α Receptor
Allosteric Modulators that Delay Preterm Birth
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Fatemeh M. Mir †§, N. D. Prasad Atmuri †§, Carine B. Bourguet †,
†
‡
‡
Jennifer Rodon Fores , Xin Hou , Sylvain Chemtob , William D. Lubell
†*
†Département de Chimie, Université de Montréal, C.P. 6128 Succursale
Centre-Ville, Montréal H3C 3J7 QC, Canada
‡Centre Hospitalier Universitaire Sainte-Justine Research Center,
Montréal H3T 1C5, QC, Canada
ABSTRACT: Peptide mimicry employing a combination of aza-amino acyl
proline and indolizidinone residues has been used to develop allosteric
modulators of the prostaglandin F2α receptor. Systematic study of the
N-terminal phenylacetyl moiety, and the conformation and side chain
functions of the central turn dipeptide residue has demonstrated the
sensitive relationships between modulator activity and topology.
Examination of aza-Gly-Pro and aza-Phe-Pro analogs 2a and 2b in a murine
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
preterm labor model featuring treatment with lipopolysaccharide
demonstrated their capacity to extend significantly (>20 h) the average
time of delivery offering new prototypes for delaying premature birth.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION:
Complementary methods to mimic relevant secondary structures offer
potential
for
parallel
investigation
of
structure-activity
relationships in peptide-based drug discovery. Paired utility for type
II′ -turn mimicry has specifically been exhibited by aza-amino acyl
2
proline and indolizidin-2-one amino acid (I aa) scaffolds in several
biologically active peptides.1 For example, 10- and 7-fold greater
potency was observed respectively on replacement of the Gly-Pro
2
dipeptide by aza-Gly-Pro and I aa in calcitonin gene-related peptide
antagonists.1a Moreover, increased caspase-9 mediated apoptotic cell
death has been exhibited relative to the parent Ala-Val-Pro-Ile Smac
(second mitochondria-derived activator of caspase) protein mimic by
analogs in which the Val-Pro dipeptide was swapped for aza-amino acyl
2
1b, 1c
proline and I aa residues.
Pertinent to the present study, similar
activity and efficacy in inhibiting myometrial contractions by way of
modulation of the prostaglandin-F2α (PGF2α) receptor (FP) has been
2
exhibited by the I aa analog PDC113.824 (S-1a) and its aza-amino acyl
proline counterparts 2ac (Figure 1).1d To better seize the advantages
2
of the paired utility of aza-amino acyl proline and I aa peptide
derivatives, a series of diversity-oriented methods are now presented
2
ACS Paragon Plus Environment

Page 3 of 128
Journal of Medicinal Chemistry
1
2
for synthesizing analogs of the related turn mimics in S-1a and 2 to
conceive novel FP modulators.2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In a program targeted on the development of novel tocolytic (labor
suppressing) agents that can delay labor and inhibit preterm birth,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
modulators S-1a and 2ac have been valuable prototypes and probes for
2
mediating FP signalling and myometrial contractions. For example, I aa
analog S-1a inhibited selectively PGF2α-induced myometrial contractions
3
in a mouse model delaying labor up to 42 h. Examination of the action
of S-1a has revealed an allosteric mechanism implicating biased
signalling.3 In the absence of the endogenous agonist PGF2α, S-1a is
inactive. Without competing for the binding site of the orthosteric
ligand, S-1a modulated the exchange rate of PGF2α with tritium-labeled
PGF2α. Downstream of the FP receptor, S-1a potentiated PGF2α-mediated
activation of protein kinase C and mitogen-activated protein kinase
(ERK1/2) signalling, but reduced Gα12-dependent activation of RhoA/ROCK
signalling leading to actin remodeling and contraction of human
myometrial cells.3
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
Figure 1. I aa and aza-amino acyl proline FP modulators S-1a and 2ac,
and related counterparts
2
Replacement of the I aa scaffold with aza-amino acyl-L-proline
analogs gave azapeptides 2ac, which exhibited similar effects on
myometrial contraction and ERK1/2 signalling; however, varying
influences on RhoA/ROCK signalling contingent on the aza-residue side
chain.1d The latter result was particularly interesting for two reasons.
In the first place, the complementarity of aza-amino acyl-L-proline and
I2aa residues was highlighted because earlier attempts failed to provide
4
ACS Paragon Plus Environment

Page 5 of 128
Journal of Medicinal Chemistry
1
2
2
active analogs from replacement of the I aa scaffold with alternative
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
indolizidin-9-one
and
quinolizidinone
amino
acid
residues.
Furthermore, modification of the aza-amino acid side chain offered
potential to modulate biased signalling to ideally create alternative
probes for studying FP receptor signalling towards the development of
improved tocolytic agents.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The healthcare drivers for developing tocolytic prototypes such
as S-1a and 2ac include the growing significance of preterm birth
before 37 weeks of pregnancy, and the associated socioeconomic
consequences. Preterm birth is correlated with 60-80% of all perinatal
deaths,4 and long-term neonatal and infant healthcare outcomes,
including neurodevelopmental handicap, chronic respiratory illness,
5
infections, and long-term impairment. In spite of advances in knowledge
of risk factors and medical interventions to reduce perinatal death,
success has been limited in the prediction and prevention of preterm
labor, the rate of which has risen globally, such that 15 million babies
are estimated to be born prematurely per year.6 Healthcare costs
connected to preterm birth are significant: >$26.2 billion in 2005 in
7
the USA alone amounting to $51,600 per premature birth. Tocolytic drugs
currently used in the clinical setting to block uterine contractions
have marginal efficacy and at times are associated with significant
8
adverse effects to either the mother and/or fetus. Although they may
extend gestation for the 48 hours needed to administer corticosteroids
to the parturient for fetal surfactant maturation, for many patients,
gestation needs to be prolonged for longer periods.
Prevention of
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
preterm birth remains a major health concern associated with serious
short- and long-term complications to infants in industrialized and
agrarian countries worldwide.
In the pursuit of improved tocolytics to inhibit preterm labor and
probes to better understand the importance of FP mediated signalling
pathways in labor, a set of diversity-oriented methods for making
azapeptide and indolizidin-2-one amino acids have been developed and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
used to prepare a series of analogs of S-1a and 2ac. In the case of
2
the I aa scaffold found in S-1a, methods have been conceived that
illustrate the value of 5-iodoindolizidin-2-ones, which were prepared
9
by transannular cyclization of unsaturated macrocycles. In contrast to
the corresponding 6-iodopyrroloazepinone counterpart,9a which served
effectively in S 1 chemistry to install a variety of side chains onto
N
the seven membered cycle,10 attempts to perform substitution chemistry
on the 5-iodoindolizidin-2-ones 16 and 17 resulted in elimination to
the corresponding unsaturated indolizidin-2-ones 18 and 19 (Scheme 1).
The latter have however provided valuable intermediates for the
synthesis
stereochemistry (e.g., R- and S-1a) and substituents on both the five
e.g., R- and S-10) and six (e.g., 5b–e) membered rings. In parallel,
of
indolizidin-2-ones
having
different
ring
fusion
(
the aza-amino acyl proline dipeptide of 2ac has been elaborated on the
aza-residue using a combination of submonomer chemistry in which side
chains were introduced by alkylation of an aza-glycine residue (e.g.,
6
cf),11 as well as diversification of the acetylene of an aza-
6
ACS Paragon Plus Environment

Page 7 of 128
Journal of Medicinal Chemistry
1
2
propargylglycine (azaPra) residue in solution (e.g., 6gl and 79). In
addition, to explore modification of the proline ring system, a solid-
phase approach has been introduced for the assembly of modulators in
which hydroxyproline has served to prepare a diverse set of analogs
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(e.g., 1113) using so called proline editing.12
The influences of analogs 113 have been studied on spontaneous
and PGF2α-induced contractions of the myometrium in mouse tissue removed
immediately after parturition.
The limited activity of many of the
analogs reflects significantly the relevance of conformation and the
impact of substituents on biological activity. Moreover, the
significant potency of the aza-Gly-Pro and aza-Phe-Pro derivatives 2a
and 2b as effective counterparts of S-1a has been further demonstrated
in a mouse model for preterm labor. In sum, the reported study has
highlighted novel methods for using the dual combination of aza-amino
2
acyl proline and I aa scaffolds as complementary tools for studying
peptide-based drug design and further illustrates the promise of aza-
Gly-Pro 2a and aza-Phe-Pro 2b as prototypes for delaying preterm birth.
RESULTS AND DISCUSSION:
2
Parallel development of methods to study the I aa and aza-amino
acyl proline components of S-1a and 2ac has focused on both the
conformation and substituents of theses dipeptide mimics. Initially,
2
methods were developed for the modification of the I aa ring fusion and
2
six-membered ring system. Considering that the I aa six-membered ring
would occupy the same position as the aza-residue of the aza-amino acyl
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
proline dipeptide, methods were also employed to diversify the
semicarbazide side chain. Finally, approaches were used for modifying
2
the five-membered rings of the I aa and Pro components.
5-Iodoindolizidine-2-ones 16 and 17 were respectively synthesized
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
via transannular cyclizations of 9-membered unsaturated lactams 14 and
5 (Scheme 1).9a,13 Phenylacetamide benzyl ester 14 was prepared from
1
the corresponding N-(Boc)amino methyl ester13 by a sequence featuring
treatment with HCl gas in dichloromethane, acylation using phenylacetyl
chloride and Hünig's base, saponification with LiOH, and carboxylate
alkylation with benzyl bromide and NaHCO (Experimental Section). In
3
spite of changes to an N-terminal amide and larger ester, transannular
cyclization of 14 with iodine in THF proceeded in the same fashion as
carbamate 15 with regiocontrol and diastereoselectivity to afford the
(3S,5R,6R,9S)-indolizidin-2-one 16 in 76% yield.9a
Attempts to add side chain diversity by displacement of iodides
16 and 17 were unsuccessful using various nucleophiles; instead,
elimination of the bridge head proton occurred to form enamine.
Enamines 18 and 19 were effectively prepared by iodide elimination
with triethylamine at 80 °C in acetonitrile; however, concomitant
loss of the Fmoc group necessitated protection with di-tert-
butyldicarbonate. Olefin reduction provided access to different ring
fusion stereochemistry. For example, hydride reduction of enamine
18 with sodium cyanoborohydride and catalytic acetic acid in EtOH
gave a 2:1 diasteroisomeric ratio in favor of the convex over the
concave isomers (e.g., R- and S-21: 57% and 21% yields).
8
ACS Paragon Plus Environment

Page 9 of 128
Journal of Medicinal Chemistry
1
2
3
Alternatively, hydrogenation of enamine 19 with palladium-on-carbon
in EtOH furnished selectively concave isomer S-20 in 86% yield.
Indolizidinone ring fusion stereochemistry was ascertained by NOESY
NMR experiments based on the observed transfer of magnetization
between the ring fusion and C3 protons in S-21, and confirmed by X-
ray crystallography (Figure 2).
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1: Synthesis of indolizidin-2-one analogs R-1a, S-1b, S-
1c and 5a
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Analogs of modulator S-1a were synthesized from unsaturated
-indolizidinone 18 and ring fusion stereoisomers 20 and 21.
∆5
Hydrogenolysis of benzyl ester 18 using palladium-on-carbon in EtOH
under a balloon of hydrogen for <10 min occurred with concomitant
olefin reduction to give a 1:1 mixture of acids 22 and S-23, which
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was used in the subsequent peptide coupling step.
Benzyl ester
R-21 was converted to acid R-23 under similar hydrogenolytic
conditions. Saponification of methyl ester 20 with LiOH in dioxane
gave carboxylic acid 24. The four acids 22, S-23, R-23 and 24 were
coupled to (S,S)-pyridinylalaninyl--homophenylalanine benzyl ester
(
25)1d using TBTU, HOBt, and DIEA to give protected peptides 2628
in 2667% yields.
In the interest of exploring diversity at the phenylacetamide
component as well as preparing a potential photoaffinity labeling
agent, p-azido- and p-aminophenylacetamide analogs S-1b and S-1c
were prepared by routes commencing with removal of the Boc group
from peptide 28 using HCl gas and acylation of the resulting
hydrochloride salt 29 using 2-(4-azidophenyl)acetic acid, TBTU,
HOBt, and DIEA in CH Cl to provide 4-azidophenylacetamide 30.
2
2
Hydrolysis of benzyl ester 30 with LiOH in dioxane gave acid S-1b.
Hydrogenolytic cleavages of benzyl esters 26, 27 and 30 using
palladium-on-carbon in ethanol under
a
balloon of hydrogen
10
ACS Paragon Plus Environment

Page 11 of 128
Journal of Medicinal Chemistry
1
2
3
furnished respectively modulator analogs 5a, R-1a and S-1c, which
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
were purified by preparative reverse-phase HPLC (Table 1).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2. Synthesis of 5-aryl-indolizidinone analogs 5be
2
The modification of the six-membered ring of the I aa moiety
was next explored by C-H bond activation and arylation at C5 using
palladium-catalysis (Scheme 2). Four aryl boronates were coupled to
enamine 18 using Pd(OAc) , 1,4-benzoquinone (BQ) and silver oxide
2
in THF at 80 °C to provide 31be in 4055% yields. Hydrogenolytic
cleavages of benzyl esters 31be, respective couplings of the
resulting acids 32be to dipeptide 25, and benzyl ester cleavages
as discussed for the synthesis of 5a gave the 5-aryl analogs 5be,
which were purified by preparative reverse-phase HPLC (Table 1).
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthesis of aza-Pra-Pro and aza-Phe-Pro peptides 2c
and 6df
2
In parallel to efforts to modify the six-membered ring of the I aa
residue, a set of methods was employed to append a series of side
chains on the aza-residue to make analogs of azapeptides 2ac.
Modification of the aza-residue side chain was first explored by
the synthesis and alkylation of aza-glycinyl proline t-butyl ester
34 using propargyl bromide, as well as p-methyl-, p-nitro- and
pentafluoro benzyl bromides (Scheme 3).11a The propargyl side chain
was selected for further diversification as discussed below. The
three benzyl groups were chosen to contrast the influences of
aromatic electron-density and hydrophobicity for activity with
those of 5-arylindolizidinones 5be. Aza-dipeptides 38cf, all were
prepared from the respective azaPra and azaPhe analogs 35c and 35df
by benzhydrylidene removal using hydroxylamine hydrochloride in
1
2
ACS Paragon Plus Environment

Page 13 of 128
Journal of Medicinal Chemistry
1
2
3
pyridine,
acylation
of
the
resulting
semicarbazide
with
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
phenylacetyl chloride, and tert-butyl ester removal in a TFA/DCM
solution. They were then coupled to dipeptide 25 using iso-butyl
chloroformate and N-methylmorpholine to furnish azapeptide benzyl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
esters 39cf, which after saponification with LiOH in dioxane and
RP-HPLC purification afforded aza-amino acyl prolyl peptides 6df
(Table 1).
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 4. Aza-triazole-alanine, aza-Asp and aza-Lys analog
synthesis by azaPra diversification
With azaPra peptide 39c in hand, diversity-oriented routes
were used to prepare a broader group of side chain analogs including
aza-1,2,3-triazole-3-alanine, aza-aspartate and aza-lysine analogs
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
9 to study the influences of charge and hydrogen-bonding at the
side chain for activity (Scheme 4). A set of six aza-1,2,3-triazole-
-alanines 40gl was synthesized from azaPra peptide 39c by copper-
3
catalyzed azide-alkyne cycloadditions. Among various methods
explored, 1,3-dipolar cycloadditions were best accomplished with
sodium azide and aryl azides using CuI and DIPEA in AcOH.14 After
benzyl ester saponification, triazole-alanine peptides 6gl were
purified by HPLC (Table 1). The azaPra peptide 39c was converted to
aza-aspartate
41
in
89%
yield
using
an
15
OsO /NaIO /hexamethylenetetramine-mediated oxidation.
Hydrolysis
4
4
of benzyl ester 41 and HPLC purification gave aza-Asp peptide 7.
Finally, in Mannich-like additions on the acetylene moiety,16
azaPra peptide 39c reacted in dioxane at 80 °C with copper iodide,
formaldehyde and a set of six secondary amines: dimethylamine,
pyrrolidine,
piperidine,
morpholine,
diallylamine
and
methylbenzylamine. After saponification as described above, the set
of aza-lysines 8mr was isolated by HPLC in 40 to 80% yields.
Acetylenes 8m and 8n were reduced using catalytic hydrogenation
14
ACS Paragon Plus Environment

Page 15 of 128
Journal of Medicinal Chemistry
1
2
3
with palladium-on-carbon to afford more flexible aza-Lys analogs 9m
and 9n.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
The five-membered ring of the I aa residue was modified by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
allylic oxidation of 5,6-dehydroindolizidin-2-one 18 with selenium
dioxide and TBHP in dioxane to give a separable mixture of 5- and
7
-hydroxy-dehydroindolizidin-2-ones 43 and 44 (Scheme 5). Olefin 43
was reduced using sodium cyanoborohydride and catalytic acetic acid
in EtOH at 80 °C to give a 3:1 mixture of convex and concave
diastereomers (e.g., R- and S-45, 44% and 26% yields) after
chromatography. The regio- and stereochemistry of 5- and 7-
hydroxyindolizidin-2-ones 4345 were assigned using COSY and NOESY
NMR spectroscopic experiments and X-ray crystallography of R-45
(
Figure 2). Employing
a
similar sequence of benzyl ester
hydrogenolysis and
couplings to dipeptide 25, 7-
hydroxindolizidinones R-46 and S-46 were respectively converted to
acids R-10 and S-10, which were purified by preparative reverse-
phase HPLC (Table 1).
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 5. Synthesis of 7-hydroxy-indolizidinones R- and S-10
In parallel, a set of proline residue analogs of azapeptides
2
ac was prepared using 4-hydroxyproline. The related 4-hydroxy
(Hyp), 4-fluoro- (Flp) and 4-aminoproline (Amp) analogs were
generated to study influences of the ring pucker, hydrophobicity
and substituents on activity.12,17 A solid-phase route was developed
featuring modification of Hyp azapeptides (R)-54bd (Scheme 6).
16
ACS Paragon Plus Environment

Page 17 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 6. Solid-phase synthesis of 4R- and 4S-Hyp-, Flp- and Amp-
azapeptides 1113
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N-Fmoc-Homophenylalanine (Fmoc-hPhe-OH) was coupled to Wang
resin by way of its symmetrical anhydride to provide -amino ester
resin 48. After capping unreactive sites with acetic anhydride,
linear tripeptide resin 49 was synthesized by Fmoc removals,
couplings of N-(Fmoc)pyridylalanine and (4R)-Fmoc-Hyp-OH, and
hydroxyl group silylation. Central to the success of the solid-
phase synthesis of azapeptides was the effective introduction of
the aza-residue using different N-Fmoc-aza-amino acid chlorides.18
To avoid formation of oxadiazolone in the synthesis of the aza-
glycinyl-proline analogs,19 the 4-ethoxybenzyl side chain was
employed as a temporary protecting group, which was removed during
cleavage of the azapeptide from the resin with TFA. The benzyl and
4-ethoxybenzyl side chains were added to the aza-residue by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reductive
aminations
of
semicarbazones
formed
from
the
corresponding aldehydes and fluorenylmethyl carbazate 50a in EtOH,
using palladium hydroxide-on-carbon in THF.18 Propargyl carbazate
50b was synthesized by alkylation of 50a with propargyl bromide and
lithium hydroxide in DMF.20 Activation of carbazates 50bd with
phosgene in toluene gave the corresponding N-Fmoc-aza-amino acid
chlorides, which were coupled to tripeptide resin 49.18 After aza-
amino acylation, the Fmoc group was removed from 52bd and the aza-
residues were phenylacetylated using the corresponding anhydride in
dichloromethane. The 4R-hydroxyl group was liberated from resins
18
ACS Paragon Plus Environment

Page 19 of 128
Journal of Medicinal Chemistry
1
2
3
53bd using tetra-n-butylammonium fluoride (TBAF) in THF and
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
converted to the 4S-alcohol S-54bd employing a two-step process
featuring Mitsunobu reaction with 4-nitrobenzoic acid, PPh3 and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
diisopropyl
azodicarboxylate
(DIAD)
in
THF,
followed
by
12
nitrobenzoate removal without Fmoc cleavage using NaN in MeOH.
3
With 4R- and 4S-hyroxyproline azapeptides R- and S-54bd in
hand, syntheses were pursued to make the diasteromeric 4S- and 4R-
Flp and Amp azapeptides R- and S-55bd and 57bd (Scheme 6).
Fluorination of 4R- and 4S-hydroxyprolines R- and S-54bd was
accomplished using diethylaminosulfur trifluoride (DAST) in
dichloromethane via S 2 chemistry with inversion of stereochemistry.
N
Aminoprolines S- and R-57bc were prepared from alcohols R- and S-
4bc by activation as the corresponding p-nitrophenylsulfonates,12
5
followed by S 2 displacement using NaN . Attempts to synthesize aza-
N
3
p-ethoxyphenylalaninyl azidoprolines S- and R-56d using the same
protocol were however unsuccessful, likely due to interactions
between the electron rich aromatic ring of the p-ethoxybenzyl group
and Hyp ring. Prolyl amide- interactions are known to favor cis-
amide bond isomers,21 which in the case of 56d may have prevented
activation of the hydroxyl group as a p-nitrophenylsulfonate. To
circumvent this obstacle, the 4S- and 4R-N-Fmoc-azidoprolines were
synthesized in solution, coupled onto the resin and elongated with
N-Fmoc-aza-4-ethoxyphenylalaninyl chloride and phenyl acetic
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
anhydride to obtain azidoproline peptides S- and R-56d. Azides S-
and R-56bd were reduced with tris(2-carboxyethyl)phosphine (TCEP)
to provide the corresponding amines S- and R-57bd. Finally, resin
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cleavage was performed using 95:5 TFA/H O to provide Hyp, Flp and
2
Amp azapeptides R- and S-1113ac after HPLC purification (Table
1).
The stereochemical assignments of bicycles 16, 21 and 43–45
were made using 2D NMR spectroscopy and X-ray crystallography
Supporting Information (SI)]. After the ring protons in the bicycle
[
were assigned using COSY experiments to ascertain through-bond
correlations, configuration assignments at the amino acid derived
α-carbons were correlated to the newly generated ring fusion, iodide
and alcohol bearing carbons using NOESY experiments to determine
through-space nuclear Overhauser effects (Figure 2).
Purity at
RT (min) in
MS [M+1]/[M+Na]
m/z
entry
Compounds
214/254
nm (%)
CH
3
OH
CH
3
CN
m/z (obs.)
(
calc.)
2
h
d
1
2
3
Phenylacetyl-(3S,6R,9S)-I aa-Pal-hPhe (R-1)
5.1
5.7
4.7
5.3
-
≥ 96
≥ 96
≥ 96
626.2973
689.2807
663.2902
626.2981
689.2817
663.2903
2
c
d
4-Azidophenylacetyl-(3S,6S,9S)-I aa-Pal-hPhe (S-1b)
4-Aminophenylacetyl-(3S,6S,9S)-I aa-Pal-hPhe (S-1c)
2
6.4 c
6.1 b
6.7 c
5.9f

2
d
d
d
h
4
5
6
7
Phenylacetyl-(3S,9S)-5- -I aa-Pal-hPhe (5a)
5.0
6.0
8.1
6.7
≥ 96
≥ 96
≥ 96
≥ 96
646.2636
722.2949
730.3235
725.3082
646.2641
722.2951
730.3262
725.3104

2
Phenylacetyl-(3S,9S)-5-Ph- -I aa-Pal-hPhe (5b)
Phenylacetyl-(3S,9S)-5-(4-methoxyphenyl)- -I aa-Pal-hPhe (5c)
Phenylacetyl-(3S,9S)-5-(4-cyanophenyl)- -I aa-Pal-hPhe (5d)

2

2
6.0 h
20
ACS Paragon Plus Environment

Page 21 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6

2
10.7 c
8.9d
8
9
Phenylacetyl-(3S,9S)-5-(4-trifloromethylphenyl)- -I aa-Pal-hPhe (5e)
Phenylacetyl-aza-(4-Me)Phe-Pro-Pal-hPhe (6d)
≥ 96
784.2952
784.2964
9.5g
7.3c
10.3 a
5.8e
4.4f
6.7g
8.9 b
7.7g
6.5h
5.1b
6.3d
5.4 b
5.0 d
6.0 d
6.3 d
4.7 d
6.1 d
≥ 96
≥ 99
≥ 99
≥ 99
≥ 99
≥ 97
≥ 97
≥98
705.3395
736.3086
787.2768
682.3096
758.3409
772.3566
776.3315
803.3259
705.3394
736.3091
787.2780
682.3111
758.3408
772.3581
776.3331
803.3276
1
0
2
Phenylacetyl-aza-(4-O N)Phe-Pro-Pal-hPhe (6e)
11
12
Phenylacetyl-aza-(pentafluoro)Phe-Pro-Pal-hPhe (6f)
Phenylacetyl-aza-(triazole)Ala-Pro-Pal-hPhe (6g)
1
1
1
1
3
4
5
6
Phenylacetyl-aza-(1-phenyltriazole)Ala-Pro-Pal-hPhe (6h)
Phenylacetyl-aza-(1-toluyltriazole)Ala-Pro-Pal-hPhe (6i)
Phenylacetyl-aza-[1-(4-fluorophenyl)triazole]Ala-Pro-Pal-hPhe (6j)
Phenylacetyl-aza-[1-(4-nitrophenyl)triazole]Ala-Pro-Pal-hPhe (6k)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
h
1
1
1
2
7
8
9
0
Phenylacetyl-aza-[1-(4-iodophenyl)]triazole-Ala-Pro-Pal-hPhe (6l)
Phenylacetyl-aza-Asp-Pro-Pal-hPhe (7)
Phenylacetyl-aza-[(N,N-dimethylamino)but-2-ynyl]Gly-Pro-Pal-hPhe (8m)
5.9
5.0
4.2
≥ 99
≥ 99
≥ 98
≥ 99
884.2376
659.2824
696.3504
722.3661
884.2383
659.2836
696.3509
722,3652
b
d
7.6
5.4
b
d
Phenylacetyl-aza-[(pyrrolidinyl)but-2-ynyl]Gly -Pro-Pal-hPhe (8n)
6.3d
5.8b
7.1j
2.2e
6.6 b
5.7b
5.7b
4.4d
4.4i
4.4i
4.6i
4.9 d
4.4d
6.6d
2
2
2
1
2
3
Phenylacetyl-aza-[(piperidinyl)but-2-ynyl]Gly-Pro-Pal-hPhe (8o)
Phenylacetyl-aza-[(morpholino)but-2-ynyl]Gly-Pro-Pal-hPhe (8p)
Phenylacetyl-aza-[(N,N-diallylamino)but-2-ynyl]Gly-Pro-Pal-hPhe (8q)
≥ 99
≥ 99
≥ 99
736.3817
738.3610
748.3817
736.3822
738.3620
748.3821
2
2
4
5
Phenylacetyl-aza-[(N-methyl,N-benzyl)but-2-ynyl]Gly-Pro-Pal-hPhe (8r)
Phenylacetyl-aza-( N,N-dimethyl)Lys-Pro-Pal-hPhe (9m)
Phenylacetyl-aza-[(pyrrolidinyl)butyl]Gly-Pro-Pal-hPhe (9n)
≥ 99
≥ 99
˃ 99
772.3817
700.3817
726.3974
772.3840
700.3814
726.3980
26
27
28
2
5.6 b
4.5 d
Phenylacetyl-(3S,6S,7R,9S)-7-hydroxy-I aa-Pal-hPhe (R-10)
≥ 96
664.2742
664.2712
2
b
d
Phenylacetyl-(3S,6S,7S,9S)-7-hydroxy-I aa-Pal-hPhe (S-10)
5.2
6.7
6.5
4.4
4.4
5.8
≥ 96
˃ 99
≥ 96
664.2742
617.2718
707.3188
664.2748
617.2698
707.3208
d
d
2
3
9
0
Phenylacetyl-aza-Gly-(4R)-Hyp-Pal-hPhe (R-11a)
Phenylacetyl-aza-Phe-(4R)-Hyp-Pal-hPhe (R-11b)
e
d
3
1
Phenylacetyl-aza-Pra-(4R)-Hyp-Pal-hPhe (R-11c)
5.6k
5.2d
≥ 99
655.2875
655.2892
3
3
3
3
3
3
2
3
4
5
6
7
Phenylacetyl-aza-Gly-(4S)-Hyp-Pal-hPhe (S-11a)
Phenylacetyl-aza-Phe-(4S)-Hyp-Pal-hPhe (S-11b)
Phenylacetyl-aza-Pra-(4S)-Hyp-Pal-hPhe (S-11c)
Phenylacetyl-aza-Gly-(4S)-Flp-Pal-hPhe (S-12a)
6.9 d
6.7c
8.1 d
7.6d
4.5 d
5.8d
5.2 d
6.2d
≥ 99
≥ 99
≥ 99
~82
≥ 99
≥ 99
≥ 95
≥ 97
≥ 95
≥ 99
617.2718
707.3188
655.2875
619.2675
617.2728
707.3217
655.2878
619.2697
d
d
Phenylacetyl-aza-Phe-(4S)-Flp-Pal-hPhe (S-12b)
Phenylacetyl-aza-Pra-(4S)-Flp-Pal-hPhe (S-12c)
Phenylacetyl-aza-Gly-(4R)-Flp-Pal-hPhe ( R-12a)
Phenylacetyl-aza-Phe-(4R)-Flp-Pal-hPhe ( R-12b)
Phenylacetyl-aza-Pra-(4R)-Flp-Pal-hPhe (R-12c)
Phenylacetyl-aza-Gly-(4S)-Amp-Pal-hPhe ( S-13a)
7.6
5.4
6.8
9.5
6.0
5.3
4.6
6.2
709.3144
657.2831
619.2675
709.3144
657.2831
616.2878
709.3162
657.2857
619.2678
709.3146
657.2834
616.285
d
d
d
d
38
d
d
3
4
4
9
0
1
d
d
8.3
5.5
5.2 d
8.4 d
7.7 d
5.6 d
8.4 d
7.2 d
4.9 d
5.2 d
6.1l
5.0 d
5.2 d
4.6 d
4
4
4
4
4
2
3
4
5
6
Phenylacetyl-aza-Phe-(4S)-Amp-Pal-hPhe (S-13b)
Phenylacetyl-aza-Pra-(4S)-Amp-Pal-hPhe (S-13c)
Phenylacetyl-aza-Gly-(4R)-Amp-Pal-hPhe (R-13a)
Phenylacetyl-aza-Phe-(4R)-Amp-Pal-hPhe (R-13b)
Phenylacetyl-aza-Pra-(4R)-Amp-Pal-hPhe (R-13c)
≥ 95
~91
706.3348
654.3035
616.2878
706.3348
654.3035
706.3349
654.3038
616.2852
706.3356
654.3039
≥ 95
≥ 99
≥ 99
Table 1. Purity, retention times and mass spectrometric analyses
of I aa and aza-Xaa-Pro analogs
Isolated purity determined by LC-MS analysis using gradients of X-Y% A [MeOH (0.1% FA)/B
O (0.1% FA)/] or B [MeCN (0.1% FA)/H
0% A or B/14; c30-95% A or B /14; d10-90% A or B/14/15; e40-90% A or B /12; f50-90% A or
B /14; g50-80% A or B /14, h30-90% A or B /14; i5-80% A or B /14; j30-80% A or B /12;
k40-70% A or B/15; l5-90% A or B/15.
2
H
8
2
2
O (0.1% FA)/] over Z min: a70-90% A or B /20; b20-
Previously,
3-N-(Fmoc)amino-5-iodo-indolizidinone-9-
carboxylate 17 (Scheme 1) was assigned based on observation of
magnetization transfer between the C3, C5 and C6 protons.9a 3-
Phenylacetamide 16 exhibited similar long range nuclear Overhauser
effects indicating that such modification at the 3-position
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
nitrogen had no effect on the regio- and diastereoselectivity of
the transannular cyclization.
Similarly, magnetization transfer
between the C3 and C6 protons was observed in the NOSEY spectrum of
indolizidinone (6S)-21, the stereochemical assignment of which was
confirmed by X-ray crystallography.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
I
H
N
H
N
O
O
H
H
Ph
Ph
N
H
N
H
O
OBn
O
OBn
O
O
16
S-21
X
H
H
HO
H
H
H
O
H
O H
Ph
N
H
N
Ph
N
H
N
O
OBn
H
O
O
OBn
O
R-21: X = H
R-45: X = OH
S-45
X-ray R-45
Figure 2. Assignment of relative stereochemistry by NOSEY
correlations and X-ray structures
The protons on carbons bearing hydroxyl groups were
consistently up-field of those on nitrogen bearing carbons. Similar
to the C3-protons of proline analogs,22 the C8 proton appeared
consistently up-field of the C8 proton due to an anisotropic
effect caused by the C9 carboxylate. The R-configuration of the
ring fusion C6-protons in (6R)-21 and (6R)-45 were assigned based
on the observed transfer of magnetisation with the C8 proton. The
assignment of alcohol (6R)-45 was consistent with allylic oxidation
from the less sterically hindered face of olefin 18 and confirmed
22
ACS Paragon Plus Environment

Page 23 of 128
Journal of Medicinal Chemistry
1
2
3
by X-ray crystallography; the alcohol stereochemistry of (5S)-44 is
based similarly on the facial selectivity of the oxidation. In their
X-ray crystal structures, the dihedral angle values of the backbone
within bicycles S-21 and R-45, both resembled those of the central
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
residues of an ideal type II -turn (Table 2).
Table 2. Comparison of dihedral angles from X-ray analysis and
ideal type I and II′ β-turns
i+1
°
,
i+2
°
,
i+2
,
°
i+1, °
Type -turn
2
23
Boc-(3S,6S,9S)-I aa-OMe
158
139
176
175
173
78
71
75

2
PhAc-(3S,6S,9S)-I aa-OBn (S-
144
148
21)

120
2
PhAc-7-OH-(3S,6R,7S,9S)-I aa-
OBn (R-45)

134
142
64
157
Boc-aza-Ala-Pro-NHi-Pr24
68
60
18
140

58
89

80
25
9
Pivaloyl-D-Ala-Pro-NHi-Pr25
Ideal type I -turn26
Ideal type II′ -turn 26
60
0
60
120
0
Structure activity relationship studies
Previously, the indolizidinone peptide mimic S-1a (PDC113.824)
exhibited high efficacy (98% inhibition) and potency (IC50 = 1.1
nmol/L) in a porcine ocular micro-vessel assay, and delayed labor
significantly
in
murine
models
induced
respectively
with
lipopolysaccharide and PGF2.1d,27b Modifications of S-1a by
2
replacement of the (3S,6S,9S)-I aa residue by its D-enantiomer
2
[(3R,6R,9R)-I aa], and related indolizidin-9-one, quinolizidinone,
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and pyrroloazepinone, 5,6-, 6,6- and 7,5-fused bicycles (e.g., 3a
and 4a),1d,27 and a ten-membered macrocycle system,27a as well as
examination of the enantiomer of S-1a,1d,27b all gave analogs that
exhibited no inhibition of myometrial contractions. On the other
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hand, aza-amino acyl proline analogs 2ac displayed activity
contingent on the nature of the aza-amino acid side chain, and
reduced the tension and duration of PGF2-induced myometrial
contractions with similar efficacy as S-1a.1d,27b
Moreover,
modulators possessing Gly-Pro and D-Ala-Pro residues were
previously shown to have lower activity than aza-Gly-Pro 2a.27a
Further investigation of the turn regions of S-1a and 2 was thus
pursued in parallel to provide more detailed structure activity
relationships for guiding development of novel modulators of FP as
potential tocolytic agents for inhibiting preterm labor.
The activity of S-1a in the myometrial contraction assay proved
to be very sensitive to structural modifications (Figure 3). For
example, replacement of the phenyl acetyl moiety by p-azido and p-
amino counterparts (e.g., S-1b and S-1c) diminished activity,
respectively. Changing the ring fusion stereochemistry from the
2
2
(3S,6S,9S)-I aa residue in S-1a to the (3S,6R,9S)-I aa isomer in R-1a
reduced significantly the activity, which was one third that of S-1a at 10 M (Figure S2, SI).
5
2
Flattening the ring in ∆ -I aa analog 5a improved activity relative to R-1a, but gave only half the
24
ACS Paragon Plus Environment

Page 25 of 128
Journal of Medicinal Chemistry
1
2
3
2
activity of S-1a at 1 M. A concave I aa residue was thus shown to be a prerequisite for high
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Effects of indolizidinone S-1a and azapeptide 2a analogs on mean
tension induced by PGF2. At the start of each experiments, mean tension of
spontaneous myometrial contractions are considered as a reference response.
Increase in mean tension (%) was expressed as percentages of (X/Y)-100, where X
is changes in mean tension (g) induced by PGF2 and Y is the initial reference
response (g). Results are expressed as % inhibition, calculated as (A-B)/A, where
A is the increase in mean tension induced by PGF2 in the absence of a 20 min
pretreatment with FP modulators and B is the increase in mean tension induced
by PGF2 in the presence of a 20 min pretreatment with FP modulators. Data are
representative of 5 experiments per treated group and presented as means ± SEM.
5
2
The addition of aromatic rings to the 5-position of ∆ -I aa
analog 5a demonstrated tolerance for the phenyl group in 5b, but
not for larger substituents (e.g., 4-methoxy, 4-cyano and 4-
trifluoromethoxyphenyl analogs 5c5e), which caused a complete loss
of activity. In contrast to our previously reported FP modulators,
which do not typically exhibit effect in the absence of exogenous
5
2
PGF2, 5-(4-trifluoromethoxyphenyl)-∆ -I aa 5e exhibited activity
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
in the presence of the endogenous orthosteric ligand (Figure S3,
SI), which may be due to BKCa potassium channel activation.27c The
influence of substitution on the 6-membered lactam of 5a may be
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compared with the effects of different side chains on azapeptide
1d
2
a, on which benzyl and propargyl residues were tolerated. Attempts
to introduce larger and less electron rich aromatic side chains
onto azapeptide 2a (e.g., 6dl) resulted in loss of activity
correlating with the inactivity of 5ce. In the cases of 6f and 6k,
the analogs were insoluble as the zwitterion and examined as the
sodium salts, which exhibited half the activity of 2a at 10 M
(Figure S2, SI). Examination of the activity of azapeptides 79
featuring carboxylate and basic amine side chains yielded aza-4-
piperidinobutynylglycine 8o, which exhibited similar activity as
5
a, and aza-4-(N,N-dimethylamino)butynylglycine 8m, which was
inactive at 1 M but showed about half the activity of 2a at 10 M
Figure S2, SI), demonstrating further the restrictive size
(
requirement and need for a lyophilic moiety at this residue.
The conformational preferences of aza-amino acyl proline
residues have been examined to a limited extent in dipeptide models
by X-ray crystallographic and spectroscopic methods.24,28 For example,
X-ray crystallographic analyses of N-Cbz-azaAsn(Me)-Pro-NHi-Pr, N-Cbz-azaAsp(Et)-Pro-NHi-
Pr, N-Boc-azaAla-Pro-NHi-Pr (Table 2), and N-i-Pr-azaAla-Pro-Ot-Bu, all indicated that in the solid
state, the aza-residue exhibited R-like chirality and the aza-amino acyl proline
26
ACS Paragon Plus Environment

Page 27 of 128
Journal of Medicinal Chemistry
1
2
3
dipeptide adopted respectively  and  dihedral angle values indicative of the i + 1
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and i + 2 residues of an ideal type I -turn.24, 28 Although the type I -turn has been described as
an energy minimum adopted by model azapeptides (e.g., Ac-aza-Gly-Ala-NHMe and Ac-aza-Ala-
Ala-NHMe) in computational analyses, the corresponding type II -turn was less than one kcal
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
29
higher in energy. Examination of N-i-Pr-azaAla-Pro-Ot-Bu in solution at low temperature (–90°C
in CD Cl ) by NMR spectroscopy indicated four methyl singlets corresponding to two pairs of prolyl
2
2
amide cis and trans isomers with one major trans amide conformer representing two-thirds of the
total population.28 At ambient temperature, N-i-Pr-azaAla-Pro-Ot-Bu exhibited infrared spectra in
-1
hexane and CH Cl exhibiting strong concentration independent absorptions at 3260 cm
2
2
corresponding to an intramolecular hydrogen bond involving the azaAla NH and semicarbazide
carbonyl oxygen indicating a more extended conformation.28 Potential for conformational liberty
and adoptive chirality may both account for the activity of 2ac and related aza-amino acyl proline
2
analogs, which may assume backbone geometry similar to the I aa residue in S-1a.
The influence of a ring substituent on the proline residue in
S-1a and 2 was next explored typically with limited success. For
2
example, neither (6R,7R)- nor (6S,7R)-7-hydroxyl-I aa analogs R-
and S-10a exhibited activity in the myometrial contraction assay.
Similarly, among the (2S,4R)- and (2S,4S)-4-hydroxyproline analogs
R- and S-11ac, only aza-Pra-4R-Hyp R-11c exhibited activity, which
was comparable to azapeptide 2a. Among the (2S,4R)- and (2S,4S)-
4-fluoroproline analogs R- and S-12ac, only aza-Gly-4S-Flp S-12a
exhibited activity. Moreover, aza-Gly-4R-Amp R-13a and aza-Phe-4S-
Amp S-13b retained activity among the 4-aminoproline analogs.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 128
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
-Position substituents on proline have been shown to
influence both the ring pucker and prolyl amide isomer
equilibrium.30,31 (2S,4R)-Hydroxy- and fluoroprolines exhibit C -exo
puckers and higher trans-amide populations than their (2S,4S)-
γ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
γ
30
counterparts which favor a C -endo pucker.
(4S)‐Aminoproline has
been shown to fluctuate between ring puckers contingent on pH with
γ
31
amine protonation favoring the C -endo pucker. Considering that
active analogs were observed with both (2S,4R)- and (2S,4S)-
stereochemistry contingent on the aza-residue side chain, the 4-
position substituent may influence factors other than receptor
engagement, such as cell permeability, which may influence
performance in the ex vivo myometrial contraction assay.
Figure 4. Tocolytic action of azapeptides 2a and 2b in LPS-induced preterm labor
in mice. (Left) Percentage of animals delivered following injection of LPS (B,
.4 mg/kg by intraperitoneal injection) in the presence or absence of azapeptides
a and 2b (20 mg/kg/day). Pregnant mice were treated with azapeptides 2a and 2b
20 mg/kg/day) starting on day 16 of gestation. Control mice were injected with
saline. Bars are presented at 12, 15–24, 24–48 h, and 72 h. The time in hours
refers to time of delivery following LPS treatment. (Right) Average delivery
time after LPS treatment. Data are representative of 5–6 animals per treated
group and n=10 for the saline-injected animals. Date given in (left) were compared
by Chi-Square analysis and data in (right) are presented as mean± SEM and were
compared by Independent t-tests compared. Statistical tests were performed with
GraphPad Prism 4.3 software and p value < 0.05 was considered statistically
significant. * p < 0.05 compared with saline-treated.
1
2
(
28
ACS Paragon Plus Environment

Page 29 of 128
Journal of Medicinal Chemistry
1
2
3
4
5
Finally, the tocolytic effects of azapeptides 2a and 2b were
examined in a preterm labor model in which mice near term were
treated with azapeptide and lipopolysaccharide (LPS) Escherichia
coli endotoxin, which is known to promote a general inflammatory
state resulting in prostaglandin synthesis, and induced premature
delivery.32 Following LPS injection into mice at gestational day 16,
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
all the animals tested delivered within 1224 h (Figure 4).
Pretreatment with azapeptide prior to LPS injection significantly
delayed delivery, such that by gestational day 17, <20% of treated
animals delivered. Azapeptide-treated mice did not deliver until
day 19 even after treatment with LPS. Azapeptides 2a and 2b extended
significantly (>20 h) the average time of delivery following LPS
treatment compared with untreated animals (Figure 4b). Considering
the results with azapeptides 2a and 2b in the ex vivo myometrial
contraction assay and in vivo preterm LPS model, their ability to
delay preterm labor is due in part through the inhibition of uterine
contraction.
Conclusion:
The relationships between structure and function of novel
modulators of FP has been studied towards the development of
potential tocolytics for delaying premature birth. Commencing from
the understanding that indolizidin-2-one amino acids and aza-amino
acyl proline derivatives share a common preference for mimicry of
the central residues of type II’ -turns,1 diversity-oriented
29
ACS Paragon Plus Environment